ELECTRONIC SUPPLEMENTARY CONTENT
Design, optimization, and evaluation of ezetimibe solid supersaturatable self-nanoemulsifying drug delivery for enhanced solubility and dissolution
Rajendra Narayan Dash a , Mohammed Habibuddin b * , Touseef Humaira b a Alliance Institute of Advanced Pharmaceutical & Health Sciences, Plot No.64, Survey No.145, Sardar Patel Nagar, Kukatpally, Hyderabad - 500072, Telangana, India. b Adept Pharma and Bioscience Excellence Private Limited, Corporate Office: 10-3-561/3/A/102, Vijayanagar Colony, Hyderabad - 500057, Telangana, India.
* Corresponding author
Tel.: +914066103388
Fax: +914066103388
E-mail: drhabib21@gmail.com
1

Appendix - A
Abbreviations and glossary
Solid S-SNEDDS , solid supersaturatable self-nanoemulsifying drug delivery system
HPMC-E5, hydroxypropyl methylcellulose having 5 cps viscosity
PPI, precipitation inhibitor
CCD, central composite design
DLS, dynamic light scattering d
G, global desirability
DR
15min, percentage drug release in 15 minutes
DLS, dynamic light scattering
2

Table S1 . Emulsification efficacy of various non-ionic surfactant
Oil Surfactant HLB No. of flask inversion
Captex 355
Captex 355
Captex 355
Captex 355
Captex 355
Cremophor RH40
Solutol HS15
Labrasol
Cremophor EL
Tween 80
15
15
14
13
15
10
10
15
40
35
Appendix – B
Turbidity
(NTU)
95
102
296
147
441
Transmittance
(%)
94.8
94.1
82.5
92.6
54.1
Visual observation
Spontaneous dispersion
Spontaneous dispersion
Spontaneous dispersion
Formed crystalline gel, difficult to disperse
Formed crystalline gel, difficult to disperse
3

Table S2.
Emulsification abilities of co-surfactant
Oil Surfactant Co-surfactant No. of flask inversion
Turbidity
(NTU)
Captex 355
Captex 355
Captex 355
Captex 355
Cremophor RH40
Cremophor RH40
Cremophor RH40
Cremophor RH40
Imwitor 988 3
Capmul MCM 5
Capmul PG8 12
PEG 400 16
83
103
184
220
Transmittance
(%)
Visual observation
97.7
94.1
93.1
92.5
Spontaneous dispersion
Spontaneous dispersion
Formed crystalline gel, dispersed easily
Formed crystalline gel, dispersed easily
4

Table S3.
Details of experiments performed along with outcomes during optimization of SNEDDS preconcentrate by a central composite design.
7
3
4
8
2
20
12
17
10
11
9
19
6
15
16
5
1
13
Std. Run X1
Order Order By parts
18
14
1
2
40.00
40.00
3
4
5
6
40.00
20.00
20.00
40.00
7
8
9
10
11
12
13
14
6.36
40.00
60.00
40.00
40.00
40.00
73.64
40.00
15
16
17
18
19
20
60.00
40.00
60.00
60.00
20.00
20.00
X2
20.00
40.00
73.64
40.00
40.00
6.36
20.00
40.00
40.00
40.00
40.00
20.00
20.00
40.00
40.00
40.00
60.00
60.00
60.00
60.00
X3
20.00
36.82
20.00
30.00
10.00
3.18
20.00
20.00
30.00
20.00
20.00
20.00
20.00
20.00
10.00
20.00
10.00
30.00
30.00
10.00
X1 X2
By percentage (w/w)
40.00
34.24
40.00
34.24
40.00
28.57
40.00
48.09
9.59
40.00
54.55
40.00
29.93
40.00
55.10
60.27
66.67
40.00
46.15
40.00
18.18
22.22
40.00
28.57
40.00
48.09
60.27
40.00
18.18
40.00
55.10
40.00
29.93
9.59
22.22
40.00
46.15
40.00
54.55
66.67
X3
20.00
31.52
20.00
42.86
20.00
3.83
30.14
20.00
27.27
20.00
14.97
20.00
14.97
30.14
11.11
20.00
7.69
20.00
27.27
11.11
Y1 Y2 Y3
Observed Predicted Observed Predicted Observed Predicted
44.8
41.4
47.2
31.8
12.6
12.7
13.4
3.1
98.4
99.5
99.3
103.3
47.6
42.2
40.8
81.3
34.8
51.2
99.7
46.5
34.6
46.2
120.0
130.1
241.2
46.9
74.4
49.2
23.6
31.6
47.2
36.0
57.9
91.0
19.6
47.2
112.1
47.2
32.4
47.2
135.2
132.3
214.1
47.2
80.6
32.1
50.7
19.1
13.2
12.0
12.8
55.3
7.8
14.5
85.6
12.3
9.1
13.7
102.3
125.8
214.3
13.1
35.6
14.1
6.5
7.2
X1, Captex 355; X2, Cremophor RH40 and X3, Imwitor 988; Y1, mean droplet size (nm); Y2, turbidity (NTU); Y3, transmittance (%).
13.4
8.9
31.3
58.1
-6.2
13.4
100.4
13.4
7.7
13.4
109.5
120.4
197.1
13.4
43.5
0.4
28.5
-2.8
99.5
99.6
99.3
96.6
99.8
99.7
92.3
99.3
99.1
99.6
83.2
78.5
60.0
99.3
99.3
98.9
99.8
99.9
99.3
101.3
95.5
94.2
65.2
99.3
96.6
101.7
93.6
104.4
101.6
99.3
86.8
99.3
99.5
99.3
82.8
79.5
5

Table S4.
Analysis of variance (ANOVA) results for individual models.
Source
Regression 9
Linear 3
DF a Y1
F b p c
Y2
F b p c
Y3
F b p c
16.07 ˂0.001* 28.42 ˂0.001* 11.30 ˂0.001*
6.98 0.008* 12.19 0.001* 6.20 0.012*
X1
X2
X3
Square
1
1
1
3
9.16
5.07
0.87
3.90
0.013* 13.14 0.005* 5.77
0.048* 11.44 0.007* 7.41
0.373 2.02 0.186 0.38
0.044* 10.84 0.002* 4.62
0.037*
0.022*
0.554
0.028*
X1*X1
X2*X2
1
1
5.26 0.045* 12.63 0.005* 5.17 0.046*
7.13 0.023* 22.16 0.001* 9.88 0.010*
X3*X3
Interaction
X1*X2
X1*X3
1
3
1
1
1.16 0.307 2.55 0.141 0.03 0.874
9.73 0.003* 18.16 ˂0.001* 9.93 0.002*
14.36 0.004* 34.3 ˂0.001* 14.65 0.003*
10.25 0.009* 13.27 0.005* 7.24 0.023*
X2*X3 1 4.57 0.058 6.91 0.025* 7.89 0.018*
Y1, mean droplet size (nm); Y2, turbidity (NTU); Y3, transmittance (%); X1, Captex 355% w/w; X2, Cremophor RH40% w/w, and X3, Imwitor 988% w/w; a DF, degree of freedom; b F, test for comparing model variance with residual variance; c p, the probability of seeing the observed F-value if the null hypothesis is true; * significant model term (p<0.05).
6

Table S5. ANOVA results for individual models
Response Multiple Multiple Adjusted SS
R R² R² model a
MS model b
SS MS residual c residual d
Mean droplet size (nm) 0.9671 0.9353 0.8771 45205.18 5022.80 3125.27 312.53
F e p f
16.07 ˂ 0.001
Turbidity (NTU) 0.9810 0.9624 0.9285 53285.16 5920.57 2083.58 208.36 28.41 ˂ 0.001
Transmittance (%) 0.9542 0.9105 0.8299 1765.25 196.14 173.50 17.35 11.30 ˂ 0.001 a SS model, sum of squares of whole model; b MS model, mean squares of whole model; c SS residual, sum square of residuals; d MS residual, mean square of residuals; e F, test for comparing model variance with residual variance; f p, probability of seeing the observed F-value if the null hypothesis is true.
7

Figure S1. Ternary phase diagram, Blue color region represents region of nano emulsion. Where; X1, Captex 355 (% w/w); X2, Cremophor RH40 (% w/w), and X3, Imwitor
988 (% w/w).
8

DESIGN-EXPERT Plot
Desirability
Design Points
X = A: Captex-355
Y = B: Cremophor RH-40
Actual Factor
C: Imwitor-988 = 30.00
60.00
50.00
Predict 0.99
X 30.00
Y 40.00
40.00
30.00
20.00
20.00
30.00
40.00
50.00
60.00
A: Cap tex-355
Figure S2. Global desirability of the responses such as mean droplet size (nm), turbidity (NTU), and transmittance (%).
9

Figure S3. Desirability plots for mean droplet size (nm) (a); Turbidity (NTU) (b) and transmittance (%) (c). Where; X1, Captex 355 (% w/w); X2, Cremophor RH40 (% w/w) and X3, Imwitor 988 (% w/w).
10

Figure S4. Predicted versus residual values for mean droplet size (nm) (a), turbidity (NTU) (b) and percentage transmittance (c).
11

Figure S5.
Normal probability plot for mean droplet size (nm) (a), turbidity (NTU) (b) and percentage transmittance (c).
12

Figure S6.
Histogram (raw residual) plots for mean droplet size (nm) (a), turbidity (NTU) (b) and percentage transmittance (c).
13

Figure S7. FTIR spectra of a, ezetimibe; b, physical mixture of ezetimibe and solid S-SNEDDS blank; c, solid S-SNEDDS; d, solid S-SNEDDS blank.
14

Figure S8.
Representation of self emulsification of formulation No. 15 having mean droplet size 241.2 nm (a), formulation No. 6 having mean droplet size 81.3 nm (b) and formulation No. 19 having mean droplet size 23.6 nm (c) after diluting 500 times with water.
15

300
250
200
R² = 0,9778
150
100
50
0
0 50 100 150
200 250
Figure S9. Correlation between droplet size (nm) and turbidity (NTU) of diluted formulations prepared as per CCD.
16

120
100
80
60
R² = 0,9398
40
20
0
0 50 100 150
200 250 300
Figure S10. Correlation between droplet size (nm) and transmittance (%) of diluted formulations prepared as per CCD.
17

Figure S11. Observed versus predicted values of mean droplet size (nm) (a), turbidity (NTU) (b) and percentage transmittance (c).
18

Figure S12. Zeta potential of optimized SNEDDS preconcentrate.
19

0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
d c b a
9.5
Figure S13. Chromatogram obtained from the accelerated stability study at 40 ± 2 o C/75 ± 5 % RH; Blank (a), 0 day (b), 3 months (c), 6 month (d).
20

E
D
C
B
A
I
H
G
F
-0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0
10.5
11.0
11.5
12.0
12.5
13.0
13.5
14.0
14.5
Figure S14. Overlay chromatogram obtained during development of a stability indicating HPLC method; A, blank; B, acid induced degradation (with 2M HCl for 6 h); C, base induced degradation (with 0.1M NaOH for 15 min.); D, oxidative degradation (10% v/v H
2
O
2
for 48 h); E, hydrolytic degradation (with water at 80 o C for 12 h); F, dry heat degradation (at 80 o C for 48 h); G, moist heat degradation (at 80 o C with ambient humidity for 48 h); H, photolytic degradation (under direct sunlight for 1 week) and I, standard ezetimibe (20µg mL -1 ).
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0
0,00305
-0,5
-1
-1,5
-2
0,0031 0,00315 0,0032 0,00325 0,0033 0,00335
-2,5
-3 y = -3962,3x + 9,1001
R² = 0,9992
-3,5
-4
-4,5
Figure S15. Arrhenius plot for ezetimibe degradation from solid S-SNEDDS, Where y : log of rate constants (K) obtained from different storage conditions, x : 1 / mean temperature ( o K) for various storage temperature employed.
22