Hydroxychloroquine niosomes: A new trend in topical management of oral lichen planus

International Journal of Pharmaceutics 458 (2013) 287–295

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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

Pharmaceutical nanotechnology

Hydroxychloroquine niosomes: A new trend in topical management of oral lichen planus Ehab R. Bendas a,∗ , Hamoud Abdullah a , Mohamed H.M. El-Komy b , Mohamed A.A. Kassem a a b

Department of Pharmaceutics, Faculty of Pharmacy, Cairo University, Cairo, Egypt Department of Dermatology, Faculty of Medicine, Cairo University, Cairo, Egypt

a r t i c l e

i n f o

Article history: Received 28 September 2013 Accepted 22 October 2013 Available online 30 October 2013 Keywords: Hydroxychloroquine Niosomes Pluronic F-127 gel Oral lichen planus Clinical study

a b s t r a c t The work aimed at studying a novel topical niosomal gel formulation of hydroxychloroquine for the management of oral lichen planus. Niosomes have been reported as conceivable vesicles to deliver drug molecules to the desired mucous membrane or skin layers. Hydroxychloroquine niosomes were designed using different methods of preparation. Tween 20 and cholesterol in molar ratio (1:0.5) were used. The prepared systems were characterized for entrapment efficiency, particle size and in vitro drug release. Different factors affecting the encapsulation of hydroxychloroquine in niosomes were studied vs. varying the type of surfactant, the cholesterol:surfactant molar ratio and the amount of the drug. The selected noisome formulation was dispersed in different gel formulations and evaluated according to the in vitro drug release and the physical stability. The results showed that the type of surfactant, cholesterol ratio and incorporated amount of drug altered the entrapment efficiency and the in vitro release of hydroxychloroquine from niosomes. The optimum formulation was prepared by reverse phase evaporation technique using Brij 98:cholesterol molar ratio (1:1.5) and containing 20 mg of hydroxychloroquine and incorporated in 20% w/v Pluronic F-127 gel. A double-blind, controlled clinical study was performed using two groups of patients. Group A (n = 11) who received hydroxychloroquine niosomal gel formulation, one application-a-day over 4 months showed 64.28% reduction in the size of lesions and the average score of pain was reduced from “4” to “1”. Compared to placebo group B (n = 5), who showed only 3.94% reduction in the lesion size and the average score of pain was remained “3”. Our results suggest that these niosomal formulations could constitute a promising approach for the topical treatment of oral lichen planus in short time with less side effects and no recurrence after stopping the treatment. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Drug delivery systems using particulate carriers such as liposomes or niosomes have proved to possess distinct advantages over conventional dosage forms, because the particles can act as drug reservoirs, can carry both hydrophilic drugs or hydrophobic drugs and the drug release rate can be adjusted (Handjani-Vila et al., 1979). Liposomes, consist mainly of phospholipids, prepared from double chain phospholipids (neutral or charged) (Purohit et al., 2001). Niosomes, offer an alternative to phospholipid vesicles. The hydrated mixtures of cholesterol and non-ionic surfactants give rise to these lipid vesicles (Carafa et al., 2009). Niosomes have been developed to increase the stability of liposomes (Choi et al., 2006). Niosomes have been reported to enhance

∗ Corresponding author at: Faculty of Pharmacy, Cairo University, Kasr El-Ainy Street 11562, Egypt. Tel.: +20 2 25311260; fax: +20 2 23628426. E-mail address: [email protected] (E.R. Bendas). 0378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2013.10.042

the residence time of drugs in the stratum corneum and epidermis and also to improve the penetration of the trapped substances across the skin. In addition, these systems have been reported to decrease the side effects and to give a convenient and considerable drug release (Balakrishnan et al., 2009; Schroeter et al., 2010). They are biodegradable, biocompatible, and more importantly non-immunogenic with minimum toxicity (Perini et al., 1996). Niosomes have evolved for treatment of many dreadful diseases efficiently with reduced side effects and better patient compliance. Overall, niosomes are a very effective tool for drug delivery and targeting of numerous therapeutically active moieties (Rajera et al., 2011). Lichen planus is a chronic disease affecting both the skin and oral mucosa (Scully and Carrozzo, 2008). Oral lichen planus (OLP) is a chronic inflammatory, noninfectious mucocutaneous disease (Gorouhi et al., 2007). The precise cause of oral lichen planus is unknown but it is considered as an autoimmune disease (Ismail et al., 2007). The treatment of OLP is disappointing and controversial (Scully et al., 1998). Traditional treatment consists of various

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drugs, including retinoids (Ott et al., 1996), cyclosporine (Jungell and Malmstrom, 1996), tacrolimus (Kaliakatsou et al., 2002) and fenretinide (Tradati et al., 1994). The most frequently described therapy for OLP has been the administration of topical or systemic corticosteroids (Sharma et al., 2012). Despite the therapeutic effects of topical corticosteroids, they have significant adverse effects such as fungal infections and adrenal suppression, resulting in a continuing search for novel therapies (Myers et al., 2002; Gonzalez-Moles et al., 2002; Thongprasom et al., 2013). Topical aloe vera (Salazar-Sanchez et al., 2010), topical pimecrolimus (Gorouhi et al., 2007) and oral curcuminoids (Chainani-Wu et al., 2012) are the most promising of the new treatment modalities. However, all of these medications have soothing effects, and relapses occur when they are withdrawn. Oral lesions of lichen planus and lupus erythematosus can appear to be clinically identical, even their histopathologic features are similar (Karjalainen and Tomich, 1989). Hydroxychloroquine sulfate (Plaquenil), an oral antimalarial agent, was evaluated in an open trial (10 patients) for 6 months for its ability to improve oral lichen planus. Nine of ten patients had an excellent response to therapy (Eisen, 1993). Hydroxychloroquine, a hydroxylated form of the antimalarial drug chloroquine, was first synthesized in the 1940s. Shortly thereafter, it was discovered to be as effective as, but less toxic than, chloroquine. Over the years, hydroxychloroquine has also been shown to provide beneficial effects for systemic and cutaneous lupus erythematosus conditions, rheumatoid arthritis, chronic Q fever, and several skin disorders and is now routinely used in the management of these diseases (Ben-Zvi et al., 2012; Petri, 2011). In the present investigation, niosomes have been prepared to encapsulate the hydroxychloroquine. The non-ionic surfactant vesicles have been prepared and evaluated for entrapment efficiency, particle size and in vitro drug release. Also the effect of various parameters viz. method of preparation, type of surfactant, molar ratio of cholesterol and surfactant and amount of the drug was studied on the various evaluated parameters. The developed system is clinically studied for efficient treatment of oral lichen planus. 2. Materials Hydroxychloroquine sulfate (HQ), kindly provided by Minapharm Pharmaceuticals, Cairo, Egypt. Absolute alcohol, Chloroform and Methanol, Prolabo (Paris, France). Sodium carboxymethylcellulose (CMC), methylcellulose (MC), Cholesterol, Stearylarnine, and Dicetyl phosphate, Pluronic F-127, Brij 98, Tween 20, Tween 40, Tween 65, Tween 80, Span 20, Span 40, Span 60 and Span 80, Sigma Chemical Co. (St. Louis, USA). Carbopol 934, B. F., Goodrich Chemical Company (Ohio, USA). Triethanolamine, E. Merck (Germany). Diethyl ether, MS, Laboratory Rasayan, S.D FineChem. Ltd. (Poicha, India). Sodium chloride, potassium chloride, disodinm hydrogen phosphate, potassium dihydrogen phosphate (Prolabo, France). Cellulose nitrate membrane filter, diameter pore: 0.45 ␮m, Albet Company (Spain). 3. Methods 3.1. Preparation of hydroxychloroquine niosomes Hydroxychloroquine niosomes were prepared by several techniques characterized as follows:

diethyl ether and was injected slowly (0.25 ml per min.) through a 14 gauge needle into beaker containing 5 ml of aqueous phase consisting of Phosphate buffer saline pH 7.4, in which 20 mg of HQ were dissolved which maintained at 60 ◦ C. The Phosphate buffer saline (PBS) solution consists of: 139 mM of NaCl, 2.5 mM of KCl, 8 mM of Na2 HPO4 and 1.5 mM of KH2 PO4 (Hofland et al., 1994). 3.1.2. Sonication method A mixture of Tween 20 and cholesterol was dissolved in 5 ml of solvent mixture consisting of chloroform and methanol in a ratio of 4:1 in 10 ml glass vial. The solvent was evaporated overnight. Then 5 ml of aqueous phase (PBS) containing 20 mg of HQ was added to the previous formed film. The mixture was sonicated at 60 ◦ C for 3 min. 3.1.3. Hand shaking A mixture of Tween 20 and cholesterol was dissolved in 10 ml of diethyl ether in 50-ml round-bottomed flask. Ether was removed at room temperature under reduced pressure, in a rotary evaporator (Rotavapor, Type R 110, Büchi, Switzerland). The dry film was hydrated with 5 ml of aqueous phase (PBS) containing 20 mg of HQ at 50–60 ◦ C with gentle agitation. 3.1.4. Reverse phase evaporation method (REV) Lipid solution consisting of Tween 20 and cholesterol and 10 ml of diethyl ether was mixed in beaker. Two milliliter of aqueous phase (PBS) containing 20 mg of HQ was added to the lipid solution. The mixture was sonicated at 4 ◦ C for 5 min. The ether layer was evaporated at room temperature till suspension was formed. Three milliliter of PBS was added with sonication for 30 s three times (3 × 30 s). Then the solvent was removed at room temperature, under reduced pressure, using rotary evaporator for 15 min (Pillai and Salim, 1999). 3.2. Separation of free (unentrapped) hydroxychloroquine from niosomal suspension The unentrapped HQ was separated by centrifugation at 14,000 rpm at 4 ◦ C for 30 min using cooling ultracentrifuge (Cooling Ultracentrifuge. Model 8880, Centurion Scientific Ltd., W. Sussex, UK.). The supernatant was removed, and the residue was washed twice with PBS (pH 7.4) and re-centrifuged after each washing step. 3.3. Determination of entrapment efficiency The entrapped HQ concentrations were determined as follows: the residue was collected from the centrifuge tubes and 0.1 mg of niosomes was mixed with 9.9 ml of absolute alcohol. The resultant solution was shaken well and sonicated for 5 min to obtain a clear solution. One milliliter of this solution was taken and transferred to another tube containing 9 ml of absolute alcohol. The solution was shaken and sonicated for another 5 min. The concentration of HQ in absolute alcohol was determined spectrophotometrically by measuring the absorbance at max 343 nm (Arguelho et al., 2003). The entrapment efficiency was determined relative to the original drug concentration added according to the following equation: %E =

3.1.1. Ether injection method Accurately weighed quantities of Tween 20 and cholesterol in molar ratio (1:0.5) to produce 150 ␮mol were dissolved in 20 ml of

ED × 100 TD

Where:%E: is the percent entrapment efficiency.ED: is the concentration of entrapped drug.TD: is the total drug concentration.

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3.4. Characterization of hydroxychloroquine niosomes 3.4.1. Photomicroscopic analysis Samples of HQ niosomes were examined microscopically at a magnification of 10× and 40× with a Leica image analyzer (Leica image analyzer. Model Q 55 OIW equipped with Leica DMLB microscope Cambridge, England. Connected to Camera, Model TK-C 13 80 JVC, Victor Company, Japan) which consists of binocular microscope equipped with a computerized digital camera. A drop of niosome preparation placed on a microscope slide and diluted, then was examined and photographed for morphological evaluation. 3.4.2. Particle size analysis Samples of freshly prepared HQ niosomes were placed on a microscope slide and examined by a ‘Leica image analyzer’ using special computer program to characterize the particle size and size distribution. 3.4.3. In vitro release experiments This study was carried out using a USP Dissolution tester (Apparatus I). A glass cylindrical tube (2.5 cm in diameter and 6 cm in length) containing the niosomal HQ suspension to be tested was attached instead of the basket and was tightly covered with a semipermeable membrane (0.45 ␮m pore size). The drug-loaded niosomal suspension contains 20 mg of HQ was placed in the cylindrical tube covered with a semipermeable membrane. The cylindrical tube was immersed in a 100 ml of PBS (pH 7.4), contained in the vessel of the USP Dissolution Test Apparatus. The release study was carried out at 32 ± 0.5 ◦ C and the stirring shaft was rotated at a speed of 50 rpm. The samples were withdrawn at predetermined time intervals (15 min, 30 min, 60 min, 90 min, 120 min, 180 min, 240 min, 300 min, 360 min, and 420 min). Each sample was replaced with equal volume of PBS to maintain a constant volume. The absorbance of the collected samples was measured spectrophotometrically at 343 nm (Arguelho et al., 2003) after suitable dilutions using PBS as a blank. All experiments were performed in triplicates. 3.5. Factors affecting the encapsulation of hydroxychloroquine in niosomes 3.5.1. Effect of method of preparation Niosomes were prepared according to the previous techniques. The encapsulation efficiency, particle size and drug release rates were estimated for each method of preparation. 3.5.2. Effect of type of surfactant: Reverse phase evaporation (REV) method that gave satisfied results for percent encapsulation efficiency and drug release rate was chosen for further investigations. Different types of non-ionic surfactants were used in the same concentration as Tween 20 such as: Brij 98, Tween 40, Tween 65, Tween 80, Span 20, Span 40, Span 60 and Span 80. The surfactant:cholesterol ratio was 1:0.5 and the total concentration was adjusted to be 150 ␮mol and HQ content was 20 mg in all preparations. The encapsulation efficiency, particle size and drug release rates were estimated for each type of surfactant used. 3.5.3. Effect of cholesterol:surfactant ratio Cholesterol was used to increase the rigidity of the bilayer of niosomes (Abdelkader et al., 2010). The effects of increasing cholesterol content on drug entrapment and on drug release in niosomes prepared by Reverse phase evaporation method (REV) and using Brij 98 as a surfactant were investigated. The following niosomal formulations of Brij 98 to cholesterol molar ratios were prepared

289

(1:0.5, 1:0.75, 1:1 and 1:1.5). The encapsulation efficiency, particle size and drug release rates were determined for each niosomal preparation. 3.5.4. Effect of amount of drug The effect of incorporating increasing amounts of HQ into hydrophilic phase of niosomes on percent of the encapsulation efficiency, release rates and mean particle size was investigated. The weights of HQ used were 20 mg, 30 mg and 50 mg for each 5 ml of noisomal preparation. In each preparation, the lipophilic phase consists of Brij 98:cholesterol in a molar ratio of 1:1.5 using 10 ml of diethyl ether as a solvent and 5 ml of PBS (pH 7.4) as aqueous phase in which HQ was dissolved. 3.6. Dispersion of HQ niosomes in gel formulations In order to facilitate the administration of niosomal preparations to patients and to control drug release, the prepared niosomal dispersions were incorporated in different gel formulations. The in vitro drug release and kinetic analysis of release data of hydroxychloroquine were compared for the different types of gel formulations. The types of gel formulations studied were Pluronic F-127 (20% and 30%), Methylcellulose (MC) (5% and 10%), Sodium carboxymethylcellulose (sod. CMC) (5% and 10%), and Carbopol 934 (2% and 3%). Niosomal preparations were prepared previously by reverse phase evaporation (REV) technique and then incorporated in different types of gels as follows: 3.6.1. Pluronic F-127 gels The dispersion of hydroxychloroquine niosomal suspension was incorporated into Pluronic F-127 gels (20% and 30%) prepared according to the following procedures: the specified amount of Pluronic co-polymer was slowly added to cold physiological saline (5–10 ◦ C) in a beaker. The contents were stirred gently with a magnetic stirrer bar, to assure complete dissolution. The containers were left overnight in the refrigerator. Eventually a viscous solution was formed due to the reverse gelation characteristics of the Pluronic at refrigerator temperature. For the gel to be formed, the solution should be kept at 30 ◦ C for not less than 3 h (Schmolka, 1994). 3.6.2. Methylcellulose (MC) gels The weighed quantity of methylcellulose powder was sprinkled gently in the vortex of 100 ml beaker containing boiled distilled water, and stirred mechanically at a high speed until a thin dispersion, without lumps, was formed. The solution was kept overnight in the refrigerator. The following concentrations of methylcellulose gel bases were prepared: 5 and 10% w/v. Hydroxychloroquine niosomal suspension was dispersed in the prepared gels. 3.6.3. Sodium carboxymethylcellulose (sod. CMC) gels The weighed quantity of sodium carboxymethylcellulose powder was added slowly to distilled water, to form a smooth gel and avoid incorporating air into the product. The final concentrations of the prepared gels were 5 and 10% w/v. Then, the niosomes of HQ were dispersed. 3.6.4. Carbopol 934 gels The weighed amount of Carbopol 934 resin was sprinkled gradually, into the vortex of 100 ml of distilled water, placed in a 200 ml beaker, and stirred with a mechanical stirrer at a high speed until no lumps was observed. Stirring speed was then reduced to break the foam, and then 1 ml of triethanolamine was added at once to form the gel. Carbopol gel bases were prepared at concentrations

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of 1 and 2% w/v, each containing 1 ml of triethanolamine. Then, the niosomal dispersions of HQ were incorporated.

Table 1 Effect of method of preparation of niosomes on entrapment efficiency and noisome size.

3.7. Preservation of hydroxychloroquine niosomal gels

Method of preparation

Entrapment efficiency (%E)

Niosome size (␮m) ± SE

In order to protect the product formulation and active ingredients from microbial degradation and the consumer from the infection that could be acquired through product use, oral gels have to be preserved as they are used as multi-dose preparations (Steinsvag et al., 1996). Benzalkonium chloride “Quaternary ammonium compound” was used as a preservative for all gel preparations due to its stability and effectiveness. Benzalkonium chloride was tested for its effectiveness as a preservative according to USP challenge test as follows: a sample was taken from the gel preserved with benzalkonium chloride in concentration of 0.01% w/w. This sample was challenged with various microbial cultures. The test organisms were Escherichia coli, Candida albicans, Pseudomonas aeruginosa and Staphylococcus aureus. The inoculum size was adjusted to be about 105 –106 Colony Forming Unit (C.F.U.)/ml. For the initial cultivation of the test organisms, an agar medium was selected which is favorable to vigorous growth of the respective stock culture. The bacterial cultures were incubated at 25–35 ◦ C for 24–48 h. The containers were examined at 6 h, 12 h, 24 h, 7 days, 14 days and 28 days subsequent to inoculation. The number of viable microorganisms present at each of these time intervals was determined by the plate count procedure, and the percentage change in the concentration of each microorganism during the test was calculated, using the theoretical concentrations of microorganisms present at the start of the test.

Ether injection Sonication Revere phase evaporation Hand shaking

84.60 82.35 86.40 80.50

0.32 ± 0.08 0.46 ± 0.09 0.92 ± 0.04 0.64 ± 0.10

3.8. Evaluation of hydroxychloroquine niosomal gels 3.8.1. In vitro release experiments This study was carried out using a USP Dissolution test (Apparatus I) as mentioned in Section 3.4.3. 3.8.2. Stability of hydroxychloroquine niosomal gel The stability of HQ niosomal gel was assessed by comparing the extent of HQ released before and after storage. The samples were stored at refrigeration temperature (2–8 ◦ C) and at room temperature (25 ◦ C) in sealed glass vials. Samples were withdrawn after 2, 30, 60 and 90 days. The release of HQ from niosomal gel was determined as described above in Section 3.4.3. The stability of hydroxychloroquine niosomal gel was assessed also by observation and recording the change in the physical form and flow of the gel by time through measuring the rheological properties of the gel using Brookfield viscometer, where a sample of the gel was placed in the suitable cup of the viscometer. This instrument continuously shears the material at various rates, using spindle 52. Measurements were made over the whole range of speed settings from 0.3 rpm to 30 rpm, with 30 s between each two successive speeds, and then in descending order from speed settings 30 rpm to 0.3 rpm. Samples to be tested were withdrawn after 2, 30, 60 and 90 days of storage. Shear rate (s−1 ), shear stress (dyne/cm2 ) and viscosity (centipoise) (cp) were determined from the instrument readings. 3.9. Clinical study on hydroxychloroquine niosomal gel formulation The double-blind clinical study was conducted on 16 patients of both sex and of age group 25–68 years having classical oral lichen planus. All the cases were diagnosed clinically. The patients were randomly allocated into two groups: group A which consists of 11 patients was given HQ niosomal gel formulation and group B which consists of five patients was given non-medicated niosomal

gel (Placebo group). All the patients had given informed written consent. The study was conducted in accordance with the ethical principles provided by the Declaration of Helsinki and approved by the ethical committee of faculty of Pharmacy, Cairo University. The patients were excluded from the study if they were pregnant, nursing mothers, sensitive to hydroxychloroquine or, taking medications that could interfere with the trial drug and having serious systemic illness. The patients were advised to wash their mouth thoroughly after dinner and apply the formulation to the lesion site 30 min later once daily, so that there would not be any possible interference between the drug and food ingredients. The pain and size of the lesions were evaluated. Pain was evaluated by asking the subjects to rank the severity of their pain according to Numerical rating scales (NRS), from 0 to 10 where 0 represents “no pain” and 10 represents “worst pain imaginable”. The size of any ulcer or erosive area was measured with calipers. Outcome measures were assessed at baseline and followed up weekly during treatment for 4 months. The size of the lesions was measured and the side effects as burning or tingling sensations were reported during the study period. 3.10. Data analysis Data were analyzed with SPSS 16 software. With the purpose of comparing the data collected before and after treatment in the same group, and to compare data between the two groups, one way analysis of variance (ANOVA) was used, followed by LSD Post Hoc test. p-value of <0.05 was considered significant. 4. Results and discussion 4.1. Effect of method of preparation on encapsulation of hydroxychloroquine in niosomes and on its in vitro release Drug loading is a crucial factor in the formulation of niosome delivery system (Arunothayanun et al., 2000). To deliver a sufficient amount of drug providing therapeutic effect, a high trapping efficiency of drug in vesicular structure is required (Pavelic et al., 2001). So to formulate drug into noisomes, it is necessary to decrease the leakage of an entrapped drug (Kulkarni et al., 1995). Table 1 shows the effect of method of preparation (using Tween 20 as surfactant) on the percent entrapment efficiency of HQ in niosomes. It is obvious that reverse phase evaporation (REV) method is the most efficient method due to the highest entrapment efficiency (86.4%), followed by ether injection method (84.60%), sonication method (82.35%) and finally hand shaking method (80.5%). There is no significant difference between reverse phase evaporation (REV) method and ether injection method of preparation, but there is significant difference between REV method and other methods of preparation, using (ANOVA) statistical technique at level of significance 0.05. The release of HQ from niosomes prepared by different methods of preparation is presented graphically in Fig. 1. High percent released after 420 h was observed from niosomes prepared by reverse phase evaporation method (51.41%), followed by ether injection method (48.4%), then sonication method (30.06%),

E.R. Bendas et al. / International Journal of Pharmaceutics 458 (2013) 287–295

60

Hand shaking

REV

Sonication

90

Ether injection

Percentage HQ released

Percentage HQ released

50 40 30

20 10 0

291

(A)

Span 80

80

Span 60

70

Span 40

60

Span 20

50 40 30 20 10

0

100

200

300

400

0

500

0

100

Time, min.

200

300

400

500

Time, min. 60

and finally hand shaking method (29.06%). There is no significant difference between reverse phase evaporation REV method and ether injection method; however there is significant difference between these methods and sonication and hand shaking methods, using ANOVA statistical technique at level of significance 0.05. Hand shaking method and sonication method gave unsatisfactory percent of hydroxychloroquine released over the time of release. The largest average niosome size was 0.92 ␮m when prepared by reverse phase evaporation (REV) method, followed by 0.64 ␮m when prepared by hand shaking, followed by sonication method (0.46 ␮m), and then by ether injection method (0.32 ␮m). There is significant difference between the reverse phase evaporation (REV) method and other methods, when statistically analyzed using ANOVA test at level of significance 0.05. The presence of a particle size distribution causes a substantial acceleration of the release at early times and a marked retardation for longer times. The acceleration of the early portion of the release curve is the result of release from particles smaller than the mean size. Particle that is larger than the average size results in the retardation of the release at long times (Klose et al., 2006). From the previous results, the revere phase evaporation (REV) method is considered an ideal method for preparation of HQ niosomes, due to highest encapsulation efficiency; large particle size with smallest variation in size, which indicated by smallest standard error and suitable release pattern of the drug. This method of preparation was used in studying the other factors affecting the encapsulation of hydroxychloroquine in niosomes. 4.2. Effect type of surfactant on encapsulation of hydroxychloroquine in niosomes Table 2 shows the effect of type of surfactant on the percent entrapment efficiency. From the results tabulated it is clear that Brij 98 (HLB = 15.3) is the most efficient surfactant due to highest Table 2 Effect of type of surfactant on entrapment efficiency and noisome size. Surfactant type

HLB

Entrapment efficiency (%EE)

Niosome size (␮m) ± SE

Brij 98 Tween 20 Tween 40 Tween 65 Tween 80 Span 20 Span 40 Span 60 Span 80

15.3 16.7 15.6 10.5 15.0 8.6 6.7 4.7 4.3

87.50 86.40 82.45 82.88 83.70 81.13 80.50 79.86 80.00

0.82 ± 0.04 0.92 ± 0.04 0.87 ± 0.09 0.67 ± 0.08 0.79 ± 0.09 0.62 ± 0.05 0.58 ± 0.10 0.54 ± 0.07 0.51 ± 0.08

Percentage HQ released

Fig. 1. Effect of method of preparation on the in vitro release of HQ from niosomes prepared by Tween 20:cholesterol molar ratio (1:0.5).

(B)

Twen 80 Tween 65 Tween 40 Tween 20 Brij 98

50 40 30 20 10 0

0

100

200

300 Time, min.

400

500

Fig. 2. A: Effect of type of surfactant on the in vitro release of HQ from niosomes prepared by reverse phase evaporation technique using surfactant:cholesterol molar ratio (1:0.5). B: Effect of type of surfactant on the in vitro release of HQ from niosomes prepared by reverse phase evaporation technique using surfactant:cholesterol molar ratio (1:0.5).

encapsulation efficiency (87.5%), followed by Tween 20 (HLB = 16.7) (86.4%), Tween 80 (HLB = 15) was 83.7%, Tween 65 (HLB = 10.5) (82.88%), Tween 40 (HLB = 15.6) (82.45%), Span 20 (HLB = 8.6) (81.13%), Span 40 (HLB = 6.7) was 80.5%, Span 80 (HLB = 4.8) was 80%, then Span 60 (HLB = 4.7) was 79.86%. The release of hydroxychloroquine from niosomes prepared by reverse phase evaporation (REV) method using different types of surfactants was presented graphically in Fig. 2A and B. By changing the type of surfactant used, the amount of HQ released is markedly changed. The highest % released after 420 min was attained when using Span 20 (74.10%), followed by Span 40 (70.10%), Span 60 (69.85%), Span 80 (68.65%), Brij 98 (54.97%), Tween 20 (51.41%), Tween 40 (49.97%), Tween 65 (46.97%), and then Tween 80 (28.66%). The release values from formulations of Span 20, Span 40, Span 60, and Span 80 are higher than types of Tween, thus Span types can be used to enhance the release of HQ from niosomes. Brij 98, Tween 20, Tween 40, and Tween 65, when used as surfactant resulted in slowing of the release of hydroxychloroquine from niosomes. Tween 80 produced the slowest release. There is no significant difference between the release results of Span 40, Span 60, and Span 80, using ANOVA test at level of significance 0.05. The effect of type of surfactant (with different HLB values) on particle size of niosomes was shown in Table 2. The largest average niosome size was 0.92 ␮m when prepared using Tween 20 and the smallest particle size was 0.51 ␮m when prepared using Span 80. It is clear that by increasing HLB value, there is increase in particle size (Khazaeli et al., 2007). Brij 98 (HLB = 15.3) is considered the ideal surfactant in preparation of HQ niosomes due to high entrapment efficiency, moderate release from niosomes prepared using this surfactant. Optimum and relatively large particle size (0.82 ␮m)

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70

Table 3 Effect of surfactant:cholesterol ratio on entrapment efficiency and noisome size. Entrapment efficiency (%EE)

Niosome size (␮m) ± SE

1:0.5 1:0.75 1:1 1:1.5

84.24 86.83 87.50 90.62

0.82 ± 0.04 0.84 ± 0.06 0.85 ± 0.07 0.86 ± 0.03

with minimum size heterogeneity of the vesicles was obtained when Brij 98 was used in preparation of niosomes.

60 Percentage HQ released

Surfactant (Brij 98):cholesterol molar ratio

50 40 30

50 mg of HQ

20

30 mg of HQ

10

20 mg of HQ

0

0

100

200

300

400

500

Time, min.

4.3. Effect of surfactant:cholesterol ratio on encapsulation of hydroxychloroquine in niosomes The effect of increasing cholesterol amount on the percent entrapment efficiency of HQ in niosomes prepared by reverse phase evaporation (REV) method and using Brij 98 was shown in Table 3. The percent entrapment efficiency was increased from 84.24% to 86.83% and to 87.5% for the following, Brij 98:cholesterol molar ratios, 1:0.5, 1:0.75, and 1:1 respectively. But it is obviously appear that the formula of ratio 1:1.5 gave the highest percent entrapment efficiency (90.62%). There is no significant difference between 1st, 2nd and 3rd formulations. There is significant difference between 4th formulation and other formulations, when using ANOVA test at level of significance 0.05. Increasing of cholesterol amount results in increase in membrane rigidity and thus increasing of percentage entrapment efficiency and formation of less leaky vesicles. It is evidenced that more cholesterol is necessary to reimburse for the large head group of Brij 98 of high HLB of 15.3. The release of HQ from niosomal vesicles of different Brij 98:cholesterol molar ratios is shown in Fig. 3. The release data showed the percentage HQ released after 420 min was 54.63%, 54.97% and 52.79% and then 52.67%, for Brij 98:cholesterol molar ratios 1:0.5, 1:0.75, 1:1, and 1:1.5 respectively. The mean particle diameter for niosomes composed of Brij 98:cholesterol molar ratios (1:0.5, 1:0.75, 1:1, and 1:1.5) was, 0.82 ␮m, 0.84 ␮m, 0.85 ␮m and 0.86 ␮m respectively. There is no significant difference between the different formulations, using ANOVA test at level of significance 0.05. The addition of cholesterol increased the mean vesicle size for HQ niosomes. Brij 98:cholesterol molar ratio 1:1.5 gave the highest entrapment, largest particle size and slowest release. These results were in accordance with that reported by Pardakhty et al. (2007).

60

Percentage HQ released

50 40 Brij 98 : cholesterol (1:1.5)

30

Brij 98 : cholesterol (1:1) 20

4.4. Effect of amount of drug on encapsulation of hydroxychloroquine in niosomes The percent of entrapment efficiency was 90.62%, 92.36% and 92.71% for the following HQ amounts: 20 mg, 30 mg and 50 mg respectively. From the results obtained, there is direct relation between increasing the drug amount and increasing encapsulation efficiency. Using ANOVA test at level of significance 0.05 there is only significant difference between the formulations contains 20 mg of HQ and those contain 30 mg and 50 mg of the drug. There is no significant difference between 30 mg and 50 mg formulations. The release data was graphically presented in Fig. 4. From the results obtained, the percentage hydroxychloroquine released after 420 min are 52.67%, 55.33% and 62.80% for 20 mg, 30 mg and 50 mg of HQ respectively. By using ANOVA test at level of significance 0.05 there is no significant difference between release results of 1st and 2nd formulations. The results indicate that there is increase in release rate of HQ in 3rd formulation when increasing initial amount of drug and the difference was found to be significant between 3rd and other formulations. The effect of increasing amount of drug on particle size of hydroxychloroquine niosomes was shown in Table 4. The mean particle diameter was 0.86 ␮m, 0.88 ␮m and 0.89 ␮m for 20 mg, 30 mg and 50 mg of HQ respectively. Using ANOVA test at level of significance 0.05, there is a significant difference in the formulations containing 20 mg and 50 mg of HQ. And there is no significant difference between 20 mg and 30 mg, and also there is no significant difference between 30 mg and 50 mg of HQ. From the above results, it was concluded that increasing amount of drug has effect on entrapment efficiency, and increasing amount of drug has a direct effect on mean particle size of niosomal vesicles, and has increasing effect on release rate. The optimized formulation (Brij 98:cholesterol molar ratio 1:1.5) with 20 mg of HQ prepared by reverse phase evaporation technique showed the most favorable release of the drug and was found to be the superlative formulation. The microscopic image of this formulation was displayed in Fig. 5.

Brij 98 : cholesterol (1:0.75)

10 0

Fig. 4. Effect of amount of drug on the in vitro release of HQ from niosomes prepared by reverse phase evaporation technique using Brij 98:cholesterol molar ratio (1:1.5).

Brij 98 : cholesterol (1:0.5) 0

100

200

300 Time, min.

400

500

Fig. 3. Effect of cholesterol on the in vitro release of HQ from niosomes prepared by reverse phase evaporation technique using Brij 98 as surfactant.

Table 4 Effect of amount of drug on entrapment efficiency and noisome size. Amount of hydroxychloroquine (mg)

Entrapment efficiency (%EE)

Niosome size (␮m) ± SE

20 30 50

90.62 92.36 92.71

0.86 ± 0.03 0.88 ± 0.04 0.89 ± 0.06

E.R. Bendas et al. / International Journal of Pharmaceutics 458 (2013) 287–295

45

(A)

40

Percentage HQ released

293

35 30 25 Carbopol 3% Carbopol 2% CMC 10% CMC 5%

20

15 10 5 0 100

0

200

300

400

500

Time, min. 60 Fig. 5. Photomicrograph of HQ niosomes prepared by reverse phase evaporation technique using Brij 98:cholesterol molar ratio (1:1.5).

4.6. In vitro release of hydroxychloroquine from niosomal gel The results of the in vitro release of HQ from niosomes dispersed in different gel bases are shown in Fig. 6A and B. It is obvious that the release of HQ from 20% pluronic gel occurred in two phases, an initial rapid release of the drug that lasted for 2–3 h, followed by a sustained release phase. This release pattern might be due to a combination of the release of free, surface-bound and encapsulated drug through the micellar network channel structures of the gel. Statistically there is a significant difference between release of the drug from pluronic 20% gel base and from other formulae using ANOVA test at level of significance 0.05. But there is no significant difference between the release of the drug from pluronic 30% gel, CMC 5% and Carbopol 2% gel formulations. The release of HQ from different niosomal gel formulations during the first 420 min followed diffusion controlled mechanism. The release rate constants were 2.870 mg% min−1/2 , 2.472 mg% min−1/2 , 1.856 mg% min−1/2 , 2.220 mg% min−1/2 , 2.037 mg% min−1/2 , 1.955 mg% min−1/2 , 2.011 mg% min−1/2 , and 1.824 mg% min−1/2 for Pluronic 20%, Pluronic 30%, MC 5%, MC 10%, CMC 5%, CMC 10%, Carbopol 2% and Carbopol 3% niosomal gel formulations, respectively. The results indicate that incorporation of HQ niosomes in Pluronic 20% gel results in suitable and feasible release rate of HQ. The release rate constant of drug from niosomal suspension formulation consisting of 20 mg of HQ and Brij 98:cholesterol molar ratio (1:1.5) was 2.99 mg% min−1/2 that was so close to that of HQ niosomes dispersed in 20% pluronic gel formulation (2.870 mg% min−1/2 ). 4.7. Stability of hydroxychloroquine niosomal gel Fig. 7, shows the release pattern of HQ from niosomal Pluronic gel after storage for 90 days at 4 ◦ C and 25 ◦ C. The difference in the

Percentage HQ released

According to USP testing for effectiveness of preservatives, the results showed that benzalkonium chloride in concentration of 0.01% w/w had a bacteriostatic effect and was effective as preservative in the gel formulation examined, as the concentrations of viable bacteria were reduced to not more than 0.1% of the initial concentrations by the 14th day; the concentrations of viable yeasts and molds remained at or below the initial concentrations during the first 14 days; and the concentration of each test microorganism remained at or below these designated levels during the remainder of the 28-day-test period.

50 40

30 MC 10% MC 5% Plournic 30% Plournic 20%

20 10 0

0

100

200 300 Time, min.

400

500

Fig. 6. A: Effect of the gel type on the in vitro release of HQ from niosomes prepared by reverse phase evaporation technique using Brij 98:cholesterol molar ratio (1:1.5). B: Effect of the gel type on the in vitro release of HQ from niosomes prepared by reverse phase evaporation technique using Brij 98:cholesterol molar ratio (1:1.5).

release was non-significant when stored at 4 ◦ C when compared to the release from the freshly prepared gels, but was significant when stored at 25 ◦ C. The formulation and manufacture of a gel system is not complete without an evaluation of the stability of that system. A gel formulation that is unstable or not suitable for marketing under normal circumstances would exhibit some irreversible change in its rheological properties of sufficient magnitude to cause it to be unacceptable in its final use. Nevertheless, apparent viscosity measurements determined at a single shear rate could be useful for comparative purposes, as with different batches or stored samples over time (Fu et al., 2010). There is no significant difference between viscosity results before and after storing of niosomal Pluronic gel 70 60 Percentage HQ released

4.5. Effectiveness of benzalkonium chloride as preservative

(B)

50 90 days at 25 °C 90 days at 4 °C 60 days at 25 °C 60 days at 4 °C 30 days at 25 °C 30 days at 4 °C 2 days at 25 °C 2 days at 4 °C

40 30 20 10 0

0

100

200

300

400

500

Time, min. Fig. 7. Effect of the storage conditions and storage time on the in vitro release of HQ from niosomes dispersed in pluronic F-127 (20% gels) prepared by reverse phase evaporation technique using Brij 98:cholesterol molar ratio (1:1.5).

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Fig. 8. Photographic images of erosive oral lichen planus before (A) and after (B) treatment with HQ niosomal gels for 1.5 months in 33-year-old female.

Fig. 9. Photographic images of erosive oral lichen planus before (A) and after (B) treatment with HQ niosomal gels for 2 months in 45-year-old male.

Table 5 Demographic data of patients participated in the study.

Total number of patients Males Females Average age (years) Average size of ulcerated area (cm ± SD) Average score of pain

HQ niosomal gel

Placebo

11 2 9 44.73 2.00 ± 1.07 4

5 2 3 48.00 2.02 ± 0.97 3

at 4 ◦ C and at 25 ◦ C (using ANOVA test at level of significance 0.05). These results are in accordance with viscosity rheograms in which Pluronic exhibited pseudoplastic flow with thixotropic behavior, which is indication of its stability. So, Pluronic F-127 (20%) was chosen as the ideal gel base for subsequent in vivo studies. 4.8. Clinical efficiency of hydroxychloroquine niosomal gels The demographic details of the 16 patients consented to take part in this study are presented in Table 5.

Data regarding the evaluation of the two major parameters (size of lesions and pain) are displayed in Table 6. It is obvious that the size of lesions was reduced from 2.00 ± 1.07 cm to 0.65 ± 0.82 cm in the group who treated with HQ niosomal gel, with 64.28% reduction in the size of lesions. While there is only 3.94% reduction in the lesion size in the Placebo group, as the size of lesions was reduced from 2.02 ± 0.97 cm to 1.92 ± 0.87 cm. Statistically, topical HQ had significant effect on the size of lesions of oral lichen planus when formulated as niosomal gel. There is significance difference in the size of lesions when comparison was established between the group of patients treated with HQ niosomal gel (before and after treatment) (p = 0.002). There is significant difference between the results of the group treated with HQ niosomal gel (after treatment) and the Placebo group (before treatment) (p = 0.012). Also, there is significant difference between the results of the group treated with HQ niosomal gel (after treatment) and the Placebo group (after treatment) (p = 0.019). There is no significant difference between the results of group of patients treated with Placebo (before and after treatment) and there is no significant difference between the results of the group treated with HQ (before treatment) and the Placebo (before or after treatment), as p-values > 0.05.

Table 6 The size of lesions and score of pain before and after treatment using hydroxychloroquine niosomal gel and placebo. Parameter

Average size of lesions (cm ± SD) % of reduction of size of lesions Score of pain

HQ niosomal gel

Placebo

Pre-treatment

Post-treatment

Pre-treatment

Post-treatment

2.00 ± 1.07

0.65 ± 0.82 64.28% 1

2.02 ± 0.97

1.92 ± 0.87 3.94% 3

4

3

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Pain severity was decreased in the treatment group as the average score of pain was reduced from “4” before treatment to “1” after treatment, where three patients were pain-free (27.3%) after treatment, compared to the Placebo group where the average score of pain was remained “3” after application as before application. The follow up was for 4 months. The results of therapeutic achievement were reported after 5–6 weeks of treatment in most of patients as indicated by photos displayed in Figs. 8 and 9. After completion of treatment with HQ niosomal gel in the majority of patients for 4 months, no pain and no recurrence were reported. Eight patients (72.7%) noticed relief of all symptoms with no side effects when completed the treatment with HQ niosomal gel, while three patients (27.3%) experienced side effects as burning and tingling at the end of the treatment period. 5. Conclusion In the current study, the results revealed that the formulation variables significantly affect the encapsulation of hydroxychloroquine in niosomes. The type of surfactant, the surfactant:cholesterol molar ratio and the amount of the drug also affect the loading capacity and the in vitro release rate of hydroxychloroquine from niosomes. Regarding the fact that hydroxychloroquine can treat cutaneous conditions orally, this study suggests the innovative strategy of treatment of oral lichen planus by using topical hydroxychloroquine niosomes. The outcomes of the clinical study publicized the potential of hydroxychloroquine niosomal gel in topical treatment of oral lichen planus in short time with less side effects and no recurrence after stopping the treatment. References Abdelkader, H., Ismail, S., Kamal, A., Alany, R.G., 2010. Preparation of niosomes as an ocular delivery system for naltrexone hydrochloride: physicochemical characterization. Pharmazie 11, 811–817. Arguelho, M.L., Andrade, J.F., Stradiotto, N.R., 2003. Electrochemical study of hydroxychloroquine and its determination in plaquenil by differential pulse voltammetry. J. Pharm. Biomed. Anal. 2, 269–275. Arunothayanun, P., Bernard, M.S., Craig, D.Q., Uchegbu, I.F., Florence, A.T., 2000. The effect of processing variables on the physical characteristics of non-ionic surfactant vesicles (niosomes) formed from a hexadecyl diglycerol ether. Int. J. Pharm. 201, 7–14. Balakrishnan, P., Shanmugam, S., Lee, W.S., Lee, W.M., Kim, J.O., Oh, D.H., Kim, D.D., Kim, J.S., Yoo, B.K., Choi, H.G., Woo, J.S., Yong, C.S., 2009. Formulation and in vitro assessment of minoxidil niosomes for enhanced skin delivery? Int. J. Pharm. 377, 1–8. Ben-Zvi, I., Kivity, S., Langevitz, P., Shoenfeld, Y., 2012. Hydroxychloroquine: from malaria to autoimmunity. Clin. Rev. Allergy Immunol. 42, 145–153. Carafa, M., Marianecci, C., Rinaldi, F., Santucci, E., Tampucci, S., Monti, D., 2009. Span and Tween neutral and pH-sensitive vesicles: characterization and in vitro skin permeation. J. Liposome Res. 9, 332–340. Chainani-Wu, N., Collins, K., Silverman Jr., S., 2012. Use of curcuminoids in a cohort of patients with oral lichen planus, an autoimmune disease. Phytomedicine 19, 418–423. Choi, M.J., Kim, J.H., Maibach, H.I., 2006. Topical DNA vaccination with DNA/Lipid based complex. Curr. Drug Deliv. 3, 37–45. Eisen, D., 1993. Hydroxychloroquine sulfate (Plaquenil) improves oral lichen planus: an open trial. J. Am. Acad. Dermatol. 28, 609–612. Fu, S., Thacker, A., Sperger, D.M., Boni, R.L., Velankar, S., Munson, E.J., Block, L.H., 2010. Rheological evaluation of inter-grade and inter-batch variability of sodium alginate. AAPS PharmSciTech 11, 1662–1674. Gonzalez-Moles, M.A., Morales, P., Rodriguez-Archilla, A., Isabel, I.R., GonzalezMoles, S., 2002. Treatment of severe chronic oral erosive lesions with clobetasol

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