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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 Gulati Dean LSAMS
Introduction ,[object Object],[object Object],[object Object],[object Object],[object Object]
Topical treatment of acne vulgaris ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Problems occured in topical treatment of acne ,[object Object],[object Object]
Remedy for problem persist in topical treatment of acne Proposed structure of niosomes Andrew.cmu.edu/jamesv/Research lab.html
Niosomes as effective carrier in topical drug  delivery ,[object Object],[object Object],[object Object],[object Object]
Aim and Objectives ,[object Object],[object Object],[object Object],[object Object]
EXPERIMENTAL
Characterization of drugs Sigma Aldrich catalog. CAS (94-36-0) 3, 580B pp., 2924.  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  1 H-NMR signals was in agreement with their reference values. The taken  1 H-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 R f  value 0.854 was found in (Hexane: EtoAc = 50:50) R f   value 0.977 was found in  (Hexane: EtoAc = 50:50)
Standard plot of tretinoin in methanol
Standard plot of benzoyl peroxide in ethanol
Partition coefficient of drugs ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],Kim et al., 2002. UNEP publication 1-110
Multicomponent scanning analysis of mixture of tretinoin and benzoyl peroxide 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.
Incompatibility studies between drugs ,[object Object]
FTIR spectra of incompatible studies between drugs (a) Benzoyl peroxide (b) Tretinoin  (c) Mixture of BPO and tretinoin
UV scan analysis for incompatibilty studies between drugs
Antimicrobial susceptibility testing BPO was tested for antimicrobial activity against S taphylococcus epidermidis  using nutrient agar growth media Photograph of zone of inhibition of  Staphylococcus epidermidis  using BPO as antibacterial agent
MIC determination of BPO ,[object Object],[object Object],[object Object]
Table for MIC of BPO against  Staphylococcus epidermidis 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
Chomnawang et al., 2005.  J.  Ethnopharmacol. 101, 330-333.
Niosomes preparation ,[object Object],[object Object],[object Object],[object Object],Manosroi et al., 2003. Colloids and Surfaces B: Biointerfaces 30, 129-138.
Photograph of Niosomes at 100X magnification
Characterizations of niosomes ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Transmission Electron Microscopy TEM photograph of niosomes (negative staining) TEM photograph of niosomes (without staining)
Particle size analysis (PCS)
Zeta Potential Analysis
Encapsulation efficiency of niosomes Note: Stock solution of benzoyl peroxide is 15 mg/ml. Percent encapsulation  efficiency is mean of triplicate experiment. Sr. No. Niosomal Formulation loaded with Benzoyl peroxide  Hydration Media % Encapsulation Efficiency 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 tretinoin is 4 mg/ml. Percent encapsulation efficiency is mean  of triplicate experiment. Sr. No. Niosomal Formulation loaded with tretinoin Hydration Media % Encapsulation Efficiency 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 %
Effect of total lipid concentration on encapsulation  efficiency on niosomes Sr. No Total lipid (mg/ml) % Encapsulation efficiency of Tretinoin Total lipid (mg/ml) % Encapsulation efficiency 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 - -
DSC analysis of niosomal formulation ,[object Object],[object Object],[object Object],Hathout et al., 2007. AAPS PharmSciTech. 8(1), E1-E12.
Ex vivo  permeation study of antiacne niosomal gel ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
Result: 5.04 ± 0.014µg/cm 2  was permeated amount of BPO from niosomal gel in 24 h.
Result: 6.25 ± 0.14µg/cm 2  was permeated amount of tretinoin from niosomal gel in 24 h
Comparison of  ex vivo  permeation studies of antiacne cream, niosomal gel and alcoholic solution ,[object Object],[object Object],Formulation Permeated amount of Tretinoin Permeated amount of BPO Antiacne Niosomal Gel 6.25 ± 0.14µg/cm 2  in 24 h 5.04 ± 0.014µg/cm 2  in 24 h Antiacne  Cream 6.60 ± 0.13µg/cm 2  in 8 h 7.91 ± 0.023 µg/cm 2  in 24 h Alcoholic solution 7.72 ± 0.16µg/cm 2  in 6 h 12.18 ± 0.013 µg/cm 2  in 6 h
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
In vitro  retention study S. No. Formulation Drug retained (µg) 1. Anti acne Cream 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. Alcoholic solution Tretinoin 2.68± 0.33 Benzoyl Peroxide 59.98 ± 0.50
Stability studies of niosomal formulation Stability profile of niosomal formulation loaded with BPO Gupta et al., 2007.   Trop. J. Pharm. Res. 6 (2), 687-693. Time  (Days)  Amount of  BPO (mg)  at room temperature Amount of  BPO (mg) at refrigeration temperature Amount of  BPO (mg) at 45°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
Stability profile of niosomal formulation loaded with tretinoin 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
In vivo  evaluation of antiacne activity of niosomal gel  ,[object Object],[object Object],[object Object],[object Object],Ito et al., 1985. J. Invest. Dermatol. 85, 249-254.   Vogel et al., 2002. Pharmacological Assay. 2nd edition, pp.,1339-1340.
Formulations used for  in vivo  study Composition of niosomal gel Composition of antiacne cream (w/o) Nanda, S., et al., 2005.  Skin Cosmetic. Birla Publication Pvt ltd, pp, 301.  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 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
Histological Observations ,[object Object],[object Object],[object Object],[object Object],[object Object]
Results of Biopsy Report ,[object Object],[object Object],[object Object],At time of acne induction  Observation Control sample Treated pinna Sebaceous gland, epidermis, follicular and sebaceous duct epithelia Normal Hyperplasia Unit of comedo present at day 7 th  and 28 th   Single unit Several units Volume of sebaceous secretory unit 5% of sectioned surface 6% of sectioned surface
Light micrograph of normal rabbit ear pinna (control sample) at 0 day at 10 X
Light micrograph of oleic acid treated rabbit ear pinna at 10X (treated pinna) at 7 th  day
Light micrograph of oleic acid treated rabbit ear pinna at 10 X (treated pinna) at 28 th  day
Light micrograph of treated rabbit ear pinna further treated with antiacne niosomal gel for 14 days at 10X magnification
Light micrograph of treated rabbit ear pinna further treated with antiacne cream for 14 days at 10X magnification
Conclusions ,[object Object],[object Object],[object Object]
Topical Niosomal Preparation Presentation  1

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Topical Niosomal Preparation Presentation 1

  • 1. 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 Gulati Dean LSAMS
  • 2.
  • 3.
  • 4.
  • 5. Remedy for problem persist in topical treatment of acne Proposed structure of niosomes Andrew.cmu.edu/jamesv/Research lab.html
  • 6.
  • 7.
  • 9. Characterization of drugs Sigma Aldrich catalog. CAS (94-36-0) 3, 580B pp., 2924. 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 1 H-NMR signals was in agreement with their reference values. The taken 1 H-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 R f value 0.854 was found in (Hexane: EtoAc = 50:50) R f value 0.977 was found in (Hexane: EtoAc = 50:50)
  • 10. Standard plot of tretinoin in methanol
  • 11. Standard plot of benzoyl peroxide in ethanol
  • 12.
  • 13. Multicomponent scanning analysis of mixture of tretinoin and benzoyl peroxide 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.
  • 14.
  • 15. FTIR spectra of incompatible studies between drugs (a) Benzoyl peroxide (b) Tretinoin (c) Mixture of BPO and tretinoin
  • 16. UV scan analysis for incompatibilty studies between drugs
  • 17. Antimicrobial susceptibility testing BPO was tested for antimicrobial activity against S taphylococcus epidermidis using nutrient agar growth media Photograph of zone of inhibition of Staphylococcus epidermidis using BPO as antibacterial agent
  • 18.
  • 19.
  • 20. Table for MIC of BPO against Staphylococcus epidermidis 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
  • 21. Chomnawang et al., 2005. J. Ethnopharmacol. 101, 330-333.
  • 22.
  • 23. Photograph of Niosomes at 100X magnification
  • 24.
  • 25. Transmission Electron Microscopy TEM photograph of niosomes (negative staining) TEM photograph of niosomes (without staining)
  • 27.
  • 29. Encapsulation efficiency of niosomes Note: Stock solution of benzoyl peroxide is 15 mg/ml. Percent encapsulation efficiency is mean of triplicate experiment. Sr. No. Niosomal Formulation loaded with Benzoyl peroxide Hydration Media % Encapsulation Efficiency 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 %
  • 30. Note: Stock solution of tretinoin is 4 mg/ml. Percent encapsulation efficiency is mean of triplicate experiment. Sr. No. Niosomal Formulation loaded with tretinoin Hydration Media % Encapsulation Efficiency 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 %
  • 31. Effect of total lipid concentration on encapsulation efficiency on niosomes Sr. No Total lipid (mg/ml) % Encapsulation efficiency of Tretinoin Total lipid (mg/ml) % Encapsulation efficiency 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 - -
  • 32.
  • 33.
  • 34.
  • 35.
  • 36.
  • 37. Result: 5.04 ± 0.014µg/cm 2 was permeated amount of BPO from niosomal gel in 24 h.
  • 38. Result: 6.25 ± 0.14µg/cm 2 was permeated amount of tretinoin from niosomal gel in 24 h
  • 39.
  • 40.
  • 41. 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
  • 42.
  • 43. In vitro retention study S. No. Formulation Drug retained (µg) 1. Anti acne Cream 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. Alcoholic solution Tretinoin 2.68± 0.33 Benzoyl Peroxide 59.98 ± 0.50
  • 44.
  • 45. Stability studies of niosomal formulation Stability profile of niosomal formulation loaded with BPO Gupta et al., 2007. Trop. J. Pharm. Res. 6 (2), 687-693. Time (Days) Amount of BPO (mg) at room temperature Amount of BPO (mg) at refrigeration temperature Amount of BPO (mg) at 45°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
  • 46.
  • 47. Stability profile of niosomal formulation loaded with tretinoin 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
  • 48.
  • 49.
  • 50. Formulations used for in vivo study Composition of niosomal gel Composition of antiacne cream (w/o) Nanda, S., et al., 2005. Skin Cosmetic. Birla Publication Pvt ltd, pp, 301. 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 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
  • 51.
  • 52.
  • 53. Light micrograph of normal rabbit ear pinna (control sample) at 0 day at 10 X
  • 54. Light micrograph of oleic acid treated rabbit ear pinna at 10X (treated pinna) at 7 th day
  • 55. Light micrograph of oleic acid treated rabbit ear pinna at 10 X (treated pinna) at 28 th day
  • 56. Light micrograph of treated rabbit ear pinna further treated with antiacne niosomal gel for 14 days at 10X magnification
  • 57. Light micrograph of treated rabbit ear pinna further treated with antiacne cream for 14 days at 10X magnification
  • 58.