A Pharmaceutical Study On An Anti-Acne Drug

Transcription

1 A Pharmaceutical Study On An Anti-Acne Drug Thesis Presented by: Samar Mohamed Nasralla Abdelrahaman Master of Pharmaceutical Science For the Degree of Doctor of Philosophy in Pharmaceutical Science ʺPharmaceuticsʺ Under The Supervision of Prof. Dr. Mohamed Ahmed El-Nabarawi Professor of Pharmaceutics and Industrial Pharmacy, Ex-Vice for Community Service and Environmental Development, Chairman of Pharmaceutics and Industrial Department Faculty of pharmacy-cairo University Associate. Prof. Dr. Rehab Nabil Shamma Associate Professor of Pharmaceutics and Industrial Pharmacy Faculty of Pharmacy-Cairo University Submitted to Department of Pharmaceutics and Industrial Pharmacy Faculty of Pharmacy Cairo University (2018)

2 (Abstract) Acne is the most common dermatologic disorder affecting approximately 85% of the teenagers. Dapsone (DPS) suppresses the growth of pathogenic bacteria such as streptococci, staphylococci, pneumococci, mycobacteria, and Propionibacterium acnes (P. acnes) which is the main bacteria causing acne. Moreover DPS has anti-inflammatory activity, which results in successful treatment of acne vulgaris. Vesicular systems play an important role in drug delivery as they can reduce drug toxicity and modify drug pharmacokinetics and bioavailability. The oral chronic use for DPS results in many side effects such as methmoglobinemia and hemolytic anemia. Topical DPS gel, it is a novel option for treating acne vulgaris but as any conventional form has many side effects such as skin irritation, redness, dry skin, burning and itching. Vesicular system release the drug in a sustained manner, thus decreases the different side effects. The work in thesis is divided into three chapters: Chapter Ι: Preparation, characterization, in-vitro and ex-vivo investigation of DPSloaded bilosomes. Chapter ІІ: Preparation, characterization in-vitro and ex vivo investigation of DPSloaded invasomes. Chapter ІІІ: In-vivo skin deposition study of the DPS systems and histopathological examination. From these chapters, it could be concluded that each of the optimized formulations, namely, (BIL 1 ), which was prepared using Span 60: cholesterol: SDC (5:1:0.25) belonging to chapter I and (INV 2 ), which prepared with Limonene which concentration 1.5% belonging to chapter II, demonstrated promising anti-acne systems and have good storage stability. Moreover, the ex-vivo permeation study confirmed the superiority of the prepared system in delivering the drug through the skin layers and succeeded to show 4- fold higher amount of DPS retained in the skin when compared to the drug alcoholic solution. Key words: Dapsone, invasomes, bilosomes, bile salts, in-vivo

3 Introduction Approximately 95% of the population suffers at some point in their lifetime from acne vulgaris[1]. It is the most common dermatologic disorder affecting approximately 85% of teenagers[2]. Acne Vulgaris is a chronic inflammatory follicular disorder of the skin, occurring in specialized pilosebaceous units consisting of the follicular canal with its rudimentary hair, and the group of sebaceous glands that surround and open on to the follicle[3-5]. Having acne can give rise to feelings of embarrassment, loss of self-esteem, and depression, as well as physical symptoms (such as soreness and pain) associated with individual lesions. The pathophysiology of acne includes abnormal proliferation and differentiation of keratinocytes, increased sebum production, hyper proliferation of Propionibacterium acne, and inflammatory response initiated by bacterial antigens and cytokines [6-10]. Novel drug delivery systems have been used to reduce the side effect of drugs mainly cutaneous irritation, erythema, dryness, peeling, and scaling, remain major problems used in the topical treatment of acne. Topical treatment of acne with active pharmaceutical ingredients (API) makes direct contact with the target site before entering the systemic circulation which reduces the systemic side effect of the parenteral or oral administration of drug[11]. The anti-acne drug-loaded vesicular and particulate delivery systems (liposomes, polymeric microspheres, and solid lipid nanoparticles (SLN), hydrogel, aerosol, fullerenes and so topical treatment are advantageous compared to conventional available topical delivery system, so

4 encapsulation of anti-acne drugs in vesicular and particulate delivery systems represents an innovative and alternative approach for acne treatment. Controlled drug release from those novel carrier based delivery systems and subsequent biodegradation are essential for developing successful formulations, where the drug release mechanism of these systems involves desorption of adsorbed drug, diffusion through the carrier matrix, erosion, and combination of erosion and diffusion method[12, 13]. DPS has antimicrobial properties[14]. Sulfones were found to suppress the growth of various pathogenic bacteria such as streptococci, staphylococci, pneumococci, mycobacteria, and other strains. The mechanism of action of topical DPS in the treatment of acne vulgaris may result from a combination of both anti-inflammatory and antimicrobial effects, as DPS showed wide variations in anti-inflammatory efficacy. DPS also has some antibacterial activity against Propionibacterium acnes (P. acnes). Owing to its antimicrobial activities, DPS is clearly playing a role in the treatment of certain infectious diseases[15]. DPS gel is a novel option for treating acne vulgaris that may be operating via anti-inflammatory mechanisms. Efficacy in acne has been demonstrated in phase 3 and long-term studies. It has undergone rigorous evaluation for safety with no evidence of increased hemolytic risk even in G6PD-deficient patients[16].

5 Reviews 1. Ho, V., Schacter, D., and Miller, R., Acne management for the 90s: current treatment guidelines. The Canadian Journal of Diagnosis, : p Arora, M. K., Yadav, A., and Saini, V., Role of hormones in acne vulgaris. Clin Biochem, (13): p Bloom, D. F., Is acne really a disease?: a theory of acne as an evolutionarily significant, high-order psychoneuroimmune interaction timed to cortical development with a crucial role in mate choice. Med Hypotheses, (3): p Jain, A. and Basal, E., Inhibition of Propionibacterium acnes-induced mediators of inflammation by Indian herbs. Phytomedicine, (1): p Lucky, A. W., Cullen, S. I., Funicella, T., Jarratt, M. T., Jones, T., and Reddick, M. E., Double-blind, vehicle-controlled, multicenter comparison of two 0.025% tretinoin creams in patients with acne vulgaris. J Am Acad Dermatol, (4): p. S Yamaguchi, N., Satoh-Yamaguchi, K., and Ono, M., In vitro evaluation of antibacterial, anticollagenase, and antioxidant activities of hop components (Humulus lupulus) addressing acne vulgaris. Phytomedicine, (4): p Leyden, J., Shalita, A., Hordinsky, M., Swinyer, L., Stanczyk, F. Z., and Weber, M. E., Efficacy of a low-dose oral contraceptive containing 20 microg of ethinyl estradiol and 100 microg of levonorgestrel for the treatment of moderate acne: A randomized,

6 placebo-controlled trial. J Am Acad Dermatol, (3): p Kawashima, M., Harada, S., Loesche, C., and Miyachi, Y., Adapalene gel 0.1% is effective and safe for Japanese patients with acne vulgaris: a randomized, multicenter, investigator-blinded, controlled study. J Dermatol Sci, (3): p Huber, J. and Walch, K., Treating acne with oral contraceptives: use of lower doses. Contraception, (1): p Yang, D., Pornpattananangkul, D., Nakatsuji, T., Chan, M., Carson, D., Huang, C. M., and Zhang, L., The antimicrobial activity of liposomal lauric acids against Propionibacterium acnes. Biomaterials, (30): p Vyas, A., Kumar Sonker, A., and Gidwani, B., Carrier-based drug delivery system for treatment of acne. ScientificWorldJournal, : p Castro, G. A. and Ferreira, L. A., Novel vesicular and particulate drug delivery systems for topical treatment of acne. Expert Opin Drug Deliv, (6): p Korting, H. C. and Schafer-Korting, M., Carriers in the topical treatment of skin disease. Handb Exp Pharmacol, 2010(197): p Coleman, M. D., Dapsone: modes of action, toxicity and possible strategies for increasing patient tolerance. Br J Dermatol, (5): p Grunwald, M. H. and Amichai, B., Dapsone - the treatment of infectious and inflammatory diseases in dermatology. Int J Antimicrob Agents, (3): p

7 16. Maddin, S., Dapsone 5% Gel: A new option in topical therapy for acne. skin therapy letter, (8): p. 1-8.

8 Aim of work DPS has dual activity, an anti-bacterial activity activity against (P. acnes), in addition to its anti-inflammatory activity, which makes it the drug of choice for treatment of acne vulgaris. DPS topical gel is a novel option for treating acne vulgaris, but as any convential form has many side effects such as skin irritation, redness, dry skin, burning and itching. Topical conventional dosage forms are limited in their ability to achieve sustained release or drug targeting. Vesicular system offers sustained release systems so decreases the different side effects. Using vesicular systems will lead to different advantages: 1- Overcoming the skin barrier, and increase drug concentration in epidermis and dermis layers of the skin. 2- Increasing the local activity while diminishing percutaneous absorption of the drug. 3- Vesicle act as drug localizer not only drug transporters. 4- The follicular delivery is done via vesicle as demand for localized drug therapy at the level of the hair follicles which is very important in dermatological disorders such as acne vulgaris and alopecia because of the similarity in lipid composition to the epidermis. 5- Vesicle can enhance dermal and transdermal delivery while reducing systemic absorption so avoid many side effects. Thus the aim of this work was the development of a topical vesicular delivery system of DPS, capable of delivering the drug at the pathological site, thus producing skin concentrations of the drug with reduced side effects. Improved DPS topical system with a high degree of skin retention

9 and prolonged maintenance in the target area at a therapeutic level could be promising treatment of acne with improved therapeutic response. The work in thesis is divided into three chapters: Chapter Ι: Preparation, characterization, in-vitro and ex vivo investigation of DPS-loaded bilosomes. Chapter ІІ: Preparation, characterization in-vitro and ex vivo investigation of DPS-loaded invasomes. Chapter ІІІ: In-vivo skin deposition study of DPS systems and histopathological examination.

10 Summery Acne is the most common dermatologic disorder affecting approximately 85% of the teenagers. Dapsone (DPS) suppresses the growth of pathogenic bacteria such as streptococci, staphylococci, pneumococci, mycobacteria, and Propionibacterium acnes (P. acnes) which is the main bacteria causing acne. Moreover DPS has anti-inflammatory activity, which results in successful treatment of acne vulgaris. Vesicular systems play an important role in drug delivery as they can reduce drug toxicity and modify drug pharmacokinetics and bioavailability. The oral chronic use for DPS results in many side effects such as methmoglobinemia and hemolytic anemia. Topical DPS gel, it is a novel option for treating acne vulgaris but as any conventional form has many side effects such as skin irritation, redness, dry skin, burning and itching. Vesicular system release the drug in a sustained manner, thus decreases the different side effects. The work in thesis is divided into three chapters: Chapter Ι: Preparation, characterization, in-vitro and ex-vivo investigation of DPS-loaded bilosomes This chapter deals with preparation and evaluation of DPS-loaded bilosomes according to the thin film hydration technique. DPS, Span 60 and cholesterol were dissolved in 10 ml chloroform, then evaporated at 60 C under reduced pressure using a rotary evaporator for 30 min until a thin film was formed, then hydrated with 10 ml distilled water containing the bile salt for 1 h to form a dispersion of DPS-loaded bilosomes. The dispersion was filtered through filter paper to remove any free DPS particles. The prepared DPS-loaded bilosomes were evaluated according to a full factorial design. The drug encapsulation efficiency (Y 1 ), PS (Y 2 ), % DPS released after 2 h

11 (Y 3 ) and 24 h (Y 4 ) also %RE after 24 h(y 5 ) were selected as independent variables. The bile salt type (SDC, SC, STC) (X 1 ), the bile salt concentration (0.25and 0.5 M) (X 2 ) and molar ratio of Span 60 : Cholesterol (5:1 and 10:1) (X 3 ) were assessed as 3 factors. The optimization process was performed using Design-Expert. The optimization process was performed for X 1 (type of bile salt), X 2 (concentration of bile salt) and X 3 (Molar ratio of Span 60: cholesterol) using the following target ranges; maximum EE% (Y 1 ), minimum PS (Y 2 ), maximum % DPS released after 2 h (Y 3 ), maximum % DPS released after 24 h (Y 4 ) and maximum RE% of DPS released after 24 h (Y 5 ). One system (BIL 1 ) was found to obey this criterion which was prepared using Span 60: cholesterol: SDC (5:1:0.25) with the desirability value of This optimized system was selected for further examinations such as zeta potential, morphological examination, thermal analysis (DSC and X-ray diffraction), and finally Ex-vivo permeation studies using rat skin, in comparison with the drug alcoholic solution. Results showed that the optimized DPS-loaded bilosomes were spherical in shape and carrying high negative charge, as confirmed by a zeta potential value of mv. Moreover, the ex-vivo permeation study confirmed the superiority of the prepared system in delivering the drug through the skin layers and succeeded to show 1.5-fold higher amount of DPS retained in the skin when compared to the drug alcoholic solution. Chapter ІІ: Preparation, characterization in-vitro and ex vivo investigation of DPS-loaded invasomes. This chapter deals with preparation and evaluation of DPS-loaded invasomes according to the thin film hydration technique. DPS, PC and terpene were dissolved in 10 ml methanol/chloroform (2:1, v/v), then evaporated at 60 C

12 under reduced pressure using a rotary evaporator for 30 min until a thin film was formed, then hydrated with 10 ml of ethanolic water mixture (3% v/v) for 1 h to form a dispersion of DPS-loaded invasomes. The dispersion was filtered through filter paper to remove any free DPS particles. The prepared DPS-loaded invasomes were evaluated according to a full factorial design. The drug encapsulation efficiency (Y 1 ), PS (Y 2 ), %DPS released after 2 h (Y 3 ) and 24 h (Y 4 ) also %RE after 24 h (Y 5 ) were selected as independent variables. The terpene type (Limonene, Cineole, Citral, Fenchone) (X 1 ), the terpene concentration (0.5%, 1.5% and 3%) (X 2 ) were assessed as 2 factors. The optimization process was performed using Design-Expert software. The optimization process was performed for X 1 (type of terpene), X 2 (concentration of terpene) using the following target ranges; maximum EE% (Y 1 ), minimum PS (Y 2 ), maximum % DPS released after 2 h (Y 3 ), maximum % DPS released after 24 h (Y 4 ) and maximum RE% of DPS (Y 5 ). One system was found to obey this criterion which was INV2, which prepared with Limonene which concentration 1.5%, the desirability, was This optimized system was selected for further examinations such as zeta potential, morphological examination, thermal analysis (DSC and X-ray diffraction), and finally ex-vivo permeation studies using rat skin, in comparison with the drug alcoholic solution. Results showed that the optimized DPS-loaded invasomes were spherical in shape and carrying high negative charge, as confirmed by a zeta potential value of mv. Moreover, the ex-vivo permeation study confirmed the superiority of the prepared system in delivering the drug through the skin layers and succeeded to show 4-fold higher amount of DPS retained in the skin when compared to the drug alcoholic solution.

13 Chapter ІІІ: In-vivo skin deposition study of the DPS systems and histopathological examination. The two selected DPS-loaded systems from chapters 1 and 2 were tested for their ability to retain DPS in rat skin and the results were compared with that that of DPS alcoholic solution. The concentration of DPS was determined using a validated HPLC analysis method. In-vivo skin deposition of DPS after topical application both systems were measured. BIL 1 and INV 2 showed about 2.5fold higher than DPS alcoholic solution in the amount of DPS retained in the skin after 10 h treatment. Moreover, the prepared systems were evaluated for any skin damage using histological evaluation. Results confirmed that the prepared systems were non-irritant, with no histopathological changes or any inflammation were observed in the rat skin treated with any of the optimized systems when compared to the control group.

14 Conclusions 1. DPS-loaded bilosomes were successfully prepared using the thin film evaporation technique, according to a full factorial design. 2. Twelve DPS-loaded bilosomes were prepared by using three types of bile salt, Span 60and cholesterol. 3. The EE% of DPS-loaded bilosomes prepared using SDC significantly showed highest entrapment efficiency compared to other bile salts. 4. The EE% of DPS-loaded bilosomes significantly decreased by increasing the concentration of bile salts, and by increasing the Span 60: cholesterol molar ratio from 5:1 to 10:1. 5. DPS-loaded bilosomes prepared using SDC had the largest PS among the prepared bilosomes, while those prepared using STC had the smallest particle size. 6. The PS of DPS-loaded bilosomes significantly increased by increasing the molar ratio Span 60: cholesterol. 7. DPS-loaded bilosomes prepared using SDC showed the maximum drug release over other DPS-loaded bilosomes prepared by other bile salts over 24 h. 8. All DPS- loaded bilosomes followed Fickeian transport with values. R 2 the in-vitro release for all DPS-loaded bilosomes showed Koresomeyer-Peppas release kinetics. 9. The optimization was done using Design-Expert software and one DPS-bilosomal system was selected with the desired results which was BIL 1 containing Span 60: cholesterol: SDC (5:1:0.25).

15 10. The optimized DPS-loaded bilosomes (BIL 1 ) had negative zeta potential value of more than ±30 mv, indicating the stability of DPSloaded suspension formed. 11. Morphological examination of the optimized DPS-loaded bilosomes (BIL 1 ) using TEM showed uniform and spherical with no aggregation. 12. DSC and XRD evaluation of the lyophilized optimized DPS-loaded bilosomes (BIL 1 ) lyophilized blank bilosomes (BIL 1 ) and other pure components confirmed the encapsulation of DPS within the bilosomes in an amorphous form. 13. Storage for 3 months at 4 C had no significant difference (p value <0.05) in the EE% and PS of the stored optimized DPS-loaded bilosomes. 14. Ex-vivo permeation through rat skin showed that the prepared optimized DPS-loaded bilosomes permeated the SC, showing 1.5 fold higher DPS retained in the skin layers, compared to the alcoholic solution. 2- DPS-loaded invasomes were successfully prepared using the thin film evaporation technique. 3- Twelve DPS-loaded invasomes were prepared using four types of terpenes, PC and ethanol. 4- The EE% of DPS-loaded invasomes prepared using Limonene showed significantly the highest entrapment efficiency compared to other terpenes. 5- The EE% of DPD-loaded invasomes increased significantly by increasing the concentration of terpenes.

16 6- DPS-loaded invasomes prepared using Citral had the largest PS among the tested invasomes, while DPS-loaded invasomes prepared with Limonene had the smallest particle size. 7- The PS of DPS-loaded invasomes significantly increased by increasing the concentration of terpenes. 8- DPS-loaded invasomes prepared using Limonene 0.5% showed the maximum in-vitro release over other DPS-loaded invasomes prepared by other terpenes over 24 h. 9- Limonene prepared DPS-loaded invasomes showed and maximum RE% after 24 h over other DPS-loaded invasomes prepared by other terpenes. 10- The kinetic analysis of the in-vitro release for all DPS-loaded invasomes showed a Fickian transport. The in-vitro release for (INV 1, INV 2, INV 4, INV 5, INV 6, INV 9, INV 10, INV 11 and INV 12 ) was according to Koresomeyer-Peppas release kinetics, but other (INV 3, INV 7 and INV 8 ) followed diffusion kinetics. 11- The optimization process was done using Design-Exper software and one DPS-loaded invasomes had the desired results which was INV Optimized DPS-loaded invasomes had a negative zeta potential value of more than ±30 mv, indicating the stability of DPS-loaded suspension formed. 13- Morphological examination of the optimized DPS-loaded invasomes using TEM showed uniform and spherical. 14- DSC and XRD evaluation of the lyophilized optimized invasomes were done and confirmed the encapsulation of DPS within the invasomes in an amorphous form.

17 15- Storage for 3 months at 4 C showed no significant difference in the EE% and PS of the stored optimized DPS-loaded invasomes. 16- Ex-vivo results are presented show a significant difference in the DPS permeation between 10% DPS ethanolic solution and INV 2, showing 4fold higher DPS retained in the skin layers, compared to the alcoholic solution. 17- Skin deposition investigation was done for optimized DPS-loaded bilosomes (BIL 1 ), optimized DPS-loaded invasomes (INV 2 ) and DPS alcoholic solution to investigate their ability in the deposition of DPS base into various strata of hairless mouse skin. 18- DPS showed higher in-vivo skin deposition from the investigated DPS-loaded invasomes (INV 2 ) during the study period when compared to DPS-loaded bilosomes (BIL 1 ) and DPS-alcoholic solution. 19- The skin deposition parameters are collectively determined for DPS-loaded bilosomes (BIL 1 ) and compared against deposition parameters produced by topical application of ethanolic DPS solution. 20- DPS showed higher in-vivo skin deposition from the investigated DPS-loaded invasomes during the study period compared to DPS solution, as INV 2 showed about 2.5 fold higher, when compared to the drug solution. 21- The skin deposition parameters are collectively determined for DPS-loaded invasomes (INV 2 ) and compared against deposition parameters produced by topical application of ethanolic DPS solution.

18 22- Histopathological investigations were performed to assess the safety of the DPS-loaded bilosomes and invasomes on the skin. 23- Skin sections did not receive treatment and sections treated with DPS-loaded bilosomes (BIL 1 ) and DPS-loaded invasomes (INV 2 ) showed normal histological structures characterized by absence of defects or inflammation. The structure of epidermis, dermis, subcutaneous adipose tissue and musculature were both intact, while the compartments were similar to that of normal skin tissue.