Percentage Yield and Saturated Solubility
Prepared batches (B1-B9) were evaluated for percentage yield and saturated solubility and the data are summarized in Table 4. The percentage yield was found to be more than 90% in all batches (Table 4) and was highest with B9. On the other hand, the saturated solubility of RXN significantly improved with the addition of carriers (PEG 4000 and Neusilin US2), and also showed improvement with increase in carrier content. The highest RXN solubility noticed was 0.041 mg/mL, which is 3.4 times higher as compared to solubility of pure drug in water (0.012 mg/mL). This improvement in solubility of RXN could be due to the potential of hydrophilic PEG 4000 (carrier) to improve the wettability of the drug (44) as well as the Neusilin US2 (adsorbate), which increases the surface area of the formulation (45, 46).
Table 4
Percentage yield and saturation solubility of different solid dispersion adsorbate batches
Batch No.
|
% Yield
|
Solubility (mg/mL)
|
B1
|
95.83±1.02
|
0.026±0.005
|
B2
|
96.03±1.68
|
0.029±0.001
|
B3
|
95.84±0.86
|
0.038±0.006
|
B4
|
95.84±0.23
|
0.027±0.006
|
B5
|
97.45±1.45
|
0.038±0.003
|
B6
|
96.90±0.54
|
0.041±0.007
|
B7
|
96.83±0.49
|
0.023±0.001
|
B8
|
97.54±1.05
|
0.028±0.004
|
B9
|
98.23±0.36
|
0.031±0.002
|
Flow Characteristics of SDA
The results of angle of repose, bulk density, tapped density, Carr’s index and Hausner’s ratio of different batches of SDA are presented in Table 5. The data here signifies all the prepared batches possess adequate flow properties and could be used for compression of tablets.
Table 5
Flow characteristics of different solid dispersion adsorbate batches
Batch
No.
|
Angle of repose (°)
|
Bulk density (g/cm3)
|
Tapped density (g/cm3)
|
Carr’s index
(%)
|
Hausner’s ratio
|
B1
|
27.78±0.16
|
0.68±0.05
|
0.79±0.07
|
13.85±1.03
|
1.15±0.01
|
B2
|
21.64±0.11
|
0.43±0.02
|
0.53±0.03
|
18.12±0.10
|
1.21±0.01
|
B3
|
17.25±0.29
|
0.42±0.02
|
0.46±0.03
|
9.25±0.65
|
1.09±0.01
|
B4
|
20.92±0.11
|
0.53±0.03
|
0.59±0.04
|
10.82±0.80
|
1.11±0.01
|
B5
|
20.41±0.21
|
0.46±0.03
|
0.51±0.03
|
9.96±0.58
|
1.10±0.01
|
B6
|
17.73±0.31
|
0.43±0.02
|
0.51±0.05
|
9.62±0.65
|
1.17±0.08
|
B7
|
22.07±0.24
|
0.79±0.07
|
0.94±0.09
|
16.15±1.47
|
1.18±0.02
|
B8
|
21.40±0.18
|
0.57±0.04
|
0.75±0.07
|
22.78±0.44
|
1.22±0.11
|
B9
|
18.10±0.14
|
0.43±0.02
|
0.50±0.05
|
9.62±0.64
|
1.11±0.07
|
Characterization of SDA
FTIR
The FTIR spectra of pure RXN, PEG 4000, Neusilin US and SDA are presented in Figure 1. The prominent peaks of various functional group of RXN were observed at 3351 cm−1 (N-H stretch), 1736 cm−1 (C=O stretch carbonyl group), 1646 cm−1 (C=O stretch ester group), 1516 cm−1 (N-O stretch), 828 cm−1 (Benzene stretch). From the figure, it is observed that the infrared spectrum of pure drug and SDA have significant difference in the absorption peaks intensity. A slight shift towards the lower wavelength or broadening was noticed in few peaks.
XRD
The diffraction spectra of RXN, PEG 4000, Neusilin US2 and solid dispersion of RXN adsorbate are shown in Figure 2. The graph of pure RXN showed high crystalline nature of the drug with main diffraction peaks. The X-ray diffractogram of SDA didn’t show any characteristic peaks which are observed in pure drug diffraction patterns, which indicates transformation of crystalline to 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 pertaining to improvement in dissolution of RXN.
DSC
The DSC thermogram of pure drug, Neusilin US2, PEG 4000 and SDA are presented in Figure 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.
SEM
SEM images were captured to observe the surface morphology of the SDA (Figure 4). The SEM of the RXN powder were found to be irregular in shape as well as exist as crystalline particles in the aggregates. It has been described that Neusilin US2 is porous in nature with numerous inter and intra particle apertures exists on its surface that can contribute enormous surface area and eventually helps in greater adsorption (47). Further, the SEM images in Figure 4 also demonstrated total adsorption of PEG 4000 and RXN on the surface of Neusilin US2. This observation confirms that the particles in the prepared SDA did not aggregate and possess free flowing properties.
Evaluation 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 results of immediate release SDA tablets are shown in Table 6. Thickness of SDA tablets ranged from 3.10±0.02 to 3.48±0.02 mm while the diameter of tablets was found to be in the range of 8.10 ± 0.001 to 8.19 ± 0.001 mm, hence all tables are in the acceptable range. Similarly, all formulations showed uniformity of weight within the pharmacopoeia limits for uncoated tablets. Hardness of SDA tablets ranged from 2.41±0.20 to 2.83±0.28 kg/cm2, which assures the tablets withstand during handling and transportation and also can bear the wear and tear. Friability was in the range of 0.32±0.02 to 0.48±0.05%, which ensures acceptable resistance by tablets to withstand mechanical shocks and can withstand wear and tear. Disintegration time was ranging between 25.33±0.57 to 44.23±1.52 sec. Batches B1, B4 and B7 which contain no adsorbate showed more disintegration time (44, 41 and 43 sec), rest of batches which contain adsorbate showed less disintegration time (Table 6). From the results it was clear that the presence of adsorbate in formulation decreases disintegration time due to increased surface area. The percentage drug content of all the batches of SDA were found to be between 96.15 – 99.60%, which are in acceptable range.
Table 6
Evaluation of physicochemical properties of immediate release SDA tablets
Batch
No.
|
Average weight (mg)
|
Thickness (mm)
|
Hardness
(kg/cm2)
|
% Friability
|
Disintegration time (sec)
|
Drug content (%)
|
B1
|
302±4.02
|
3.10±0.02
|
2.83±0.28
|
0.48±0.05
|
44.23±1.52
|
101.12±1.62
|
B2
|
301±4.24
|
3.43±0.01
|
2.60±0.17
|
0.53±0.04
|
34.33±0.57
|
102.35±1.45
|
B3
|
301±4.10
|
3.33±0.01
|
2.50±0.26
|
0.58±0.01
|
26.00±2.64
|
100.52±1.04
|
B4
|
303±3.74
|
3.48±0.02
|
2.70±0.20
|
0.46±0.02
|
41.33±1.52
|
102.45±1.24
|
B5
|
302±3.19
|
3.31±0.01
|
2.55±0.14
|
0.40±0.01
|
29.33±2.08
|
104.12±0.65
|
B6
|
300±2.97
|
3.44±0.01
|
2.41±0.20
|
0.32±0.02
|
25.33±0.57
|
103.14±1.68
|
B7
|
303±3.27
|
3.19±0.01
|
2.73±0.12
|
0.45±0.03
|
43.66±2.08
|
104.68±0.63
|
B8
|
302±3.34
|
3.41±0.01
|
2.63±0.20
|
0.34±0.04
|
31.00±2.64
|
105.12±1.64
|
B9
|
300±2.70
|
3.14±0.01
|
2.56±0.21
|
0.32±0.02
|
27.66±0.57
|
104.26±1.38
|
In Vitro Dissolution
Drug release profile of all the SDA tablets was shown in Figure 5. The release patterns of all batches showed fast dissolution. Batches (B3, B5, B6, B8 and B9) exhibited more than 85% dissolution in 10 min. In all the batches concentration of carrier (X1) and concentration of adsorbate (X2) played an important role in drug dissolution rate. The pure drug showed weak dissolution rate (p < 0.001) due the absence of carrier and adsorbate and hence the low solubility of RXN in aqueous phase resulted in the slow release of the drug. Batches B1, B4, and B7 showed decrease in dissolution rate compared to other batches due the absence of adsorbate in those batches but they have more dissolution rate than pure drug, which confirmed the formation of solid dispersion. Batches B2, B5, B8, B10 and B11 showed increased percentage drug release due to the presence of adsorbate, which helped in increasing the surface area of formulation and thereby increasing dissolution. Batches B3, B6 and B9 showed increased percentage drug release due to the higher concentration of adsorbate in those batches. The enhancement in the dissolution of RXN from SDA tablets formulations can be ascribed due to several factors like enhanced surface area offered for drug release, improvement in aqueous solubility of the drug, and increased wettability of the drug particles. The immediate sinking of the particles was noted during the dissolution studies.
Fitting Data to the Model
A 2-factor, 3-level experimental design was utilized as the RSM needed 9 experiments. The responses observed with all the nine batches were concurrently fit to a quadratic model using design expert software version 11.0. The independent variables and response of dependent variables are shown in Table 7. The best fit model observed was the quadratic model. A positive number indicates an effect that favors the optimization, on the other hand, a minus number suggests that the relation between factor and response is negative. It is evident that both the independent variables, viz., the amount of carrier (X1) and the amount of adsorbate (X2) have positive effects on the responses, viz., t85 (time required for 85% drug release) and saturated solubility.
Table 7
Design layouts with respective observed response
Batch
No.
|
X1 amount of carrier
|
X2 amount of
adsorbate
|
Y1 = t85%
(min)
|
Y2 =saturated solubility (mg/mL)
|
B1
|
-1
|
-1
|
16.10
|
0.026
|
B2
|
-1
|
0
|
15.20
|
0.029
|
B3
|
-1
|
+1
|
10.15
|
0.038
|
B4
|
0
|
-1
|
15.30
|
0.027
|
B5
|
0
|
0
|
9.40
|
0.038
|
B6
|
0
|
+1
|
9.10
|
0.041
|
B7
|
+1
|
-1
|
14.45
|
0.023
|
B8
|
+1
|
0
|
9.30
|
0.028
|
B9
|
+1
|
+1
|
8.55
|
0.031
|
Data Analysis of Y1 (Time Required for 85% Drug Release)
The observed values of t85 for all the 9 batches varied from 8.55 to 16.10 min. The outcome certainly indicated that Y1 is deeply affected by the amount of carrier and amount of adsorbate for study. The batch 9 showed the highest t85 and batch 1 gave the minimum t85. The response (Y1) obtained for two independent variables was exposed to multiple regression to get quadratic polynomial equation:
Y1 (Full model) =+10.62 -1.53X1 -3.01X2 +0.0125X1X2 +1.03X12 +0.975X22
The above equation clearly illustrates the broad range of numbers for different coefficients. The coefficient value for independent variable X1 (-1.53) signifies the positive response on the dependent variable Y1, so increasing carrier amount led to decrease in time required for 85% drug release. This is due to the potential of hydrophilic PEG 4000 to improve the wettability of the drug as described in the literature (44). Similarly, the coefficient value for X2 (-3.01) shows the positive response on drug release, i.e. on increasing adsorbate amount leads to decrease in the time required for 85% drug release. The possible explanation for this observation could be due to the potential of Neusilin US2 to enhance the dissolution rate by increasing the effective surface area (46) and/or conversion of crystalline RXN to amorphous state (48) and/or favored rapid diffusion of molecularly dispersed drug through the pores (28). The regression coefficients having P value < 0.05 are considered as highly significant. The term X2 with P value < 0.05 was significant in contributing to prediction of time required for 85% drug release. The reduced equation for RXN can now be written as:
Y1 (Reduced model) =+10.62 -3.01X2
The independent variable X2 (amount of Neusilin US2) was found significant (P < 0.05) in affecting Y1 (t85%). Figure 6a shows contour plot and 2b exhibits 3d surface response plot of time required for 85% drug release. It is clearly observed from the graphs that as the amount of carrier and amount of adsorbate increases, the time required for 85% drug release decreases.
Data Analysis of Y2 (Saturated Solubility)
The observed values of saturated solubility for all 9 batches varied from 0.023- 0.041 mg/mL. The results clearly showed that Y2 is also affected by the amount of carrier and amount of adsorbate in the study. Batch 6 showed highest solubility (0.041 mg/mL) and batch 7 gave minimum solubility (0.023 mg/mL). The response (Y2) obtained for two independent variables was subjected to multiple regression to get quadratic polynomial equation:
Y2 (full model) =+0.0358-0.0018X1+0.0057X2-0.0010X1X2-0.0062X12-0.0007X22
It is evident from the above equation that there are wide range of values for various coefficients. The X2 and X12 (P<0.05) were found to be significantly affecting response Y2. The coefficient value for independent variable X1 (-0.0018) showed the negative response on the dependent variable Y2, i.e. on increasing carrier amount from 4 mg to 6 mg, it leads to increase in saturated solubility but further increasing from 6 mg to 8 mg, it decreases saturated solubility. The coefficient value for X2 (0.0057) indicates the positive effect on response Y2, i.e. on increasing adsorbate amount leads to marginal increase in saturated solubility. The terms X2 and X12 having P value < 0.05 were significant in contributing in estimation of particle size. The reduced equation for Y2 can now be described as:
Y2 (Reduced model) =+0.0358+0.0057X2+0.0001X12
Figure 7a shows 2D contour plot and 3b exhibits 3D response surface plot of saturated solubility. It is clearly observed from the graphs that the amount of carrier initially increases the saturated solubility up to 6 mg and then decreases while the amount of adsorbate increases saturated solubility also increases.
Overlay and Checkpoint Analysis
The overlay plot helped 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. The Figure 8 showed the favored ranges of the variables and their responses are as follows: time required for 85% release (9.27 min) and saturated solubility (0.0395 mg/mL). Also, the design space provided a range for the factors: amount of carrier (4–8) and amount of adsorbate (0-3).
Optimization
The optimized batch was selected from an overlay plot of response variables and various solutions were analyzed. The formulation composition with amount of carrier and amount of adsorbate were observed to fulfil the required properties of an optimum formulation of immediate release parameter. The composition of optimized solid dispersion tablets is given in Table 8. The solid dispersion for the final formula was composed of 0.435 g of RXN, 6 g of PEG 4000 and 3 g of Neusilin US2.
Table 8
Formulation table of optimized batch of solid dispersion tablets
Ingredients
|
Quantity (mg)
|
Solid dispersion adsorbate
|
216
|
Crospovidone
|
15
|
Micro crystalline cellulose
|
64
|
Talc
|
2.5
|
Magnesium stearate
|
2.5
|
Total weight
|
300 mg
|
Evaluation of Optimized SDA
The optimized SDA of RXN was prepared and evaluated for flow property. The angle of repose of optimized formula was found to be 17.25±0.29o, which show excellent flow, the Hausner’s ratio was found to be 1.17±0.08, which show excellent flow and Carr’s index was found to 9.20±0.65, which also depicts excellent flow property. Saturated solubility study was performed on SDA of RXN. The saturated solubility was found to be 0.041±0.0007 mg/mL in distilled water which was more than the pure drug because of the presence of Neusilin US2 in the batch which increased the surface area of the particles and hence the solubility of the formulation also enhanced. The drug content of optimized SDA was found to be 98.61%±1.18, which was in the acceptable range and confirms the integrity of the drug in the formulation. The optimized SDA of RXN was evaluated for percentage yield in the ratio obtained through overlay plot and was found to be 95.84±0.86%.
Evaluation of Optimized SDA Tablet
The optimized SDA tablet had been evaluated for its average weight (300±2.97 mg), thickness (3.44±0.01 mm), hardness (2.41 kg/cm2), friability (0.32±0.02%) and disintegration time (26±2.64 sec). All the parameters were in the acceptable range. In vitro drug release profile of pure RXN, immediate release SDA tablet, marketed product and directly compressible tablet are shown in figure 9. From the in vitro release patterns, it was clear that, optimized SDA tablet showed the fastest dissolution rate (P < 0.0001) as compared to other tested formulations. The optimized batch showed more than 85% dissolution in 10 min. The enhancement in the dissolution of RXN from SDA tablets can be due to several factors like enhanced drug surface area available for release, an improved wettability of the drug particles leading to an increased aqueous solubility of the drug.
Cell Viability Test
The cytotoxicity exhibited by the RXN SDA tablet and suspension containing RXN are shown in Figure 10. MTT assay was employed for cytotoxicity studies on Caco-2 cells. Cell viability of SDA tablet and suspension was evaluated at five different concentrations: 20 µM, 40 µM, 60 µM, 80 µM and 100 µM. Drug containing formulations exhibited a weak inverse correlation between concentration and cell viability. The highest concentration (100 µM) of RXN SDA tablet and RXN suspension exhibited a 95% cell viability indicating absence of cytotoxicity. RXN suspension exhibited similar cytotoxicity as that of RXN SDA, suggesting that the developed formulation did not precipitate any additional toxicity.
Pharmacokinetic Study
Based on the plasma concentration-time profiles, corresponding pharmacokinetic parameters were determined and tabulated in Table 9. The Tmax for RXN SDA tablets was 2 h, whereas the value for RXN suspension was 3h. The rapid rate of absorption for RXN SDA tablets relative to RXN suspension could likely to provide a quick onset of action. This result can be attributed to the increase in dissolution rate and solubility of RXN from the SDA tablets in the gastrointestinal tract, allowing rapid absorption of the drug. The Cmax values for RXN suspension, and RXN SDA tablets were found to be 262.32 ± 32.71 ng/mL, and 628.83 ± 42.60 ng/mL respectively. Cmax values for RXN SDA tablets were 2.4 times more than RXN suspension indicating better release from formulation and consequent drug absorption in the systemic circulation. Mean AUC0−24 values achieved with RXN suspension, and RXN SDA tablets indicate significant (P < 0.00001) enhancement of bioavailability from RXN SDA tablets (Table 9). The overall relative bioavailability enhancement noticed with RXN SDA tablets was ~3 folds higher than the RXN suspension. Higher Cmax and AUC achieved with test formulation indicated enhancement in oral bioavailability. The results of this study show a good correlation with the in vitro release studies. The outcomes of this study may be extrapolated towards the possibilities of dose reduction upon the administration of the test formulation. Similar observation was noticed in earlier studies with other drugs as well (49).
Table 9
Pharmacokinetic parameters for different rivaroxaban formulations after administration in Sprague-Dawley rats
Parameters
|
Rivaroxaban suspension
|
Rivaroxaban solid dispersion tablet
|
Tmax (h)
|
3.0
|
2.0
|
Cmax (ng/mL)
|
262.32 ± 32.71
|
628.83 ± 42.60
|
AUC 0−24 (ng.h/mL)
|
1414.09 ± 192.18
|
3945.37 ± 300.71
|
AUC 0−∞ (ng.h/mL)
|
1462.00 ± 207.35
|
4257.14 ± 311.15
|
Relative bioavailability
|
-
|
2.79 folds
|
Pharmacodynamic 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. In the present study, bleeding time was markedly prolonged by all the formulations in comparison to control. The bleeding time noticed with RXN SDA tablets (189.00 ± 7.70s) was significantly higher (P < 0.00001) than RXN suspension (126.00 ± 6.52s) and vehicle (68.00 ± 9.08s). Overall, the data here demonstrated higher prolongation of bleeding time with RXN SDA tablets.
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. 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 higher percentage of platelet aggregation noticed in RXN SDA tablets can be ascribed to the improved bioavailability of RXN, which ultimately increases the adenosine concentration and potentiates the antiplatelet action. The data are in agreement with previous pharmacodynamic studies (50) asserting the clinical benefit of the developed novel formulation of RXN.