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Topical Niosomal Preparation Containing Combination of Benzoyl peroxide and Tretinoin:

Formulation and Evaluation For Antiacne Activity

Submitted by: Under the supervision of:Ankush Gupta Mr. Narendra Kumar Pandey

Senior Lecturer (Pharmaceutics)Dr. Monica GulatiDean LSAMS

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Introduction

Acne vulgaris

Topical treatment of acne vulgaris

Problems with topical treatment

Vesicle approach for topical treatment

Benefits of vesicle approach in treatment of acne vulgaris

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Topical treatment of acne vulgaris

Topical retinoids Gollnick et al., 2003. Dermatology 206, 29-36

Benzoyl peroxide (BPO) Lookingbill et al., 1997. J. Am. Acad. Dermatol. 37, 590-595

Azealic acid Spellman et al., 1998. Clinical Therapeutics 20, 4, 711-721

Erythromycin Lesher, et al., 1985. J. Am. Acad. Dermatol. 12, 526-531

Clindamycin Gratton et al., 1982. J. Am. Acad. Dermatol. 7, 50-53

Combination therapies Glass et al., 1999. Dermatology 199, 242-247

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Problems occured in topical treatment of acne

Skin barrier problem Choi et al., 2005. Skin Pharmacol. Physiol.18, 209-219

Adverse effect of drugs Kim et al., 2002. UNEP publication 1-110

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Remedy for problem persist in topical treatment of acne

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Proposed structure of niosomes Andrew.cmu.edu/jamesv/Research lab.html

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Niosomes as effective carrier in topical drug deliveryAvoid skin barrier properties Choi et al., 2005. Skin Pharmacol.

Physiol. 18, 209-219

Provide continuous drug delivery at targeted site for long time period Manconi et al., 2003. Int. J. Pharm. 260, 261-272

Non immunogenic, biodegradable, biocompatible Fang et al., 2001. Int. J. Pharm. 215, 91-99

No storage problem Manosroi et al., 2003. Colloids and Surfaces B: Biointerfaces 30, 129-138

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Aim and Objectives•To prepare topical niosomal gel of combination of BPO and

tretinoin to reduce the side effect of drugs associated with the topical treatment

•To retain more amount of drug at affected site for long time which would be beneficial for the treatment of acne vulgaris

•Formulation and evaluation for antiacne activity of prepared niosomal gel

•Objective of the present study was to increase the therapeutic index of formulation by decreasing the dose of drugs using vesicular approach

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EXPERIMENTAL

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Characterization of drugs

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Sr. No.

Method Drugs

Tretinoin Benzoyl peroxide

1 UV Spectroscopy

348.6 nm (reported 352 nm) 234.8 nm (reported 235 nm)

2 IR Spectroscopy

Characterstic band at 1600.9 cm-1 for C=O str and 2945.4 cm-1 O-H str

Characterstic band at 1759.4 cm-1 for C=O str ester and 1226.7 cm-1 C-O str

3 NMR Spectroscopy

The taken 1H-NMR signals was in agreement with their reference values.

The taken 1H-NMR signals was in agreement with their reference values.

4 Melting point 179°C (reported 180-181°C) 103°C (reported 104-106°C)

5 TLC analysis Rf value 0.854 was found in

(Hexane: EtoAc = 50:50)

Rf value 0.977 was found in (Hexane: EtoAc = 50:50)

Sigma Aldrich catalog. CAS (94-36-0) 3, 580B pp., 2924.

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Standard plot of tretinoin in methanol

Std.plot of Tretinoin at 348.6 nm y = 0.0918xr2 = 0.9991

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 2 4 6 8 10

conc.(µg/ml)

Ab

s

1111

Standard plot of benzoyl peroxide in ethanol

Std plot of BPO in ethanol at 234.8 nm

y = 0.2069x-0.0253r2 = 0.9984

0

0.2

0.4

0.6

0.8

1

1.2

0 1 2 3 4 5 6

conc (µg/ml)

Ab

s

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Partition coefficient of drugs

Partition coefficient were calculated using shake flask

method

Calculated values in octanol / water

5.01 (Tretinoin)

3.80 (Benzoyl peroxide)

Reported values in octanol / water

5.032 (Tretinoin)

3.83 (Benzoyl peroxide)

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Kim et al., 2002. UNEP publication 1-110

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Multicomponent scanning analysis of mixture of tretinoin and benzoyl peroxide

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Absorbance of tretinoin at 234.8 nm was -0.059 and absorbance of BPO at 348.6 nm was -0.026. The results shows that there was no interference in the mixture of both drugs.

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Incompatibility studies between drugs

Studied by FTIR spectroscopy and UV scan analysis

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FTIR spectra of incompatible studies between drugs (a) Benzoyl peroxide (b) Tretinoin

(c) Mixture of BPO and tretinoin

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UV scan analysis for incompatibilty studies between drugs

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Antimicrobial susceptibility testing

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BPO was tested for antimicrobial activity against SBPO was tested for antimicrobial activity against Staphylococcus taphylococcus epidermidis epidermidis using nutrient agar growth mediausing nutrient agar growth media

Photograph of zone of inhibition of Staphylococcus epidermidis using BPO as antibacterial agent

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Zone of inhibition of Staphylococcus epidermidis using benzoyl peroxide

12.4

13.4

14.4

15.4

16.4

17.4

50 250 450 650 850 1050 1250 1450 1650 1850 2050

Conc. (µg/ml)

Zone

of i

nhib

ition

(mm

)

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MIC determination of BPO

MIC was determined by microdilution or broth dilution method

Measured broth was taken in sterlized test tube, added bacterial suspension BPO solution in it and incubated at 37±1°C for 24 hrs aerobically using incubator shaker

After 24 hrs, growth of Staphylococcus epidermidis was measured as function of turbidity at 660 nm using UV spectrophotometer

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Table for MIC of BPO against Staphylococcus epidermidis

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Sr. No.

Conc. (µg/ml)

Stock Solution taken (µl)

Bacterial suspension(µl)

Broth taken (µl)

Total broth (µl)

Average of abs

S.D.

1. 0 0 100 4900 5000 0.675 ±0.004

2. 20 100 100 4800 5000 0.127 ±0.0015

3. 22 110 100 4790 5000 0.122 ±0.001

4. 24 120 100 4780 5000 0.119 ±0.0011

5. 26 130 100 4770 5000 0.118 ±0.0005

6. 28 140 100 4760 5000 -0.059 ±0.001

7. 30 150 100 4750 5000 -0.052 ±0.0015

8. 32 160 100 4740 5000 -0.047 ±0.0015

9. 34 170 100 4730 5000 -0.043 ±0.001

10. 36 180 100 4720 5000 -0.040 ±0.0011

11. 38 190 100 4710 5000 -0.038 ±0.0015

12. 40 200 100 4700 5000 -0.035 ±0.0011

2121

Chomnawang et al., 2005. J. Ethnopharmacol. 101, 330-333.

MIC of Benzoyl peroxide against Staphylococcus epidermidis

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 5 10 15 20 25 30 35 40

Conc. (µg/ml)

Ab

sorb

an

ce

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Niosomes preparation

Prepared by lipid hydration method

Various weight ratio were tried to prepare niosomal formulation

Non ionic surfactant and cholesterol ratio (207:52 or 138:52 mg ratio) was found the best formulation on the basis of encapsulation efficiency

Vesicles were observed at 100x magnification using photomicroscope

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Manosroi et al., 2003. Colloids and Surfaces B: Biointerfaces 30, 129-138.

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Photograph of Niosomes at 100X magnification

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Characterizations of niosomes Morphology analysis by transmission electron microscopy

Particle size analysis or PCS

Zeta potential

Encapsulation efficiency

Ex vivo permeation study

Comparison of ex vivo permeation study of niosomal gel, cream and alcoholic solution

In vitro retention study

Stability studies

In vivo studies

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2525

Transmission Electron Microscopy

TEM photograph of niosomes (negative staining)

TEM photograph of niosomes (without staining)

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Particle size analysis (PCS)

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2727

2828

Zeta Potential Analysis

2929

Encapsulation efficiency of niosomes

Sr. No. Niosomal Formulation loaded with Benzoyl peroxide

Hydration Media

% EncapsulationEfficiency

1. Span 60 : CH (69: 35) weight (mg) ratio Water 52.6 ±0.45 %

2. Span 60 : CH (69: 35) weight (mg) ratio Saline 51.1 ±0.34 %

3. Span 60: CH (138: 52) weight (mg) ratio Water 98.75 ±1.25 %

4. Span 60: CH (138: 52) weight (mg) ratio Saline 94.86 ±0.56 %

5. Span 60: CH (207: 52) weight (mg) ratio Water 92.4 ±0.49 %

6. Span 60: CH (207: 52) weight (mg) ratio Saline 89.06 ±0.76 %

Note: Stock solution of benzoyl peroxide is 15 mg/ml. Percent encapsulation Note: Stock solution of benzoyl peroxide is 15 mg/ml. Percent encapsulation efficiency is mean of triplicate experiment.efficiency is mean of triplicate experiment.

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Sr. No. Niosomal Formulation loaded with tretinoin

HydrationMedia

% EncapsulationEfficiency

1. Span 60 : CH (69: 35) weight (mg) ratio Water 24.5 ±0.65 %

2. Span 60 : CH (69: 35) weight (mg) ratio Saline 43.25 ±0.35 %

3. Span 60: CH (138: 35) weight (mg) ratio Water 48.25 ±0.82 %

4. Span 60: CH (138: 35) weight (mg) ratio Saline 74.00 ±0.72 %

5. Span 60: CH (138: 52) weight (mg) ratio Water 52.05±0.85 %

6. Span 60: CH (138: 52) weight (mg) ratio Saline 86.45±0.54%

7. Span 60: CH (207: 52) weight (mg) ratio Water 74.75 ±0.34 %

8. Span 60: CH (207: 52) weight (mg) ratio Saline 96.25 ±0.56 %

9. Span 60: CH (276: 52) weight (mg) ratio Water 61.25 ±0.63 %

10. Span 60: CH (276: 52) weight (mg) ratio Saline 70.75 ±0.43 %

Note: Stock solution of tretinoin is 4 mg/ml. Percent encapsulation efficiency is mean Note: Stock solution of tretinoin is 4 mg/ml. Percent encapsulation efficiency is mean of triplicate experiment.of triplicate experiment.

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Effect of total lipid concentration on encapsulation efficiency on niosomes

Sr. No Total lipid(mg/ml)

% Encapsulationefficiency ofTretinoin

Total lipid(mg/ml)

% Encapsulationefficiency of BPO

1 10.4 43.25 ±0.35 10.4 52.6 ±0.45 %

2 17.3 74.00 ±0.72 19 98.75 ±1.25 %

3 19 86.45±0.54 25.9 92.4 ±0.49 %

4 25.9 96.25 ±0.56 -- --

5 32.8 70.75 ±0.43 -- --

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Effect of total lipid concentration on % encapsulation efficiency of tretinoin

43.25

53.25

63.25

73.25

83.25

93.25

10.4 15.4 20.4 25.4 30.4

Total lipid (mg/ml)

% E

ncap

sula

tion

effic

ienc

y

3333

Effect of total lipid concentration on encapsulation efficiency of BPO

50

60

70

80

90

100

0 5 10 15 20 25 30

Total lipid (mg/ml)

% E

ncap

sula

tion

effic

ienc

y

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DSC analysis of niosomal formulation

Determined the gel lipid phase transition temperature of niosomes

The hydrocarbon chains in ordered bilayer of vesicle were transformed from rigid gel to facile liquid crystal

It was found to be 56-58°C for prepared niosomal formulation

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Hathout et al., 2007. AAPS PharmSciTech. 8(1), E1-E12.

3535

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Ex vivo permeation study of antiacne niosomal gel

• Performed by using Franz diffusion

cell

• Rat skin (occulsive condition)

• Study time (24 hr)

• Effective diffusion area (2.54 cm2)

• Sample withdrawn (3 ml)

• Graph plot between permeated

amount µg/cm2 versus time intervals

3737

Result: 5.04 ± 0.014µg/cmResult: 5.04 ± 0.014µg/cm22 was permeated amount of BPO from niosomal gel in 24 h. was permeated amount of BPO from niosomal gel in 24 h.

Permeation profile of BPO from niosomal gel

0.000

1.000

2.000

3.000

4.000

5.000

0 2 4 6 8 10 12 14 16 18 20 22 24 26

Time (h)

Per

mea

ted

am

ou

nt

µg

/cm

2

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Result: 6.25 ± 0.14µg/cm2 was permeated amount of tretinoin from niosomal gel in 24 h

Permeation profile of tretinoin from niosomal formulation

-0.5

0.5

1.5

2.5

3.5

4.5

5.5

6.5

0 2 4 6 8 10 12 14 16 18 20 22 24 26

Time (h)

Per

mea

ted

am

ou

nt

µg

/cm

2

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Comparison of ex vivo permeation studies of antiacne cream, niosomal gel and alcoholic solution

On the basis of permeation studies the order of sustained release from formulation are

gel > cream > alcoholic solution

Formulation Permeated amount of Tretinoin

Permeated amount of BPO

Antiacne Niosomal Gel 6.25 ± 0.14µg/cm2 in 24 h 5.04 ± 0.014µg/cm2 in 24 h

Antiacne Cream 6.60 ± 0.13µg/cm2 in 8 h 7.91 ± 0.023 µg/cm2 in 24 h

Alcoholic solution 7.72 ± 0.16µg/cm2 in 6 h 12.18 ± 0.013 µg/cm2 in 6 h

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Permeation profile of BPO and tretinoin from cream, alcoholic solution and niosomal gel

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2

4

6

8

10

12

14

0 2 4 6 8 10 12 14 16 18 20 22 24 26

Time (hr)

Perm

eate

d am

ount

µg/

cm2 Release of tretinoin from

cream

Relese of BPO from cream

Release of tretinoin fromalcoholic solution

release of BPO fromalcoholic solution

Release of tretinoin fromniosomal formulation

Release of BPO fromniosomal gel

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Comparison of permeation flux of cream, niosomal gel and alcoholic solution

Formulation Tretinoin flux BPO flux

Alcoholic solution 0.837±0.037 1.530±0.001

Antiacne cream 0.811±0.008 0.195±0.001

Antiacne niosomal gel 0.390±0.012 0.436±0.000

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4242

Comparison of permeation flux of various formulation

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Alcoholicsolution(BPO)

Alcoholicsolution

(Tretinoin)

Cream(BPO)

Cream(Tretinoin)

Niosomal gel(BPO)

Niosomal gel(Tretinoin)

Formulations

Perm

eatio

n flu

x (µ

g/cm

2 /h)

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In vitro retention study

S. No. Formulation Drug retained (µg)

1. Anti acneCream

Tretinoin 11.54± 0.21

Benzoyl Peroxide 68.85± 0.40

2. Niosomal Gel

Tretinoin 15.54± 0.33

Benzoyl Peroxide 143.78± 0.40

3. Alcoholicsolution

Tretinoin 2.68± 0.33

Benzoyl Peroxide 59.98 ± 0.50

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Drug retained by skin

0

20

40

60

80

100

120

140

Alcoholicsolution

(Tretinoin)

Cream(Tretinoin)

Nisomal gel(Tretinoin)

Alcoholicsolution(BPO)

Cream(BPO)

Nisomal gel(BPO)

Various formulations

Dru

g re

tain

ed in

ski

n (μ

g)

Drug retained by skin

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Stability studies of niosomal formulation

Time (Days)

Amount of BPO (mg) atroomtemperature

Amount of BPO (mg)at refrigerationtemperature

Amount of BPO (mg) at45°C

1 15 15 15

3 14.55 14.7 14.25

7 14.4 14.55 13.66

14 14.25 14.25 12.75

30 13.95 14.1 11.4

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Stability profile of niosomal formulation loaded with BPOStability profile of niosomal formulation loaded with BPO

Gupta et al., 2007. Trop. J. Pharm. Res. 6 (2), 687-693.

4646

Stability profile of niosomal formulation loaded with benzoyl peroxide

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11.5

12

12.5

13

13.5

14

14.5

15

1 3 7 14 30

Days

Dru

g re

tain

ed (m

g) Room temperature

Refrigerated temperature

45 degree celcius

4747

Stability profile of niosomal formulation loaded with Stability profile of niosomal formulation loaded with tretinointretinoin

Time (Days)

Amount of Tretinoin (mg) at room temperature

Amount of Tretinoin (mg) at refrigeration temperature

Amount of Tretinoin (mg) at 45°C

1 3.85 3.85 3.85

3 3.73 3.77 3.65

7 3.69 3.73 3.5

14 3.65 3.65 3.27

30 3.58 3.61 2.92

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Stability profile of niosomal formulation loaded with tretinoin

2

2.2

2.4

2.6

2.8

3

3.2

3.4

3.6

3.8

1 3 7 14 30

Days

Dru

g re

tain

ed (m

g)

Room temperature

Refrigerated temperature

45 degree celcius

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In vivo evaluation of antiacne activity of niosomal gel

•Rabbit ear model was used

•New Zealand White Rabbits were used for study

•Protocol of antiacne activity evaluation was approved by

Institutional Animal Ethics Committee vide approval No-

954/ac/06/CPCSEA/09/09

•This model used to study comedo formation to assess the

comedogenecity of dermatological preparation and to

evaluate the potential of antiacne drugs

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Ito et al., 1985. J. Invest. Dermatol. 85, 249-254.

Vogel et al., 2002. Pharmacological Assay. 2nd edition, pp.,1339-1340.

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Formulations used for in vivo study

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Sr. No Ingredients % w/w

1. Tretinoin 0.020

2. Benzoyl peroxide

0.600

3. Carbopol 934 1.2

4. Sodium hydroxide

Up to pH 5.

5. Water q.s to 100

Composition of niosomal gel

Sr. No Ingredients % w/w

1. Bees wax 4

2. Paraffin 10

3. White petrolatum 16.8

4. Mineral oil 55

5. Tretinoin 0.025

6. Benzoyl peroxide

2.5

7. Glycerin 1

8. Water 10.67

Composition of antiacne cream (w/o)

Nanda, S., et al., 2005. Skin Cosmetic. Birla Publication Pvt ltd, pp, 301.

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Histological Observations

•Hyperplasia of epidermis, follicular and sebaceous duct epithelia and sebaceous gland

•Comedo formation

•Units of comedo presents

•Volume of sebaceous secretory unit

•Comedonal dilatation of follicle

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Results of Biopsy Report

Light micrograph of rabbit ear pinna section of control and treatedanimals revealed the following observation:

After treatment for 14 days with antiacne preparations the volume of sebaceous secretory units reduced to 50% and no comedonal dilatation of follicle were seen when compared with control and 7th and 28th day samples

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Observation Control sample Treated pinna

Sebaceous gland, epidermis, follicular and sebaceous duct epithelia

Normal Hyperplasia

Unit of comedo present at day 7th and 28th

Single unit Several units

Volume of sebaceous secretory unit

5% of sectioned surface

6% of sectioned surface

At time of acne induction

5353

Light micrograph of normal rabbit ear pinna (control sample) at 0 day at 10 X

5454

Light micrograph of oleic acid treated rabbit ear pinna at 10X (treated pinna) at 7th day

5555

Light micrograph of oleic acid treated rabbit ear pinna at 10 X (treated pinna) at 28th day

5656

Light micrograph of treated rabbit ear pinna further treated with antiacne niosomal gel for 14

days at 10X magnification

5757

Light micrograph of treated rabbit ear pinna further treated with antiacne cream for 14 days at

10X magnification

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Conclusions

Maximum amount of drugs was retained in skin layers in case of niosomal gel formulation

Niosomal gel maintained the therapeutic drug concentration in affected site for prolong time which would be beneficial for the topical therapy

In vivo studies revealed that niosomal gel increases the therapeutic index of formulation by decreasing the dose of BPO in the formulation approximately 4 fold than antiacne cream.

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5959