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Chapter 6 LORNOXICAM PRONIOSOMAL DELIVERY
Ph. D. Thesis Page | 140
6.1 INTRODUCTION
Lornoxicam is a member of oxicam group of NSAID with potent analgesic and anti-
inflammatory effects. Major pharmacological uses of lornoxicam are symptomatic
treatment of pain associated with rheumatoid arthritis, osteoarthritis and it has shown
proven efficacy in the management of perioperative and postoperative pain management
related to gynaecological, orthopaedic, dental and abdominal surgeries. The mechanism
of action of lornoxicam, is primarily due to the inhibition of prostaglandin biosynthesis
through the inhibition of the cyclooxygenase enzymes (COX1 and COX2). Lornoxicam
is commercially available as immediate release tablets, rapid-release tablets and
parenteral formulation for intravenous and intramuscular use. Although lornoxicam
possesses potent analgesic and anti-inflammatory activities, its short duration of action
owing to rapid elimination limits its usefulness. Moreover, lornoxicam is characterized
by poorly soluble drug in the acidic environment of the stomach which leads to local
toxicity at the site of aggregation, and oral use in clinic is often limited because of its
potential to cause adverse effects such as gastrointestinal disorders and cardiovascular
risks1. Transdermal delivery of lornoxicam will be an ideal alternative for avoiding or
reducing these side effects and targeting the inflammatory region. Moreover, the
physicochemical characteristics (e.g. molecular weight, n-octanol/water partition
coefficient) of lornoxicam are similar to the ideal properties of a molecule able to
effectively penetrate the skin. To overcome skin barrier proniosome is one of the good
vesicular approach. Therefore, lornoxicam can be a good candidate for transdermal drug
delivery2. So in the present work an attempt was made to study the transdermal
proniosomal delivery of lornoxicam.
6.2 MATERIALS AND EQUIPMENTS
6.2.1 LIST OF MATERIALS USED
Materials Company
Lornoxicam Gift sample from Zydus Cadila, Ahmedabad
Sodium Chloride Central Drug House Pvt. Ltd., India
Potassium dihydrogen phosphate Central Drug House Pvt. Ltd., India
Chapter 6 LORNOXICAM PRONIOSOMAL DELIVERY
Ph. D. Thesis Page | 141
Sodium Hydroxide Central Drug House Pvt. Ltd., India
Soya Lecithin Gift sample from CP Kelco UK Ltd, UK
Cholesterol Gift sample from CP Kelco UK Ltd, UK
S 60 Central Drug House Pvt. Ltd., India
Pottasium dihydrogen orthophosphate Central Drug House Pvt. Ltd., India
Syringe filter 0.22 µm Merck India Ltd., India
Dialysis membrane Merck India Ltd., India
Membrane filter 0.22µm Merck India Ltd., India
Double distilled water In house distillation assembly
6.2.2 EQUIPMENTS
Same as section 4.2.2.
6.3 IDENTIFICATION AND ESTIMATION OF LORNOXICAM
6.3.1 IDENTIFICATION OF LORNOXICAM BY MELTING POINT ANALYSIS
The procedure for determination is same as section 4.3.1.
Table 6.1: Melting point determination of Lornoxicam
Melting point Standard drug3 Procured drug
225-230 °C 228-232 °C
The melting point of lornoxicam was found to be 228-232 °C. This matches with the
standard melting temperature range 225-230 °C indicating the identity of lornoxicam
(Table 6.1).
6.3.2 IDENTIFICATION OF LORNOXICAM BY FTIR SPECTROS COPY
FTIR spectra of drug in KBr pellets at moderate scanning speed between 4000-400
cm-1 was made. The spectra of standard and procured lornoxicam are shown in Figure 6.1
and Figure 6.2. The peak related to the functional group present in standard and procured
lornoxicam are given in Table 6.2.
The procured drug shows similarity with the reported value of functional groups present
in standard drug, which indicates purity and identity of lornoxicam.
Chapter 6 LORNOXICAM PRONIOSOMAL DELIVERY
Ph. D. Thesis Page | 142
Figure 6.1: FTIR spectra of standard Lornoxicam3
Figure 6.2: FTIR spectra of procured Lornoxicam
Chapter 6
Ph. D. Thesis
Table 6.2: FTIR spectra of standard and procured Lornoxicam
Functional group present
C=C stretching
-NH
-C-N
Aromatic
-C-X
S=O
CO stretch
6.3.3 IDENTIFICATION OF
Drug solution was prepared in phosphate
Visible spectrophotometer in the range of 200nm to 400nm. The
376 nm4. UV spectrum of lornoxicam is shown in Figure 6.3.
LORNOXICAM PRONIOSOMAL DELIVERY
Ph. D. Thesis
.2: FTIR spectra of standard and procured Lornoxicam
Wave Number (cm-1)
Standard Peak Region
Standard drug Procured drug (from
3100-3000 3067.06
1640-1550 1580.39
1350-1000 1323.36, 1185.64, 1092.83
900-690 828.83
785-540 764.87, 726.7, 688.7
766.56, 721.24, 688.46
1030-1060 1055.33
1700-1600 1646
IDENTIFICATION OF LORNOXICAM BY UV SPECTRUM
Drug solution was prepared in phosphate buffer pH 7.4 and scanned in a UV
Visible spectrophotometer in the range of 200nm to 400nm. The λ
UV spectrum of lornoxicam is shown in Figure 6.3.
Figure 6.3: UV spectrum of Lornoxicam
LORNOXICAM PRONIOSOMAL DELIVERY
Page | 143
.2: FTIR spectra of standard and procured Lornoxicam
Procured drug (from Figure 6.2)
3067.23
1595.81
1327.75, 1187.94, 1085.73
832.13
766.56, 721.24, 688.46
1040.41
1646.91
UV SPECTRUM
and scanned in a UV
Visible spectrophotometer in the range of 200nm to 400nm. The λmax was found to be
Chapter 6 LORNOXICAM PRONIOSOMAL DELIVERY
Ph. D. Thesis Page | 144
6.3.4 ESTIMATION OF LORNOXICAM BY UV SPECTROMETRIC
ANALYTICAL PROCEDURE
� Preparation of Standard Curve
The calibration curve of lornoxicam was obtained by dissolving the drug in phosphate
buffer pH 7.4 and measuring the absorbance of the resulting aliquots at 376 nm, using
phosphate buffer pH 7.4 as blank. Concentrations of 5.6 to 22.4µg/ml were prepared
by suitable dilution of the stock solutions with phosphate buffer pH 7.4. The
absorbance of the resulting aliquots was measured at 376 nm using UV
spectrophotometer. A graph of Concentration vs. Absorbance was plotted.
The linear regression data obtained from the calibration curve showed a linear
relationship over the concentration range of 5.6 to 22.4µg/ml. The experiment was
performed in triplicate and based on average absorbance; the equation for the best
line fit was generated. The results of standard curve preparation are shown in Table
6.3 and Figure 6.4.
Figure 6.4: Standard curve of Lornoxicam in phosphate buffer pH 7.4
Table 6.3: Standard Curve of Lornoxicam in Phosphate buffer pH 7.4
Concentration (µg/ml) Absorbance (nm)
0 0
05.6 0.2295 ± 0.032
08.4 0.3147 ± 0.033
0
0.22950.3147
0.42460.4929
0.6130.677
0.771
y = 0.033x + 0.024R² = 0.995
0
0.2
0.4
0.6
0.8
0 4 8 12 16 20 24
Abs
orba
nce
(nm
)
Concentration (µg/ml)
Standard curve of Lornoxicam
Chapter 6 LORNOXICAM PRONIOSOMAL DELIVERY
Ph. D. Thesis Page | 145
11.2 0.4246 ± 0.037
14.0 0.4929 ± 0.041
16.8 0.613 ± 0.042
19.6 0.677 ± 0.039
22.4 0.771 ± 0.038
Each result is the mean ± S.D (n = 3)
6.4 PHYSICOCHEMICAL PARAMETERS
6.4.1 PHYSICOCHEMICAL PARAMETERS OF LORNOXICAM
6.4.1.1 Solubility measurements
The procedure for determination is same as section 4.4.1.1. The solubility of drug
was determined by spectrophotometric method of analysis at 376nm wavelength.
6.4.1.2 Partition coefficient determination
The procedure for determination is same as section 4.4.1.2.
6.4.1.3 pKa determination
The procedure for determination is same as section 4.4.1.3.
� Result of physicochemical properties of lornoxicam is shown in Table 6.4.
Table 6.4: Physicochemical properties of Lornoxicam
Parameter Result
Water Solubility (mg/ml) 0.04 ± 0.49
0.9% w/v NaCl Solubility (mg/ml) 0.11 ± 0.45
Partition Coefficient Log p 1.84 ± 0.69
Dissociation Constant pKa 4.71 ± 2.04
Each result is the mean ± S.D (n = 3)
6.4.2 PHYSICOCHEMICAL PARAMETERS OF GOAT SKIN
The procedure for the determination and result are same as section 4.4.2.
Chapter 6 LORNOXICAM PRONIOSOMAL DELIVERY
Ph. D. Thesis Page | 146
6.5 EXPERIMENTAL SETUP
6.5.1 PREPARATION OF LORNOXICAM PRONIOSOMES
The procedure for preparation of proniosomes is same as section 4.5.1. The effect
of surfactant type was studied. The composition of different formulations (F1–F8) is
listed in Table 6.5. The procedure for the evaluation and result are given in section 6.6
and 6.7, respectively. The amount of ingredient taken was on the bases of previous
literature5.
(Composition in mg)
6.5.2 EXPERIMENTAL DESIGN
A four-factor three-level Box–Behnken design was used to explore the quadratic
response surfaces and for constructing a second-order polynomial models using Design
Expert software (9.0.3). The design matrix shows 29 experimental runs, for which the
nonlinear computer generated quadratic model is defined as
Y = b0 + b1X1 + b2X2 + b3X3 + b4X4 + b12X1X2 + b13X1X3 + b14X1X4 + b23X2X3 +
b24X2X4 + b34X3X4 + b11X12 + b22X2
2 + b33X32 + b44X4
2
Table 6.5: : Batch formulation for effect formulation for effect Effect of different surfactant on EE % and drug permeation
Ingredient Formula no.
F1 F2 F3 F4 F5 F6 F7 F8
Lornoxicam 5 5 5 5 5 5 5 5
S 20 180 --- --- --- --- --- --- ---
S 40 --- 180 --- --- --- --- --- ---
S 60 --- --- 180 --- --- --- --- ---
S 80 --- --- --- 180 --- --- --- ---
T 20 --- --- --- --- 180 --- --- ---
T 40 --- --- --- --- --- 180 --- ---
T 60 --- --- --- --- --- --- 180 ---
T 80 --- --- --- --- --- --- --- 180
Lecithin 180 180 180 180 180 180 180 180
Cholesterol 20 20 20 20 20 20 20 20
Chapter 6 LORNOXICAM PRONIOSOMAL DELIVERY
Ph. D. Thesis Page | 147
where Y is the measured response associated with each factor level combination; b0 is a
constant; b1, b2, b3 and b4 are linear coefficients, b12, b13, b14, b23, b24 and b34 are interaction
coefficients between the three factors; b11, b22, b33 and b44 are quadratic coefficients
computed from the observed experimental values of Y from experimental runs; and X1,
X2, X3 and X4 are the coded levels of independent variables. The terms X1X2 and X12 (I =
1, 2, or 3) represent the interaction and quadratic terms, respectively. The concentration
range of S 60 (X1), cholesterol (X2), lecithin (X3), and lornoxicam (X4) were used to
prepare the 29 formulations and the respective observed responses are given in Table 6.6.
The Coded and uncoded values of independent variables are given in Table 6.7.
Table 6.6: Variables in Box–Behnken design for preparation of lornoxicam
proniosomes.
Factor Level used, actual coded
Low (-1) Medium (0) High (+1)
Independent variables (mg)
X1 = S 60
X2 = cholesterol
X3 = lecithin
X4 = lornoxicam
70
5
90
5
90
10
100
10
110
15
110
15
Dependent variable
Y1 = vesicle size (nm)
Y2 = EE %
Y3 = flux (µg/cm2/h)
Targets
Minimum
Maximum
Enhance
Table 6.7: Coded and uncoded values of independent variables
Formulation
No.
Independent variables
Coded values Uncoded values
X1 X2 X3 X4 X1 X2 X3 X4
1 0 -1 0 1 90 5 100 15
2 0 -1 0 -1 90 5 100 5
3 1 1 0 0 110 15 100 10
Chapter 6 LORNOXICAM PRONIOSOMAL DELIVERY
Ph. D. Thesis Page | 148
4 1 0 0 -1 110 10 100 5
5 1 -1 0 0 110 5 100 10
6 1 0 1 0 110 10 110 10
7 0 0 0 0 90 10 100 10
8 0 0 1 1 90 10 110 15
9 1 0 0 1 110 10 100 15
10 0 -1 1 0 90 5 110 10
11 -1 0 -1 0 70 10 90 10
12 -1 1 0 0 70 15 100 10
13 0 0 0 0 90 10 100 10
14 0 0 0 0 90 10 100 10
15 -1 0 0 -1 70 10 100 5
16 -1 0 1 0 70 10 110 10
17 0 0 -1 1 90 10 90 15
18 0 1 0 1 90 15 100 15
19 0 0 -1 -1 90 10 90 5
20 0 -1 -1 0 90 5 90 10
21 1 0 -1 0 110 10 90 10
22 0 1 0 -1 90 15 100 5
23 -1 -1 0 0 70 5 100 10
24 0 1 1 0 90 15 110 10
25 0 0 0 0 90 10 100 10
26 0 0 0 0 90 10 100 10
27 -1 0 0 1 70 10 100 15
28 0 0 1 -1 90 10 110 5
29 0 1 -1 0 90 15 90 10
30* 0.5 0.5 -0.5 -0.5 100 12.5 95 7.5
31* -0.5 -0.5 0 0 80 7.5 100 10
32* 0 0 0.5 0.5 90 10 105 12.5
*Check point batches
Chapter 6 LORNOXICAM PRONIOSOMAL DELIVERY
Ph. D. Thesis Page | 149
6.6 EVALUATION OF PRONIOSOMES
6.6.1 LORNOXICAM EE%
The procedure for the evaluation of EE% is same as section 4.6.1. The lornoxicam
concentration in the resulting solution was assayed by UV spectrophotometer at 376nm.
The percentage of drug encapsulation was calculated by the following equation:
EE% = [(Ct – Cf) / Ct] X 100
where Ct is the concentration of total lornoxicam, and Cf is the concentration of free
lornoxicam. The result is shown in section 6.7.1 and 6.7.2.
6.6.2 VESICLE SIZE AND ZETA POTENTIAL ANALYSIS
The procedure for the determination of vesicle size and zeta potential is same as
section 4.6.2. The result is shown in section 6.7.2 and 6.7.3, respectively.
6.6.3 MICROSCOPICAL EXAMINATION
6.6.3.1 Optical microscope
The procedure for the determination of morphology by optical microscope is
same as section 4.6.3.1. The result is shown in section 6.7.4.1.
6.6.3.2 TEM
The procedure for the determination of morphology by TEM is same as section
4.6.3.2. The result is shown in section 6.7.4.2.
6.6.4 DSC
The procedure for the DSC study is same as section 4.6.4. The result is shown in
section 6.7.5.
6.6.5 IN-VITRO RELEASE TEST
The procedure for the in-vitro release test and data analysis is same as section 4.6.5.
Weight amount of optimized proniosomal gels (containing the equivalent of 5 mg
Chapter 6 LORNOXICAM PRONIOSOMAL DELIVERY
Ph. D. Thesis Page | 150
lornoxicam) were loaded into the donor compartments. The amount of drug released from
the bases was determined spectrophotometrically at 376nm. The result is shown in
section 6.7.6.
6.6.6 EX-VIVO PERMEATION
The procedure for the ex-vivo permeation and data analysis is same as section 4.6.6.
Weight amount of proniosomal gels (containing the equivalent of 5, 10 or 15 mg
lornoxicam – as per design formulation values) were loaded into the donor
compartments. The amount of drug released from the bases was determined
spectrophotometrically at 376nm. The result is shown in section 6.7.2.
6.6.7 RATE OF SPONTANEITY (HYDRATION)
The procedure for the determination of rate of spontaneity is same as section 4.6.7.
The result is shown in section 6.7.7.
6.6.8 PHYSICAL STABILITY
The procedure for the determination of rate of spontaneity is same as section 4.6.8.
Lornoxicam retained in proniosomes = (Entrapped lornoxicam after storage
/ Entrapped lornoxicam before storage) x 100
The result is shown in section 6.7.8.
6.6.9 IN VIVO STUDIES
6.6.9.1 Assessment of anti-inflammatory effect
The procedure for the assessment of anti-inflammatory effect is same as section
4.6.9.1. Flexilor Tablet (4mg), Glenmark, is used as marketed product of lornoxicam. In
proniosomal formulation and marketed tablet weight taken was equivalent to 0.57mg of
lornoxicam. The result is shown in section 6.7.9.1.
6.6.9.2 Assessment of antinociceptive activity
The procedure for the assessment of antinociceptive activity is same as section
4.6.9.2. Weight of tablet and proniosomal formulation taken was equivalent to 0.33mg of
lornoxicam. The result is shown in section 6.7.9.2.
Chapter 6 LORNOXICAM PRONIOSOMAL DELIVERY
Ph. D. Thesis Page | 151
6.7 RESULTS AND DISCUSSION
6.7.1 LORNOXICAM EE%
The procedure for the evaluation of EE% is given in section 4.6.1. The effect of
surfactant type on EE is discussed below.
6.7.1.1 Effect of surfactant type
Table 6.8 shows the result of EE% of lornoxicam from different surfactant. S 60
shows the significant higher EE% compared to other surfactants (p < 0.05).
Table 6.8: Effect of different surfactant on EE % of Lornoxicam
Formula no. EE %
F 1 (S 20) 93.04 ± 1.23
F 2 (S 40) 90.34 ± 1.92
F 3 (S 60) 98.18 ± 2.27
F 4 (S 80) 95.05 ± 1.61
F 5 (T 20) 77.37 ± 0.99
F 6 (T 40) 71.02 ± 2.11
F 7 (T 60) 78.49 ± 1.84
F 8 (T 80) 82.14 ± 1.89
Each result is the mean ± S.D (n = 3)
So, on the bases of EE% S 60 was chosen for further experimental study.
6.7.2 EXPERIMENTAL DESIGN
The procedure to carry out experimental design is given in 6.5.2. Based on the four-
factor three-level Box–Behnken design, the results of dependent variables are observed in
Table 6.9, 6.10, 6.11, 6.12 and Figure 6.5, 6.6.
Table 6.9: ex-vivo permeation of design formulation F1 to F10
T* F1 F2 F3 F4 F5 F6 F7 F8 F9 F10
0 0 0 0 0 0 0 0 0 0 0
1 53.44 ±
5.44
73.34 ±
7.89
95.11 ±
10.41
88.12 ±
9.78
64.71 ±
7.74
62.23 ±
6.61
114.12 ±
12.43
101.04
± 11.23
117.23
± 10.44
47.43
± 3.56
2 133.28
± 10.32
180.92
± 15.65
213.44
± 20.47
218.93
± 23.15
162.39
± 15.29
155.93
± 14.69
259.82 ±
26.79
248.92
± 22.35
262.45
± 25.57
121.3
4 ±
11.42
Chapter 6 LORNOXICAM PRONIOSOMAL DELIVERY
Ph. D. Thesis Page | 152
3 207.56
± 14.55
295.44
± 27.52
355.76
± 34.58
353.34
± 36.73
268.29
± 24.62
249.45
± 25.38
417.44 ±
40.65
395.47
± 36.69
431.21
± 44.31
194.4
5 ±
18.63
4 292.34
± 22.78
315.21
± 29.33
492.12
± 48.67
474.24
± 45.55
383.52
± 36.89
358.62
± 34.83
586.32 ±
56.72
536.83
± 49.48
585.79
± 55.67
268.5
1 ±
24.74
6 365.89
± 35.36
428.79
± 40.67
628.34
± 64.41
603.56
± 58.27
477.41
± 45.62
497.14
± 45.94
749.35 ±
75.88
671.45
± 57.82
748.67
± 70.78
334.2
8 ±
32.49
8 449.93
± 42.56
546.52
± 55.75
765.23
± 75.59
718.23
± 70.92
582.49
± 57.57
537.42
± 54.24
910.21 ±
82.34
802.92
± 68.37
894.59
± 85.98
448.6
8 ±
38.74
12 518. 32
± 50.28
646.12
± 62.89
854.87
± 84.79
806.54
± 80.14
645.66
± 65.69
614.52
± 60.37
1064.71
± 94.43
917.42
± 81.22
1115.9
7 ±
91.23
497.7
2 ±
44.87
18 575.21
± 57.98
716.35
± 70.32
939.24
± 95.17
872.53
± 85.86
696.49
± 72.27
704.43
± 70.23
1146.22
± 101.14
993.75
± 88.53
1153.6
3 ±
100.32
531.1
3 ±
49.36
24 639.18
± 65.62
798.24
± 80.38
1010.2
2 ±
100.36
979.38
± 93.23
720.16
± 77.82
712.27
± 73.34
1223.17
± 114.46
1066.3
2
±95.56
1298.5
7 ±
112.79
595.3
2 ±
54.42
Each result is the mean ± S.D (n = 3); * T- Time (hr)
Table 6.10: ex-vivo permeation of design formulation F11 to F20
T* F11 F12 F13 F14 F15 F16 F17 F18 F19 F20
0 0 0 0 0 0 0 0 0 0 0
1 85.62 ±
7.85
105.41
± 11.24
112.34
± 10.34
50.88 ±
4.65
70.34 ±
8.23
67.61 ±
5.45
82.35 ±
7.54
76.45 ±
7.23
79.82 ±
8.59
59.44 ±
6.72
2 196.65
± 16.67
236.54
± 22.52
254.61
± 16.83
123.32
± 12.43
163.31
± 12.37
161.32
± 14.67
185.69
± 14.62
173.21
± 15.72
182. 23
± 16.23
143.28
± 14.25
3 314.44
± 26.33
368.23
± 33.63
396.23
± 23.43
209.38
± 22.76
268.67
± 21.56
258.45
± 22.36
296.34
± 23.47
281.66
± 26.44
287.41
± 25.72
227.84
± 22.51
4 432.4 ±
39.54
504.23
± 38.69
542.13
± 31.43
284.13
± 29.31
357.21
± 27.89
366.41
± 28.93
414.92
± 32.41
394.29
± 36.82
412.69
± 36.12
321.65
± 29.45
6 556.82
± 46.22
639.11
± 43.45
686.72
± 42.52
358.97
± 34.26
464.28
± 37.45
473.16
± 37.78
538.34
± 38.94
484.39
± 47.32
521.87
± 43.47
417.52
± 37.61
8 716.45
± 54.65
776.23
± 52.38
833.23
± 49.52
435.61
± 40.46
574.12
± 45.21
559.23
± 45.32
643.29
± 46.68
602.28
± 55.12
616.85
± 51.21
495.69
± 44.12
Chapter 6 LORNOXICAM PRONIOSOMAL DELIVERY
Ph. D. Thesis Page | 153
12 753.12
± 67.26
893.12
± 61.28
974.51
± 60.23
519.21
± 47.82
669.82
± 53.56
647.52
± 52.45
751.9 ±
54.38
707.34
± 62.23
729.38
± 59.43
581.57
± 53.71
18 852.34
± 72.53
934.56
± 67.73
1119.12
± 68.34
568.73
± 53.45
734.17
± 60.85
737.32
± 59.34
847.49
± 63.27
785.89
± 70.37
834.35
± 68.89
659.86
± 61.32
24 875.59
± 79.48
1109.34
± 75.56
1158.31
± 78.89
650.31
± 60.78
795.29
± 67.45
805.56
± 68.89
945.23
± 76.62
874.29
± 77.28
869.74
± 78.12
735.78
± 67.82
Each result is the mean ± S.D (n = 3); * T- Time (hr)
Table 6.11: ex-vivo permeation of design formulation F21 to F29 and control
T* F21 F22 F23 F24 F25 F26 F27 F28 F29 Control
0 0 0 0 0 0 0 0 0 0 0
1 40.21 ±
4.23
91.48 ±
8.23
43.66
± 4.12
109.32
± 9.61
127.89
± 11.83
121.67
± 13.28
98.38 ±
9.41
45.36
± 4.23
56.77
± 5.23
20.28 ±
5.56
2
110.45
± 10.73
204.67
± 16.56
105.77
± 9.34
245 .88
± 18.34
280.56
± 19.39
278.56
± 21.52
220.31
± 16.34
135.72
± 8.49
144.28
±
12.52
48.84 ±
8.82
3
181.59
± 16.33
321.54
± 24.51
168.69
±
15.43
397.84
± 25.32
440.12
± 26.92
442.67
± 29.45
345.87
± 23.78
212.76
±
13.26
235.98
±
17.23
75.47 ±
12.67
4
258.72
± 25.48
438.56
± 33.27
242.51
±
22.26
549.78
± 32.78
600.34
± 35.12
605.23
± 37.72
483.52
± 31.63
278.48
±
18.89
319.26
±
22.63
104.81
± 14.2
6
328.34
± 32.61
553.21
± 40.67
307.59
±
29.45
693.56
± 38.72
780.56
± 42.31
742.17
± 44.17
613.78
± 37.89
339.34
±
23.34
413.67
±
26.31
128.37
± 18.75
8
375.82
± 37.49
668.49
± 48.21
371.46
±
36.98
825.45
± 45.45
945.67
± 49.94
909.38
± 52.65
745.29
± 46.21
402.69
±
27.53
498.11
±
32.36
152.57
± 22.21
12
414.21
± 42.18
835.37
± 53.43
444.62
±
42.36
970.12
± 56.51
1090.38
± 56.85
1025.24
± 59.34
878.83
± 54.23
494.18
±
32.33
561.33
±
38.45
169.34
± 20.38
18
453.63
± 47.37
912.46
± 59.56
526.85
±
47.66
1045.34
± 63.69
1212.96
± 63.41
1165.08
± 68.53
946.93
± 60.68
539.39
±
38.76
638.28
±
43.18
188.74
± 21.89
24
520.12
± 51.42
1002.63
± 67.78
566.67
±
52.12
1195.23
± 70.42
1327.34
± 71.29
1235.11
± 77.58
1088.23
± 65.33
599.41
±
44.52
660.45
±
48.89
210.63
± 35.22
Each result is the mean ± S.D (n = 3); * T- Time (hr)
Chapter 6 LORNOXICAM PRONIOSOMAL DELIVERY
Ph. D. Thesis Page | 154
Figure 6.5: Permeation profile of design formulation F1 to F15
Figure 6.6: Permeation profile of design formulation F16 to F29, control
0
200
400
600
800
1000
1200
1400
0 2 4 6 8 10 12 14 16 18 20 22 24Cum
mul
ativ
e am
ount
per
mea
ted
(µ
g/cm
2 )
Time (hr)
Design formulation F1 to F15
F1 F2 F3 F4 F5 F6 F7 F8
F9 F10 F11 F12 F13 F14 F15
0
200
400
600
800
1000
1200
1400
1600
0 2 4 6 8 10 12 14 16 18 20 22 24
Cum
mul
ativ
e am
ount
per
mea
ted
(µg/
cm2 )
Time (hr)
Design formulation F16 to F29, Control
F16 F17 F18 F19 F20
F21 F22 F23 F24 F25
F26 F27 F28 F29 Control
Chapter 6 LORNOXICAM PRONIOSOMAL DELIVERY
Ph. D. Thesis Page | 155
Table 6.12: Observed response in Box–Behnken design for lornoxicam
proniosomal formulation using design expert
Form
ulatio
n No.
Independent variables Dependent variables
X1 X2 X3 X4 Y1 ( vesicle size)
nm
Y2 (EE) % Y3 (flux)
µg/cm2/h
F01 0 -1 0 1 411.4 ± 0.065 72.43 ± 1.16 47.78 ± 1.21
F02 0 -1 0 -1 460.2 ± 0.092 66.19 ± 1.79 50.80 ± 3.57
F03 1 1 0 0 550.8 ± 0.131 67.29 ± 1.63 26.25 ± 2.79
F04 1 0 0 -1 540.5 ± 0.120 75.21 ± 1.84 33.38 ± 1.34
F05 1 -1 0 0 505.3 ± 0.113 71.85 ± 1.99 41.97 ± 1.76
F06 1 0 1 0 545.0 ± 0.134 85.71 ± 1.73 27.66 ± 2.48
F07 0 0 0 0 401.0 ± 0.070 93.09 ± 2.22 39.79 ± 3.77
F08 0 0 1 1 482.3 ± 0.075 94.05 ± 2.04 49.49 ± 1.95
F09 1 0 0 1 525.0 ± 0.105 66.68 ± 1.23 34.34 ± 3.47
F10 0 -1 1 0 480.4 ± 0.091 74.57 ± 2.11 35.38 ± 1.40
F11 -1 0 -1 0 463.1 ± 0.076 63.08 ± 1.29 19.99 ± 2.66
F12 -1 1 0 0 466.3 ± 0.089 65.28 ± 2.33 21.87 ± 3.42
F13 0 0 0 0 421.4 ± 0.050 93.05 ± 1.33 37.44 ± 2.45
F14 0 0 0 0 417.3 ± 0.067 93.51 ± 2.50 36.39 ± 2.75
F15 -1 0 0 -1 446.8 ± 0.090 57.84 ± 1.39 25.07 ± 1.66
F16 -1 0 1 0 470.5 ± 0.088 60.83 ± 1.75 22.95 ± 1.07
F17 0 0 -1 1 475.2 ± 0.099 83.21 ± 1.78 28.69 ± 2.97
F18 0 1 0 1 520.4 ± 0.137 80.47 ± 0.93 44.82 ± 0.88
F19 0 0 -1 -1 485.0 ± 0.112 92.33 ± 2.37 52.15 ± 3.01
F20 0 -1 -1 0 457.4 ± 0.053 69.87 ± 0.96 46.21 ± 3.56
F21 1 0 -1 0 533.4 ± 0.119 70.23 ± 1.23 31.51 ± 4.12
F22 0 1 0 -1 521.8 ± 0.138 87.56 ± 0.81 43.21 ± 3.11
F23 -1 -1 0 0 412.8 ± 0.059 53.54 ± 0.67 20.87 ± 1.05
F24 0 1 1 0 532.7 ± 0.123 89.81 ± 0.45 32.38 ± 3.45
F25 0 0 0 0 430.2 ± 0.061 91.01 ± 1.81 38.53 ± 1.21
Chapter 6 LORNOXICAM PRONIOSOMAL DELIVERY
Ph. D. Thesis Page | 156
F26 0 0 0 0 406.8 ± 0.081 92.62 ± 1.04 40.86 ± 2.27
F27 -1 0 0 1 450.9 ± 0.062 64.74 ± 1.45 23.97 ± 4.01
F28 0 0 1 -1 515.2 ± 0.109 79.81 ± 1.25 29.37 ± 2.43
F29 0 1 -1 0 530.2 ± 0.107 86.66 ± 2.65 30.56 ± 4.16
F30* 0.5 0.5 -0.5 -0.5 488.78 ± 0.081 89.08 ± 2.41 37.79 ± 2.24
F31* -0.5 -0.5 0 0 397.13 ± 0.074 77.92 ± 1.89 35.13 ± 1.67
F32* 0 0 0.5 0.5 432.89 ± 0.044 92.79 ± 2.87 40.69 ± 3.02
Each result is the mean ± S.D (n = 3); *Check point batches
6.7.2.1 Optimization
The optimum lornoxicam proniosomal formulation was selected based on the
criteria of attaining the minimum vesicles size and maximum EE% and transdermal flux,
by applying the point prediction method6 (Table 6.13). Upon ‘‘trading off’’ various
response variables and comprehensive evaluation of feasibility search and exhaustive grid
search, the formulation (F19) composition with S 60 (90 mg), cholesterol (10 mg),
lecithin (90 mg), and lornoxicam (5 mg) was found to fulfill requisites of an optimum
formulation. The optimized formulation had vesicles size of 485.0 ±0.112 nm (Figure
6.7), EE of 92.33±2.37%, and the transdermal flux across rat skin is 52.15±3.01
µg/cm2/h.
Table 6.13: Optimized level of formulation by point prediction method
Optimized
formula
Optimized
level (mg)
Response Predicted
value
Experimental
value
S 60 90 Vesicle size
(nm)
489.0 485.0
Cholesterol 10 Encapsulation
efficiency (%)
89.57 92.33
Lecithin 90 Flux (µg/cm2/h) 52.58 52.15
Lornoxicam 5
Chapter 6 LORNOXICAM PRONIOSOMAL DELIVERY
Ph. D. Thesis Page | 157
Figure 6.7: Size distribution of optimized F19 formulation
6.7.2.2 Fitting of data to the model
Fitting of the data for observed responses to various models, it was observed that
the best-fitted model for all the four dependent variables was the quadratic model (Table
6.14). A positive value in regression equation for a response represents an effect that
favors the optimization (synergistic effect), whereas a negative value indicates an inverse
relationship (antagonistic effect) between the factor and the response7. From Table 6.14,
it is evident that the independent variables have positive effects on the response Y1, and
Y2. Response Y3 was positively affected by S 60 concentration, whereas cholesterol,
lecithin, and lornoxicam concentration had a parabolic effect on the response.
Table 6.14: Summary of results of regression analysis for responses Y1, Y2, and Y3
for fitting to quadratic model
Quadratic
model
R2 Adjusted R2 Predicted R2 Adeq
Precisior
SD %
CV
Response (Y1) 0.9695 0.9389 0.8604 17.57 11.66 2.44
Response (Y2) 0.9554 0.9108 0.7467 17.223 3.74 4.83
Response (Y3) 0.9638 0.9275 0.8123 18.812 2.56 7.34
Chapter 6 LORNOXICAM PRONIOSOMAL DELIVERY
Ph. D. Thesis Page | 158
6.7.2.3 Vesicles size analysis
The vesicle size of formulations was found in the range of 401.0(F7) to 550.8(F3)
nm. It was observed that the vesicle size has a direct positive relationship with the factors
X1, X2, and X3 but a negative relationship with the X4. Inclusion of cholesterol and S 60
in formulation of proniosome which interacts with lipid bilayers has the direct effect on
vesicle size (Figure 6.9a). There is an initial decrease in the average size of the vesicles
with increasing amounts of S 60. However, further increase in S 60 concentration led to
increase in the average size of vesicles, due to the formation of a micellar structure
(larger in size than vesicles) instead of the vesicles. Increasing the cholesterol content
also contributed an increase in the hydrophobicity, with a subsequent slight reduction in
vesicle size. To study the effect of particle size on EE, inversely proportional relationship
was observed between them; formulations with smaller particles size possessed higher
entrapment value. This relationship is presented by the following equation:
Size = +415.34 + 40.80 X1 + 32.89 X2 + 6.82 X3 – 8.69 X4 – 2 X1X2 + 1.05 X1X3 – 4.9
X1X4 – 5.13 X2X3 + 11.85 X2X4 - 5.77 X3X4 + 40.19 X12 + 32.60 X2
2 + 47.69 X32 + 30.73
X42
6.7.2.4 EE
From 3D-graph (Figure 6.9b), it could be inferred that the EE is significantly
affected by S 60 and cholesterol. There existed a direct relationship between the S 60 and
EE% of the drug containing vesicles. The lowest EE was found for formulation F23
(53.54%), and maximum EE was found for F8 (94.05%). The phase transition
temperatures for S 60 is 53 °C, high phase transition temperature facilitates stable vesicle
formation and improves the transdermal delivery of lornoxicam from proniosomes8.
Increasing the cholesterol content from 5 to 10mg was accompanied by a significant
increase in EE% (p < 0.05). This pattern takes place due to the ability of cholesterol to
abolish gel to liquid-phase transition of niosomal systems resulting in less leaky vesicles9.
However, further cholesterol increase resulted in significant decrease in EE% (p < 0.05).
Increasing cholesterol beyond a certain concentration may compete with the drug for the
space within the bilayers, hence excluding the drug and can disrupt the regular linear
structure of vesicular membranes9. It is observed from the experimental design that EE%
Chapter 6 LORNOXICAM PRONIOSOMAL DELIVERY
Ph. D. Thesis Page | 159
has a direct positive relationship with concentration of lecithin as revealed by the
following equation:
EE = +92.66 + 5.97 X1 + 5.72 X2 + 1.62 X3 + 0.22 X4 – 4.08X1X2 + 4.43 X1X3 - 3.86
X1X4 - 0.39 X2X3 – 3.33 X2X4 + 5.84 X3X4 – 20.18 X12 - 9.77X2
2 - 1.69 X32 – 5.40 X4
2
6.7.2.5 Ex-vivo skin permeation studies
From the below equation of flux, it was observed that the steady-state flux of
lornoxicam from proniosome was first increased with increasing S 60 concentration and
then decreased (Figure 6.9c). The flux is decreased on increasing the total lipid
concentration. A possible explanation for lower flux at a high total lipid concentration
might be the high concentration disrupted the lipid membrane so that it became leakier to
the entrapped drug which led to reduction in flux. Lornoxicam concentration had a
positive relationship with transdermal flux up to 5 mg of lornoxicam, but beyond this
concentration it showed a negative relationship with transdermal flux. Further increasing
the drug concentration up to 15 mg resulted in decrease in the transdermal flux, possibly
due to leakage of lornoxicam from vesicle bilayers at higher concentration. The
permeation profile of proniosomal formulation and control shows that proniosome
formulation (F19) presented maximum flux value (i.e., 52.15µg/cm2/h) over HPMC gel
(8.27µg/cm2/h) with ER of 6.3 through rat skin (Figure 6.6). It is clear that the
proniosome exhibited higher skin permeation compared to the conventional liposome
containing equivalent amount of risperidone. The proposed mechanism for improved
permeation from proniosomes formulation may involve disruption of the densely packed
lipids that fill the extra cellular spaces of the stratum corneum. They may enhance the
permeability of drugs through structure modification of stratum corneum. The
intercellular lipid barrier in the stratum corneum would be more permeable following
treatment with proniosome as reported by many researchers10. Additionally, adsorption
and fusion of drug load proniosomes onto the surface of the skin leads to a high
thermodynamic activity gradient of the drug in upper part of the stratum corneum
facilitating drug permeation11.
Flux = +38.60 + 5.03 X1 – 3.66 X2 – 0.99 X3 – 0.41 X4 – 4.18 X1X2 – 1.7 X1X3 + 5.2
X1X4 + 3.16 X2X3 + 1.16 X2X4 + 10.9 X3X4 – 12.27 X12 + 1.77 X2
2 – 2.7 X32 + 4.39 X4
2
Chapter 6 LORNOXICAM PRONIOSOMAL DELIVERY
Ph. D. Thesis Page | 160
Figure 6.8 quantitatively compared the resultant experimental values of the responses
with that of the predicted values.
(A)
(B)
(C)
Design-Expert® SoftwareVesicle size
Color points by value ofEncapsulation Efficiency:
94.05
53.54
Actual size (nm)
Pre
dict
ed s
ize
(nm
)
Predicted vs. Actual
400
450
500
550
600
400 450 500 550 600
Design-Expert® SoftwareVesicle size
Color points by value ofEncapsulation Efficiency:
94.05
53.54
Run Number
Res
idua
ls s
ize
(nm
)
Residuals vs. Run
-4.00
-2.00
0.00
2.00
4.00
1 5 9 13 17 21 25 29
Design-Expert® SoftwareEncapsulation Efficiency
Color points by value ofEncapsulation Efficiency:
94.05
53.54
Actual entrapment efficiency (%)
Pre
dict
ed e
ntra
pmen
t effi
cien
cy (%
)
Predicted vs. Actual
40
50
60
70
80
90
100
40 50 60 70 80 90 100
Chapter 6 LORNOXICAM PRONIOSOMAL DELIVERY
Ph. D. Thesis Page | 161
(D)
(E)
(F)
Figure 6.8: Linear correlation plots (A, C, E) between actual and predicted values and the
corresponding residual plots (B, D, F) for various responses.
Design-Expert® SoftwareEncapsulation Efficiency
Color points by value ofEncapsulation Efficiency:
94.05
53.54
Run Number
Res
idua
ls e
ntra
pmen
t effi
cien
cy (%
)
Residuals vs. Run
-4.00
-2.00
0.00
2.00
4.00
1 5 9 13 17 21 25 29
Design-Expert® SoftwareFlux
Color points by value ofFlux:
52.15
19.99
Actual flux (µg/cm2/h)
Pre
dict
ed fl
ux (µg/
cm2/
h)
Predicted vs. Actual
10
20
30
40
50
60
10 20 30 40 50 60
Design-Expert® SoftwareFlux
Color points by value ofFlux:
52.15
19.99
Run Number
Res
idua
ls fl
ux (µ
g/cm
2/h)
Residuals vs. Run
-4.00
-2.00
0.00
2.00
4.00
1 5 9 13 17 21 25 29
Chapter 6 LORNOXICAM PRONIOSOMAL DELIVERY
Ph. D. Thesis Page | 162
6.7.2.6 Effect of surfactant amount
Amount of surfactant is the important factor, responsible for vesicle formation. It
shows increase in the EE% as the concentration of S 60 increases (p < 0.05). This might
be due to the large number of niosomes formed which increases the volume of the
hydrophobic bilayer domain, and makes available housing for entrapment of lornoxicam
hydrophobic drug12. Increase in surfactant amount leads to increase in the alkyl chain
length which produces higher entrapment efficiency, and the corresponding order follows
the trend S 60 (C18) > S 40 (C16) > S 20 (C12), which is consistent with other previous
reports9.
6.7.2.7 Effect of lecithin
There was significant increase in EE% was observed with increasing lipid content
(p < 0.05) which is commonly added to increase the system stability13. This result was
substantiated by the high transition temperature of lipid used in this study which shows
rigidifying effect of cholesterol and formation of less leaky membrane bilayers14. The
presence of double bonds in the unhydrogenated phosphatidylcholine permits the chains
to bend, causing the adjacent molecule not to be tightly close to the bent phospholipid
molecule when they assemble to form the niosomal membrane; accordingly, the
membrane becomes more permeable12.
6.7.2.8 Effect of cholesterol
The EE is the most important parameter in niosomal formulations. To study the
effect of increased cholesterol on the amount of drug entrapment in niosomes, a series of
formulations were prepared with increasing cholesterol concentration (5, 10, and 15 mg)
with varying concentration of lornoxicam. The effect of cholesterol on lornoxicam
entrapment was varied according to the concentration of nonionic surfactant used (Figure
6.9b). The formulations prepared using S 60 showed the maximum entrapment efficiency
with cholesterol, as it has the longest saturated alkyl chain. Increasing cholesterol content
from 5 to 10mg leads to significant increase (p < 0.05) in the entrapment efficiency of
niosomes. The improvements in drug entrapment with increased cholesterol content and
the major reduction in drug entrapment (p < 0.05) when cholesterol content was further
Chapter 6 LORNOXICAM PRONIOSOMAL DELIVERY
Ph. D. Thesis Page | 163
increased (10–15 mg) may be due to two conflicting factors: (1) with increased
cholesterol, the bilayer hydrophobicity and stability increased and permeability
decreased, which may lead to efficiently trapping the hydrophobic drug into bilayers as
vesicles formed. (2) In contrast, higher amounts of cholesterol may compete with the
drug for packing space within the bilayer, hence excluding the drug as the amphiphiles
assemble into the vesicles15.
6.7.2.9 Effect of drug concentration
The effect of increasing lornoxicam amount on the EE in the range of 5–15 mg in the
niosomes prepared from S 60 is shown in Table 6.12. The EE of lornoxicam was
increased in proniosomes, as the drug concentration was increased from 5 to 10 mg
(Figure 6.9b). The increased EE of lornoxicam with higher amount of drug used in the
formulation could be due to the saturation of the media with lornoxicam that forces the
drug to be encapsulated into proniosomes16. However, further increase in drug
concentration from 10 to 15 mg showed a decrease in the entrapment efficiency (p <
0.05). This might be due to the fact that the saturation of the bilayers of S 60 might be
reached at 10mg of drug incorporation. Increasing the lornoxicam amount from 5 to 10
mg led to increase in the flux whereas further increase to 15 mg led to decrease in flux
value (Figure 6.9c). The reason for this decrease is that a fixed amount of vesicle
components (surfactant, cholesterol and lecithin) produce a constant number of
proniosomes of definite entrapment efficiency with precipitation of excess drug from the
formulation9,12. Results of ex-vivo skin permeation suggested that a too low or a too high
concentration of drug is not beneficial in vesicular delivery through skin and also
indicated that the possible penetration enhancing effect of drug is not mainly responsible
for improved percutaneous drug skin from vesicles. This leads to the assumption that
proniosomal formulations could enhance the solubility of certain poorly soluble drugs but
to a maximum limit after which any increase in the drug concentration leads to drug
precipitation9.
Chapter 6 LORNOXICAM PRONIOSOMAL DELIVERY
Ph. D. Thesis Page | 164
(A) Vesicle size
Design-Expert® SoftwareFactor Coding: ActualVesicle size (nm)
Design points above predicted valueDesign points below predicted value550.8
401
X1 = A: Amount of Span 60X2 = B: Amount of Cholesterol
Actual FactorsC: Amount of Lecithin = 100D: Amount of Lornoxicam = 10
5 7
9 11
13 15
70
80
90
100
110
350
400
450
500
550
600
Ves
icle
siz
e (n
m)
A: Amount of Span 60 (mg)B: Amount of Cholesterol (mg)
Design-Expert® SoftwareFactor Coding: ActualVesicle size (nm)
Design Points550.8
401
X1 = A: Amount of Span 60X2 = B: Amount of Cholesterol
Actual FactorsC: Amount of Lecithin = 100D: Amount of Lornoxicam = 10
70 80 90 100 110
5
7
9
11
13
15Vesicle size (nm)
A: Amount of Span 60 (mg)
B: A
mou
nt o
f Cho
lest
erol
(m
g)
400
450
500
5
Design-Expert® SoftwareFactor Coding: ActualVesicle size (nm)
Design points above predicted valueDesign points below predicted value550.8
401
X1 = C: Amount of LecithinX2 = D: Amount of Lornoxicam
Actual FactorsA: Amount of Span 60 = 90B: Amount of Cholesterol = 10
5 7
9 11
13 15
90
95
100
105
110
350
400
450
500
550
600
Ves
icle
siz
e (n
m)
C: Amount of Lecithin (mg)D: Amount of Lornoxicam (mg)
Chapter 6 LORNOXICAM PRONIOSOMAL DELIVERY
Ph. D. Thesis Page | 165
(B) EE
Design-Expert® SoftwareFactor Coding: ActualVesicle size (nm)
Design Points550.8
401
X1 = C: Amount of LecithinX2 = D: Amount of Lornoxicam
Actual FactorsA: Amount of Span 60 = 90B: Amount of Cholesterol = 10
90 95 100 105 110
5
7
9
11
13
15Vesicle size (nm)
C: Amount of Lecithin (mg)
D: A
mou
nt o
f Lor
noxi
cam
(m
g)
420
440460
460
460
480
480
500
5
Design-Expert® SoftwareFactor Coding: ActualEncapsulation Efficiency (%)
Design points above predicted valueDesign points below predicted value94.05
53.54
X1 = A: Amount of Span 60X2 = B: Amount of Cholesterol
Actual FactorsC: Amount of Lecithin = 100D: Amount of Lornoxicam = 10
5 7
9 11
13 15
70
80
90
100
110
40
50
60
70
80
90
100
Enc
apsu
latio
n E
ffic
ienc
y (%
)
A: Amount of Span 60 (mg)B: Amount of Cholesterol (mg)
Design-Expert® SoftwareFactor Coding: ActualEncapsulation Efficiency (%)
Design Points94.05
53.54
X1 = A: Amount of Span 60X2 = B: Amount of Cholesterol
Actual FactorsC: Amount of Lecithin = 100D: Amount of Lornoxicam = 10
70 80 90 100 110
5
7
9
11
13
15Encapsulation Efficiency (%)
A: Amount of Span 60 (mg)
B: A
mou
nt o
f Cho
lest
erol
(m
g)
50
60
70
7080
90
5
Chapter 6 LORNOXICAM PRONIOSOMAL DELIVERY
Ph. D. Thesis Page | 166
(C) Flux
Design-Expert® SoftwareFactor Coding: ActualEncapsulation Efficiency (%)
Design points above predicted valueDesign points below predicted value94.05
53.54
X1 = C: Amount of LecithinX2 = D: Amount of Lornoxicam
Actual FactorsA: Amount of Span 60 = 90B: Amount of Cholesterol = 10
5 7
9 11
13 15
90
95
100
105
110
40
50
60
70
80
90
100
Enc
apsu
latio
n E
ffic
ienc
y (%
)
C: Amount of Lecithin (mg)D: Amount of Lornoxicam (mg)
Design-Expert® SoftwareFactor Coding: ActualEncapsulation Efficiency (%)
Design Points94.05
53.54
X1 = C: Amount of LecithinX2 = D: Amount of Lornoxicam
Actual FactorsA: Amount of Span 60 = 90B: Amount of Cholesterol = 10
90 95 100 105 110
5
7
9
11
13
15Encapsulation Efficiency (%)
C: Amount of Lecithin (mg)
D: A
mou
nt o
f Lor
noxi
cam
(m
g)
85
85
90
90
5
Design-Expert® SoftwareFactor Coding: ActualFlux (mcg/cm2/h)
Design points above predicted valueDesign points below predicted value52.15
19.99
X1 = A: Amount of Span 60X2 = B: Amount of Cholesterol
Actual FactorsC: Amount of Lecithin = 100D: Amount of Lornoxicam = 10
5 7
9 11
13 15
70
80
90
100
110
10
20
30
40
50
60
Flu
x (m
cg/c
m2/
h)
A: Amount of Span 60 (mg)B: Amount of Cholesterol (mg)
Chapter 6 LORNOXICAM PRONIOSOMAL DELIVERY
Ph. D. Thesis Page | 167
Figure 6.9: 3-D surface and contour plots showing relative effects of different independent variables on (A) vesicle size, (B) EE, and (C) Flux.
Design-Expert® SoftwareFactor Coding: ActualFlux (mcg/cm2/h)
Design Points52.15
19.99
X1 = A: Amount of Span 60X2 = B: Amount of Cholesterol
Actual FactorsC: Amount of Lecithin = 100D: Amount of Lornoxicam = 10
70 80 90 100 110
11
13
15
17
19
21Flux (mcg/cm2/h)
A: Amount of Span 60 (mg)
B: A
mou
nt o
f Cho
lest
erol
(m
g)
25
25
3030
35
35
Design-Expert® SoftwareFactor Coding: ActualFlux (mcg/cm2/h)
Design points above predicted valueDesign points below predicted value52.15
19.99
X1 = C: Amount of LecithinX2 = D: Amount of Lornoxicam
Actual FactorsA: Amount of Span 60 = 90B: Amount of Cholesterol = 10
5 7
9 11
13 15
90
95
100
105
110
10
20
30
40
50
60
Flu
x (m
cg/c
m2/
h)
C: Amount of Lecithin (mg)D: Amount of Lornoxicam (mg)
Design-Expert® SoftwareFactor Coding: ActualFlux (mcg/cm2/h)
Design Points52.15
19.99
X1 = C: Amount of LecithinX2 = D: Amount of Lornoxicam
Actual FactorsA: Amount of Span 60 = 90B: Amount of Cholesterol = 10
90 95 100 105 110
5
7
9
11
13
15Flux (mcg/cm2/h)
C: Amount of Lecithin (mg)
D: A
mou
nt o
f Lor
noxi
cam
(m
g)
30
35
35
40
40
45
45
50
5
Chapter 6 LORNOXICAM PRONIOSOMAL DELIVERY
Ph. D. Thesis Page | 168
6.7.2.10 Check point analysis
Table 6.15 shows estimated and observed values of check point formulations.
Results indicate that the observed values were as expected, and the difference between
estimated and observed values was found to be insignificant (p > 0.05) for vesicle size,
EE and flux. Thus, we can conclude that the obtained mathematical equation is valid for
predicting the vesicle size, EE and flux.
Table 6.15: Estimated and observed values of check point formulations
Formul
ation
No.
Y1 ( vesicle size) nm Y2 (EE) % Y3 (flux) µg/cm2/h
Estimated
value
Observed
value
Estimated
value
Observed
value
Estimated
value
Observed
value
B30 488.26 488.78 89.55 89.08 38.68 37.79
B31 396.29 397.13 78.31 77.92 34.25 35.13
B21 432.61 432.89 93.26 92.79 41.05 40.69
6.7.3 ZETA POTENTIAL ANALYSIS
The procedure for the determination of zeta potential is given in section 4.6.2.
Figure 6.10 shows the zeta potential distribution of optimized formulation (F19), which
was -37.5 mV.
Figure 6.10: Zeta potential distribution of optimized F19 proniosome formulation
Chapter 6 LORNOXICAM PRONIOSOMAL DELIVERY
Ph. D. Thesis Page | 169
6.7.4 MICROSCOPICAL EXAMINATION
6.7.4.1 Optical microscope
The procedure for the determination of morphology by optical microscope is
given in section 4.6.3.1. After adding water to the examined samples; the microscopic
examination of the prepared proniosome gel formulations revealed proniosomal vesicular
structure with entrapped lornoxicam (Figure 6.11).
Figure 6.11: Light Microscopy of optimized F19 proniosome formulation (100X)
6.7.4.2 TEM
The procedure for the determination of morphology by TEM is given in section
4.6.3.2. The TEM micrograph of optimized F19 formulation is illustrated in Figure 6.12.
It was observed that the vesicles of the niosomes formed by hydration of the proniosomes
are almost spherical in shape and in nanometer size.
Figure 6.12: Photomicrograph of proniosomal gel derived niosomes evident by TEM
Chapter 6 LORNOXICAM PRONIOSOMAL DELIVERY
Ph. D. Thesis Page | 170
6.7.5 DSC14
The procedure for the DSC study is given in section 4.6.4. DSC thermograms of
lornoxicam, S 60, cholesterol, empty and loaded niosomes are illustrated in Figure 6.13.
Lornoxicam showed exotherm and S 60, cholesterol showed endotherms at 237.31 °C,
53.21, 148 °C, respectively, corresponding to their melting temperatures. DSC
thermogram of drug-free niosomes showed the appearance of a new sharp endothermic
peak at 108.37 °C indicating the interaction between the molecules of S 60, cholesterol
and lecithin and the formation of the double layer structure of the vesicle. However,
thermogram of lornoxicam loaded niosomes revealed a disappearance of the
characteristic exothermic lornoxicam peak, and the endotherm of the niosomal bilayer
was shifted from 108.37 to a very broad wide peak at 102.66 °C. These results suggest
the dispersion and entrapment of lornoxicam into the bilayers of niosomal vesicles.
Figure 6.13: DSC thermograms of Lornoxicam, S 60, cholesterol, empty and loaded niosomes
Chapter 6 LORNOXICAM PRONIOSOMAL DELIVERY
Ph. D. Thesis Page | 171
6.7.6 IN-VITRO RELEASE TEST
The procedure for the in-vitro release test and data analysis is given in section 4.6.5.
The comparison of in-vitro % drug release of lornoxicam of control and optimized batch
is given in Table 6.16 and Figure 6.14. Release of lornoxicam was significantly enhanced
from control compared to proniosomal gel (p < 0.05).
Table 6.16: Comparison of in-vitro % drug release of Lornoxicam
Time (Hr) Control Optimized batch
0 0 0
1 18.23 ± 1.67 8.12 ± 1.12
2 33.16 ± 2.76 12.56 ± 1.67
3 44.34 ± 3.87 17.83 ± 2.21
4 54.09 ± 4.34 22.36 ± 2.76
6 65.49 ± 4.31 26.15 ± 2.88
8 72.87 ± 5.89 31.27 ± 3.36
12 77.28 ± 5.36 36.46 ± 3.42
18 81.52 ± 5.12 41.57 ± 4.31
24 85.39 ± 5.27 45.88 ± 5.12
Each result is the mean ± S.D (n = 3)
Figure 6.14: Comparison of in-vitro % drug release of Lornoxicam
0
20
40
60
80
100
0 4 8 12 16 20 24
Cum
ulat
ivr
% d
rug
rele
ase
Time (hr)
In-vitro % drug release
Control Optimized batch
Chapter 6 LORNOXICAM PRONIOSOMAL DELIVERY
Ph. D. Thesis Page | 172
Model fitting:
Goodness of fit test for optimized batch was conducted using various models like Zero
order, First order, Higuchi, Korsmeyer-Peppas, Weibull and Hixon-Crowell. Values of
Sum of Square of Residuals (SSR) were found to be 298.10, 214.03, 36.38, 66.26, 31.33
and 238.66 for model Zero order, First order, Higuchi, Korsmeyer-Peppas, Weibull and
Hixon-Crowell model respectively. Release of F19 batch was fitted into Weibull model
showing high R square value (0.9825), least SSR value (31.33) and F value (3.91) as
compared to higuchi model as shown in Table 6.17. Thus, it can be concluded that release
of lornoxicam from proniosomal gel formulation was based on weibull diffusion
controlled mechanism. Hence, the n value is less than 0.5 which indicated diffusion
(fickian) release mechanism of optimized batch17.
Table 6.17: Model fitting for optimized batch F19
Model Name
Multiple
R
R
Square
X
variable Slope SSR
Fischer
Ratio
ZERO order 0.9224 0.8509 1.7344 10.6918 298.1059 37.2632
FIRST order 0.9524 0.9071 -0.0104 1.9533 214.0332 26.7541
Higuchi 0.9909 0.9818 9.8444 0.5639 36.3821 4.5478
Korsmeyer
– Peppas 0.9858 0.9718 0.5442 -1.0360 66.2609 8.2826
Weibull
Model 0.9912 0.9825 0.6253 -1.0282 31.3307 3.9163
Hixson -
Crowell 0.9432 0.8896 0.0333 0.1668 238.6613 29.8327
6.7.7 RATE OF SPONTANEITY (HYDRATION)
The procedure for the determination of rate of spontaneity is given in section 4.6.7.
The rate of spontaneity of optimized F19 batch was found as 8.55 x 104.
Chapter 6 LORNOXICAM PRONIOSOMAL DELIVERY
Ph. D. Thesis Page | 173
6.7.8 PHYSICAL STABILITY
The procedure for the determination of rate of spontaneity is given in section 4.6.8.
The physical appearance, vesicle size and % retention of lornoxicam was monitored for
the optimized proniosome formulation (F3) upon storage at refrigerated and room
temperature for a period of 3 months. At specific time intervals, the proniosomal gel was
hydrated to form niosomes and observed the formation of vesicles without any signs of
drug crystallization. The vesicle size and entrapment efficiency was monitored and the
results specify that there was no appreciable change in the vesicle size and % retention of
lornoxicam when stored at refrigerated temperature (Table 6.18). In contrast, the
formulation was destabilized at room temperature resulting in drug leakage with less
entrapment efficiency with time (p < 0.05). The stability studies suggest that the
proniosome formulation was reasonably more stable when stored at refrigerated
conditions compared to room temperature. Similar results were previously reported43,44,51.
Table 6.18: Physical stability of optimized proniosome formulation (F19) at room
temperature (37 °C) and refrigeration (4-8 °C)
Storage
period
(month)
Vesicle size (nm) EE % Lornoxicam
retained %
RT
(37 °C)
RG
(4-8 °C)
RT
(37 °C)
RG
(4-8 °C)
RT
(37 °C)
RG
(4-8 °C)
Freshly
prepared
485.0 ± 0.112 92.33 ± 2.37 ----
1 490.7 ± 0.08 487.1 ± 0.06 88.12 ± 0.55 91.95 ± 0.34 95.44 99.59
2 501.8 ± 0.08 489.3 ± 0.08 81.09 ± 0.89 89.75 ± 0.24 87.82 97.20
3 523.4 ± 0.06 493.7 ± 0.03 73.34 ± 0.56 87.66 ± 0.56 79.43 94.94
Each result is the mean ± S.D (n = 3); *RT – room temperature; RG - refrigeration
6.7.9 IN VIVO STUDIES
6.7.9.1 Assessment of anti-inflammatory effect
The procedure for the assessment of anti-inflammatory effect is given in section
4.6.9.1. Table 6.19 & Figure 6.15 reveal maximum increase in oedema in rats receiving
Chapter 6 LORNOXICAM PRONIOSOMAL DELIVERY
Ph. D. Thesis Page | 174
no treatment (negative control) after 4 h with mean increase value equivalent to 33.12%.
On comparing the anti-inflammatory efficiency of the selected transdermal lornoxicam
formulation to the efficiency of the oral market lornoxicam tablets, it was found that the
medicated proniosome gel formula significantly inhibited the induced oedema (p < 0.05).
It was also noticed that there was no significant difference between the negative control
group and the placebo group (p > 0.05). This might indicate that the non-medicated
preparation has no effect on its own.
Figure 6.15: Effect of transdermal Lornoxicam proniosomal gel formulation on inhibition of oedema in the hind paw of rats
Table 6.19: Effect of transdermal Lornoxicam proniosomal gel formulation on
inhibition of oedema (mean increase in oedema %)
Time
(hr)
Negative Control Positive control Oral marketed
product*
Proniosomal
gel
0 0 0 0 0
1 18.43 ± 1.93 16.38 ± 1.62 13.22 ± 1.63 4.75 ± 1.69
0
10
20
30
40
0 1 2 3 4 5 6 7 8
Mea
n in
crea
se in
oed
ema
(%)
Time (hr)
Study of anti-inflammatory effect
Negative Control Positive control
Oral marketed product Proniosomal gel
Chapter 6 LORNOXICAM PRONIOSOMAL DELIVERY
Ph. D. Thesis Page | 175
2 27.23 ± 2.02 22.92 ± 1.48 12.65 ± 1.32 4.01 ± 1.38
3 28.52 ± 2.11 24.84 ± 1.76 11.44 ± 1.15 3.48 ± 1.52
4 33.12 ± 2.23 29.03 ± 1.85 10.36 ± 1.43 2.83 ± 1.21
5 31.22 ± 1.94 25.14 ± 1.28 10.87 ± 1.28 2.08 ± 1.14
6 28.31 ± 1.84 24.27 ± 1.64 10.01 ± 1.17 2.54 ± 1.05
8 25.19 ± 1.79 22.31 ± 1.88 9.44 ± 1.29 2.14 ± 1.09
Each result is the mean ± S.D (n = 3); *Flexilor (4mg), Glenmark
6.7.9.2 Assessment of antinociceptive activity
The procedure for the assessment of antinociceptive activity is given in section
4.6.9.2. Results compiled in Table 6.20 show the number of writhes noticed and the
degree of inhibition achieved by the investigated formulation in comparison with the oral
lornoxicam market product.
The examined transdermal lornoxicam proniosome formulation significantly reduced the
number of writhes, by 70.41%, compared to the oral market product (p < 0.05), which
inhibited the acetic acid induced writhes by 65.61%. This goes exactly with the results
obtained from carrageenan induced oedema test, where the proniosome formulation
proved to be more efficient as well.
Table 6.20: Effect of different formulations on acetic acid-induced writhing in
mice
Preparation Number of writhes Percent inhibition (%)
Negative Control 61.62 ± 3.02 ---
Oral market product 21.19 ± 2.01 65.61
Proniosomal gel 18.23 ± 1.78 70.41
Each result is the mean ± S.D (n = 3)
6.8 CONCLUSION
It can be concluded from the results of the study that proniosomal vesicle with
appropriate size, reasonable EE, and higher drug permeation can be prepared. The
optimized batch F19 showed 6.3 fold enhancement compared to control, as well as, a
Chapter 6 LORNOXICAM PRONIOSOMAL DELIVERY
Ph. D. Thesis Page | 176
significantly higher therapeutic efficacy (p < 0.05) – revealed from monitoring both the
anti-inflammatory and antinociceptive effects – compared to the oral market lornoxicam
tablets of the same dose. Overall, these findings indicate that proniosomes can be
successfully used for the enhancement of skin permeation of lornoxicam.
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