6 lornoxicam proniosome - shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/44240/14/14_chapter...

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



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.



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



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



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


6.4.2 PHYSICOCHEMICAL PARAMETERS OF GOAT SKIN

The procedure for the determination and result are same as section 4.4.2.



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



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



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



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



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.



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


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



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



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


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




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



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



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



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



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%



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



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


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


94.05

53.54

Actual entrapment efficiency (%)

Pre

dict

ed e

ntra

pmen

t effi

cien

cy (%

)


40

50

60

70

80

90

100

40 50 60 70 80 90 100



(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


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)


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



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



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.



(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 Points550.8

401



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



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)



(B) EE


Design Points550.8

401



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 (%)


53.54



5 7

9 11

13 15

70

80

90

100

110

40

50

60

70

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Enc

apsu

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)



Design Points94.05

53.54



70 80 90 100 110

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B: A

mou

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erol

(m

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53.54



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Design Points94.05

53.54



90 95 100 105 110

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15Encapsulation Efficiency (%)


D: A

mou

nt o

f Lor

noxi

cam

(m

g)

85

85

90

90

5

Design-Expert® SoftwareFactor Coding: ActualFlux (mcg/cm2/h)


19.99



5 7

9 11

13 15

70

80

90

100

110

10

20

30

40

50

60

Flu

x (m

cg/c

m2/

h)




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 Points52.15

19.99



70 80 90 100 110

11

13

15

17

19

21Flux (mcg/cm2/h)


B: A

mou

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f Cho

lest

erol

(m

g)

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19.99



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13 15

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x (m

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m2/

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Design Points52.15

19.99



90 95 100 105 110

5

7

9

11

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15Flux (mcg/cm2/h)


D: A

mou

nt o

f Lor

noxi

cam

(m

g)

30

35

35

40

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45

45

50

5



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



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



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



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


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



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.



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



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



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


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



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.

REFERENCES

1. Shahzad Y, Khan Q, Hussain T, Shah SNH. Influence of cellulose derivative and ethylene glycol on optimization of lornoxicam transdermal formulation. Int J Bio Macromol 2013;61:26-32.

2. Xi H, Wang Z, Chen Y, Li W, Sun L, Fang L. The relationship between hydrogen-

bonded ion-pair stability and transdermal penetration of lornoxicam with organic amines. Eur J Pharm Sci 2012;47:325-30.

3. Ahmed MO, Al-Badr AA. Lornoxicam. In: Profiles of Drug Substances, Excipients,

and Related Methodology. Elsevier Inc; 2011. (vol 36) p. 205-37. 4. Kapil S, Rekha R, Kapil SVS. Preparation and evaluation of lornoxicam niosomal

gel. Int Res J Pharm 2012;3:378-83. 5. El-Laithy HM, Shoukry O, Mahran LG. Novel sugar esters proniosomes for

transdermal delivery of vinpocetine: Preclinical and clinical studies. Eur J Pharm Biopharm 2011;77:43–55.

6. Gannu R, Palem CR, Yamsani SK, Yamsani VV, Yamsani MR. Enhanced

bioavailability of buspirone from reservoir-based transdermal therapeutic system, optimization of formulation employing Box Behnken statistical design. AAPS Pharm Sci Tech 2010;11:976-85.

7. Chopra S, Patil GV, Motwani SK. Release modulating hydrophilic matrix systems of

Losartan potassium: optimization of formulation using statistical experimental design. Eur J Pharm Biopharm 2007;66:73-82.

8. Yoshioka T, Sternberg B, Florence AT. Preparation and properties of vesicles

(niosomes) of sorbitan monoesters (S 20 40 60 and 80) and a sorbitan trimester (Span 85). Int J Pharm 1994;105:1-6.

9. Mokhtar M, Sammour OA, Hammad MA, Megrab NA. Effect of some formulation

parameters on flurbiprofen encapsulation and release rates of niosomes prepared from proniosomes. Int J Pharm 2008;361:104-11.



10. Aboelwafa AA, Doaa AE, Aliaa NE. Comparative study on the effects of some polyoxyethylene alkyl ether and sorbitan fatty acid ester surfactants on the performance of transdermal carvedilol proniosomal gel using experimental design. AAPS Pharm Sci Tech 2010;11:1591-602.

11. Fang JY, Yu SY, Wu PC, Huang YB, Tsai YH. In-vitro skin permeation of estradiol

from various proniosomes formulation. Int J Pharm 2001;215:91-9. 12. Hao Y, Zhao F, Li N, Yang Y, Li K. Studies on a high encapsulation of colchicine by

a niosome system. Int J Pharm 2002;244:73-80. 13. Varshosaz J, Pardakhty A, Seied MHB. Sorbitan monopalmitate-based proniosomes

for transdermal delivery of chlorpheniramine maleate. Drug Deliv 2005;12:75-82. 14. El-Laithy HM, Shoukry O, Mahran LG. Novel sugar esters proniosomes for

transdermal delivery of vinpocetine: Preclinical and clinical studies. Eur J Pharm Biopharm 2011;77:43–55.

15. Balakrishnan P, Shanmugam S, Lee WS, Lee WM, Kim JO, Oh DH, et al.

Formulation and in-vitro assessment of minoxidil niosomes for enhanced skin delivery. Int J Pharm 2009;377:1-8.

16. EL-Samaligy MS, Afifi NN, Mahmoud EA. Increasing bioavailability of silymarin

using a buccal liposomal delivery system: preparation and experimental design investigation. Int J Pharm 2006;308:140-8.

17. Waddad AY, Abbad S, Yu F, Munyendo WL, Wang J, Lv H, et al. Formulation,

characterization and pharmacokinetics of Morin hydrate niosomes prepared from various non-ionic surfactants. Int J Pharm. 2013;456:446-58.

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