Eudragit RS100 based microsponges for dermal delivery of clobetasol propionate in psoriasis management

Journal Pre-proof Eudragit RS100 based microsponges for dermal delivery of clobetasol propionate in psoriasis management Neelam Devi, Sunil Kumar, Minakshi Prasad, Rekha Rao PII:

S1773-2247(19)30851-2

DOI:

https://doi.org/10.1016/j.jddst.2019.101347

Reference:

JDDST 101347

To appear in:

Journal of Drug Delivery Science and Technology

Received Date: 23 June 2019 Revised Date:

29 September 2019

Accepted Date: 21 October 2019

Please cite this article as: N. Devi, S. Kumar, M. Prasad, R. Rao, Eudragit RS100 based microsponges for dermal delivery of clobetasol propionate in psoriasis management, Journal of Drug Delivery Science and Technology (2019), doi: https://doi.org/10.1016/j.jddst.2019.101347. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

Quasi-emulsion solvent diffusion method In vivo activity Clobetasol propionate (CP) CP microsponges (CPMS)

CPMS carbopol gel

CP

CPF8 %T

FTIR

BMS

4000

3500

3000

2500

2000

1500

1000

500

0

-1

Wavenumber (cm )

DSC Orthokeratotic activity (%)

80

B

60

a

a,c

CP gel

CPMS gel

40 20 0

100

Plain gel

98 97 96 95

40

30

20

94 10

Drug content (%)

99

0

SEM

In vitro release studies

CP gel CPMS gel

Time (minutes)

Photo stability

Anti psoriatic activity of CPMS gel

1

Eudragit RS100 based microsponges for dermal delivery of clobetasol propionate in psoriasis

2

management

3

Neelam Devia, Sunil Kumara, Minakshi Prasadb, Rekha Raoa*

4

a

5

Technology, Hisar-125001, Haryana, India

6

b

7

Sciences, Hisar-125004, Haryana, India

8

*Corresponding author address:

9

Department of Pharmaceutical Sciences,

Department of Pharmaceutical Sciences, Guru Jambheshwar University of Science and

Department of Animal Biotechnology, Lala Lajpat Rai University of Veterinary & Animal

10

Guru Jambheshwar University of Science and Technology,

11

Hisar, 125001, Haryana, India.

12

E-mail address: [email protected] (R. Rao).

13 14

Abstract Clobetasol propionate (CP), a super potent dihalogenated topical corticosteroid for

15

psoriasis, displays common side effects like allergic contact dermatitis, steroid acne, skin

16

atrophy, hypo-pigmentation and systemic absorption, on topical application. Hence, entrapment

17

of CP in an appropriate carrier system could minimise the aforementioned side effects, while

18

controlling its percutaneous absorption. Therefore, motive behind current work was to fabricate

19

and evaluate CP loaded microsponges (MS). The microsponges were successfully crafted by

20

employing quasi-emulsion solvent diffusion method. For preparation of MS, organic phase

21

comprising CP, Eudragit RS 100 and dichloromethane was added to aqueous phase (polyvinyl

22

alcohol solution), while stirring. During optimization of MS formulations, factors (drug: polymer

23

ratio, aqueous and organic phase volume) affecting the physical properties of microsponges were

24

also investigated. The prepared microsponges were found to possess particle size in the range

25

12.2±8.2−45.80±12.3 µm, entrapment efficiency: 60.00±0.06 to 96.37± 0.04 % and drug release

26

: 60.60±0.13 to 92.82±0.15 %. 1

Additionaly, CP loaded microsponges were evaluated for

27

topography, thermal and photostability. Finally, optimized CPMS were incorporated into

28

Carbopol gel base, which was subsequently evaluated. In vitro release of CPMS was compared

29

with plain CP and release results were fitted into different kinetic models. CPMS formulation

30

followed zero order kinetics indicating release of drug at constant rate, with absence of initial

31

burst release.

32

therapeutic activity, with minimum toxic effects. In vivo antipsoriatic activity of CPMS gel

33

performed using mice tail model exhibited significant therapeutic efficacy in comparison to plain

34

CP gel, which was further supported by the histopathological findings.

This delivery system resulted in extended CP release and its

maximum

35 36

Keywords: Psoriasis; clobetasol propionate; dermal delivery; microsponges.

37

1. Introduction

38

Psoriasis has a psychosocial impact on patient’s quality of life. Psoriasis is an autoimmune

39

chronic skin disorder characterized by itchy, scaly and disfiguring skin lesions. It is manifested

40

by altered keratinocyte differentiation, hyper proliferation, neovascularization and increased

41

epidermal thickness [1,2]. Immunological and biochemical changes in psoriasis activate immune

42

cells which release cytokines, chemokines and growth factors, resulting in disturbances in their

43

secretions. It is also employed to compliment systemic therapy in some cases of this disorder [2].

44

For management of dermal disorders like psoriasis, topical corticosteroids are one of the oldest

45

treatment of choice, due to their anti-inflammatory, vasoconstrictive and antiproliferative actions

46

[3]. Clobetasol propionate, a prednisolone analog of superpotent corticosteroid is available

47

commercially as Dermovate, Clobex, Temovate and Cormax for management of variety of skin

48

disorders like eczema, atopic dermatitis and vitiligo, besides psoriasis (plaque type psoriasis).

49

These marketed preparations are available in the form of conventional dosage forms like gels,

50

creams, ointments, aerosol foams and lotions (CP is available as 0.05% in commercial

51

formulations) [4–8]. Despite their promising clinical efficacy, corticosteroids like CP results in

52

secondary side effects like hypopigmentation, irritation, skin atrophy, steroid acne,

53

photosensitivity and allergic contact dermatitis on dermal application [3]. For alleviation of

54

aforementioned side effects, formulation scientists have been starving towards development of

55

alternative therapeutic system for their safe and effective delivery. It is also a well-known fact

56

that topical therapy plays a vital role in the management of mild to moderate psoriasis [9].

57

Recently to improve therapeutic efficacy of CP, the use of different kinds of novel topical 2

58

carriers like microemulsion based gel for vitiligo [10], chitin nanogels for psoriasis [11],

59

nanoemulsion and lipid core nanocapsules for contact dermatitis [12], nanocapsules to study

60

follicular uptake and intrafollicular permeation of CP [13], nanostructured lipid carriers for anti-

61

inflammatory disorders [7], NLC coated with chitosan to evaluate in vitro epidermal targeting [8]

62

and lecithin chitosan nanoparticles to assess in vivo drug tolerance and anti-inflammatory

63

efficacy [14] have been proposed. A nanoemulsion for co-delivery of clobetasol propionate and

64

calcipotriol for psoriasis management have been investigated [15].

65 66

Therefore, the proposed microsponge based topical delivery system would ameliorate the side

67

effects associated with CP and provide a prolonged contact time, resulting in increased patient

68

compliance (a major challenge of topical therapy).

69 70

Microsponges are well established, promising colloidal microparticles for dermatological

71

applications [16]. Inherent features like enhanced stability and high drug pay load, besides

72

potential for reduced skin irritation, allergenicity and mutagenicity, present these carriers, an

73

attractive alternative to contemporary topical drug delivery systems [17,18]. Additional features

74

which make them rewarding option for dermal disorder includes skin targeting [19], controlled

75

release [20] and reduced transdermal penetration [21]. For effective topical delivery, a prolonged

76

contact time of active moiety on skin surface or within epidermis is needed, while preventing its

77

penetration in systemic circulation. Microsponges are capable of prolonging their contact time

78

owing to their characteristic size (5 to 300 µm) and hence, these microcarriers are vehicle of

79

choice for topical drug delivery [18,22]. However, these porous microstructures are less suitable

80

for direct application on skin. Therefore, these are loaded in topical bases, like creams, emulgels,

81

or gels for better therapeutic performance.

82

Microsponges are chiefly constituted of polymers dispersed in aqueous medium, with a suitable

83

emulsifying agent. For controlling drug release in MS, Eudragit RS 100 is commonly reported

84

polymer [23]. It is copolymer of methyl methacrylate, ethyl acrylate and metha acrylic acid ester.

85

The widely used technique to engineer MS is the quasi emulsion-solvent diffusion technique, in

86

which suitable water insoluble polymer (Eudragit RS 100) is dissolved in water immiscible

87

solvent like acetone, dichloromethane, ethanol and emulsified with aqueous phase composed of

88

hydrophilic plastsizer. The organic solvent is diffused out slowly under constant stirring, 3

89

resulting in spherical scaffold structures. Factors like active drug to polymer ratio, stirring rate,

90

organic solvent, surfactants play a vital role in regulating the performance of these microcarriers

91

[24]. Polyvinyl alcohol, Eudragit RS 100, triethylcitrate are widely used components for crafting

92

microsponges [20,23].

93 94

The proposed novel microsponge loaded gel of CP allows for enhanced residence time at the

95

skin compared to its contemporary formulations. Further, enhanced effectiveness and retardation

96

in adverse reactions may provide better platform for CP delivery. This is the first report on CP

97

microsponges based gel and its investigation in mouse tail psoriasis model to the best of our

98

knowledge.

99 100

2. Materials and methods

101

2.1. Materials

102

Clobetasol 17 propionate was purchased from Sigma Aldrich, USA. Poly vinyl alcohol (PVA),

103

Eudragit RS 100 and dichloromethane (DCM) were procured from S. D. Fine Chemicals Ltd.,

104

Mumbai, India. Carbopol 934 and triethanolamine were supplied by Loba Chemie Pvt. Ltd.,

105

Baroda, India. Triethyl citrate, Sodium dihydrogen phosphate and disodium hydrogen phosphate

106

were procured from Spectrum Chemicals Pvt. Ltd, India. The double distilled water was used

107

throughout the study.

108 109

3. Methods

110

3.1. Selection of Fabrication variables

111

Preformulation trials were considered to set physical parameters of MS by analyzing the effect of

112

CP to Eudragit ratios (1:3, 1:5, 1:7 and 1:9), PVA (50, 75 and 100 mg), dichloromethane (5, 10

113

and 15 ml) and triethyl citrate (1 %). The process variables like stirring speed was kept 500 rpm

114

and stirring time was fixed 2 hrs. Effect of these parameters on production, encapsulation

115

efficacy and average particle size was assessed. Based on preformulation results, above

116

mentioned parameters were chosen for optimization of CP microsponges [24].

117 118

3.2. Engineering of CP containing microsponges 4

119

The microsponges were engineered by quasi emulsion solvent diffusion method through an

120

internal phase that comprised of Eudragit RS-100 and triethylcitrate (1 %v/v) (TEC) dissolved in

121

DCM. Triethylcitrate was used to increase polymer plasticity [20,25]. This was, followed by the

122

addition of CP with stirring, using propeller stirrer at 500 rpm. The above dispersion was then

123

poured into PVA aqueous solution, as the external phase. After stirring for 2 hrs, microsponges

124

were fabricated due to evaporation of DCM from the matrix. The microsponges were washed

125

with distilled water, subsequently filtered and dried at 40 °C for 12 hrs. Lastly, obtained

126

microsponges were weighed in order to calculate production yield [26]. Various MS formulation

127

batches were fabricated as reported in Table 1.

128 129

3.3. Fabrication of CP microsponge loaded gel

130

In order to prepare hydrogel, Carbopol 934 (1% w/v) was dispersed in distilled water by

131

mechanical stirring for 2 hrs, to get smooth dispersion. Subsequently, it was subjected to stand

132

for half an hour, to remove entrapped air. Then, triethanolamine was added to the viscous

133

dispersion obtained, to maintain pH (7.4) and transparency of the gel [27]. At this stage, the

134

ethanolic solution of CP (50 mg) was added to the plain Carbopol gel. Similarly CP,

135

microsponges (equivalent to 50 mg of CP) were incorporated in the plain gel to obtain gel-based

136

microsponge carrier system [28].

137 138

3.4. Evaluation of CP containing microsponges

139 140

3.4.1. Differential scanning calorimetry analysis

141

Thermal analysis of CP and CPMS was studied employing DSC (Mettler-Toledo DSC 821e,

142

Switzerland). Freshly prepared samples were mounted on aluminum pans, consequently sealed

143

and run at a heating rate (10 ˚C/min) over a temperature range (40-300 ˚C) [29].

144 145

3.4.2. Fourier transform infrared (FTIR) spectroscopy

146

Fourier transform infrared spectra of CP, Eudragit RS 100, PVA, Physical mixture (Polymer and

147

CP), blank microsponges (BMS), and CPMS were obtained using FTIR (Perkin-Elmer Life and

148

Analytical Sciences, USA) over wavenumber range of 4000 to 400 cm-1. Samples were prepared

149

by KBr disc technique. Formed pellets were placed in light path and spectra were recorded [29]. 5

150 151

3.4.3. Production yield

152

Percentage production yield was determined by determining the initial weight of raw materials

153

used and final weight of microsponges and estimated by using formula mentioned below [30].

154

Production yield (PY) =

155


 

 



 

(  
)

× 100

(1)

156 157

3.4.4. Determination of encapsulation efficiency of CP containing microsponges

158

The weighed sample of drug loaded microsponges (10 mg) were kept in 10 ml phosphate buffer

159

(7.4 pH) under ultrasonication for 1 hr. Filtered samples were analyzed at 239 nm against blank,

160

using UV spectrophotometer (Varian Cary-5000, Netherlands) [31]. Encapsulation efficiency

161

was calculated for all batches using the following equation [30,32].

162

Encapsulation efficiency (%) =

163

 

× 100

(2)

164 165

Where Mact = actual CP content in quantity of MS, and Mthe = theoretical CP content in MS.

166 167

3.4.5. Scanning electron microscopy (SEM)

168

The optimized CPMS were visualized using scanning electron microscope (JSM-6100, JEOL,

169

Japan). The dried CPMS powder was coated with gold palladium under argon atmosphere at

170

room temperature and sputtering was done for seven min to record SEM [33].

171 172

3.4.6. Particle size analysis

173

Determination of the average particle size of all batches of prepared microsponges was

174

performed using particle size analyzer (Microtrac S3500-special, USA) [29,34].

175 176

3.5. In vitro drug release and kinetics of drug release

177

In vitro release of CPMS (F1-F8) was performed by using dialysis perfusion bags. The CPMS

178

(accurately weighed) equivalent to 5 mg of CP, was placed in perfusion bag made up of dialysis

179

membrane (12,000-14,000 molecular weight cut off, Spectrum Laboratories Inc., Rancho 6

180

Dominguez, Canada), sealed on both ends, and suspended in USP type II dissolution apparatus

181

(Khera Instruments Pvt. Ltd, India). The dissolution vessel had 900 ml of dissolution medium

182

(phosphate buffer, pH 7.4) at 37±0.5 °C, and stirred continuously at 100 rpm. Aliquots (5 ml

183

each) were collected periodically, at predetermined time intervals for 9 hrs and obtained samples

184

were replaced with equal amount of amount of fresh buffer to maintain sink conditions. The

185

withdrawn samples were analyzed spectrophotometrically (Varian Cary-5000, Netherlands) at

186

239 nm [23]. The percentage of CP release at various time intervals was determined from the

187

calibration curve of CP versus time was plotted [31].

188

Kinetic analysis of the CP release data was also carried out to understand CP release mechanism.

189

Release data as investigated according to various kinetic models; zero order, first order, Higuchi

190

diffusion model, and Korsmeyer-Peppas model [33].

191 192

3.6. Evaluation of CP microsponge gels

193

3.6.1. Visual inspection and pH measurement

194

Fabricated gels were visually inspected for clarity, consistency and homogeneity [32]. The

195

fabricated gel (1 g) was suspended in distilled water (50 ml) and solution pH was noted using

196

digital pH meter [35].

197 198

3.6.2. Spreadability studies

199

A sample of fabricated gel (0.1 g) was placed on plain surface and 500 gms weight was kept on

200

the gel for 5 min. Diameter of spread circles for both gels was determined and calculated as per

201

given formula [32]: Spreadability = (diameter of the spread circle - initial diameter of gel)

202

(3)

203

3.6.3. Viscosity measurement

204

Viscosity of fabricated gel was determined employing a Brookfield viscometer (DV-E

205

Viscometer version 1) with spindle no. S7 using optimum speeds -2, 3, 4, 5, 6, 10, 12, 20, 30, 50,

206

60, 100 rpm at room temperature. An average of six measurements was taken for viscosity

207

estimation [36].

208 209

3.7. Photostability and Stability investigation

7

210

The photodegradation of CP gel and CPMS gel was carried out using Ultra Violet Fluorescence

211

Analysis Cabinet (Scientech instruments, Delhi). The samples were kept at distance of 10 cm

212

from the light source for 1 h. The withdrawn samples were analyzed quantitatively by UV

213

spectrophotometer at 239 nm [32].

214

For stability investigations, optimized CPMS gel and CP gel were filled in lacquered, clean, air

215

tight containers and stored at ambient temperature. The gels were examined for any changes in

216

pH, appearance and drug content at intervals of 10, 20, 30 and 40 days. The in vitro release

217

pattern was evaluated after 40 days [32].

218 219

3.8. In vivo antipsoriatic study of CP loaded gels

220

The protocol for in vivo antipsoriatic studies was approved by Institutional Animal Ethical

221

Committee (IAEC), Guru jambheshwar University of Science and Technology, Hisar (Endst.

222

No/IAEC 247-255). In vivo antipsoriatic potential was explored using mouse tail model [37].

223

Swiss albino mice were divided into three groups (six animals in each group): (1) Control:

224

untreated; (2) CP microsponge gel: treated with CP microsponge gel (equivalent to CP 0.05

225

%w/v); (3) CP gel: treated with CP plain gel (0.05%w/v).

226

The gels (100 mg) were applied on the mouse tail once a day for two weeks. The mice were

227

humanely sacrificed by giving anesthesia (overdose of pentabarbitone sodium) followed by

228

spinal dislocation after twenty four hours from the last application of gels. Tails were removed

229

from animals and dissected to remove underline cartilage. The skin samples were properly

230

processed and consequently stained with haematoxylin and eosin dye for histopathological

231

evaluation of tail skin. Skin samples were microscopically observed for the existence of granular

232

slab in the scale regions and epidermal thickness. Orthokeratotic induction in the adult mouse tail

233

indicated parakeratotic differentiation and was quantified by measuring granular layer length (A)

234

and scale length (B) [37].

235 236

% Orthokeratosis = (A/B) × 100

(4)

237 238

% Drug Activity =

6789 :; <= >?78>7@ A??
239 240

OK = Orthokeratosis 8

GHHD6789 :; <= >I7 E<9>?
× 100

(5)

241 242

Epidermal thickness

243 244

%∆ Epidermal thickness =

QR <= >?78>7@ A???
× 100

(6)

245 246

ET = Epidermal thickness [37].

247 248

3.9. Statistical analysis

249

All measurements were carried out in triplicates and the findings were revealed as mean values ±

250

standard deviation. Statistical differences were investigated by one-way analysis of variance

251

(ANOVA) followed by tucky’s honest significant difference test for multiple comparision for in

252

vivo antipsoriatic activity and two-way ANOVA followed by Bonferroni posttests for multiple

253

comparisons for photo stability

254

software (GraphPad Software, San Diego, CA, USA). In all tests, significant differences were

255

expressed at p < 0.05 values.

and stability analysis using GraphPad Prism version 5.01

256 257

4. Results and discussion

258

4.1. Influence of formulation parameters on production yield, encapsulation efficiency,

259

particle size

260

4.1.1. Influence of drug and polymer ratio

261

Clobetasol propionate loaded microsponges were fabricated via quasi emulsion-solvent diffusion

262

technique. To determine production yield, encapsulation capacity and particle size, a varying

263

concentration of Eudragit RS 100 (matrix forming polymer), PVA (emulsifier) and DCM

264

(solvent) were employed (Table 1). It is a well documented fact that production yield,

265

encapsulation efficiency and particle size of microsponges differs on varying drug to polymer

266

ratio [29,32]. Therefore, in this study, CP amount was kept constant while other variables such

267

as, Eudragit RS 100, PVA and DCM were systematically varied to assess their possible impact.

268

From the obtained data of production yield, encapsulation efficiency and particle size (Table 2),

269

a relationship between Eudragit RS 100 ratio, PVA and DCM amount can be observed. Results 9

270

ascertained that above variables influenced the production yield, encapsulation efficiency and

271

mean size of particles of fabricated microsponges. From the Table 3, it was evident that with

272

increase in concentration of Eudragit RS 100, microsponges production yield increased with

273

movement of CP trapped inside microporous particles. The production yield for CPF1, CPF2,

274

CPF3, CPF4 was 73.57±0.06 %, 80.70±0.35 %, 93.25±0.21 % and 94.47±0.42 %, respectively.

275

The reason behind increased production yield at higher drug polymer ratio could reduce

276

diffusion of DCM (as viscosity of the medium increases with increase in polymer concentration)

277

from concentrated solution into the aqueous phase. This takes more time for droplet formation

278

and hence, improving production yield [32].

279

The drug encapsulation efficiency was also found increased in similar fashion for these batches

280

(CPF1, CPF2, CPF3 and CPF4). Formulation CPF8 displayed maximal encapsulation efficiency

281

(96.37± 0.04 %), which was followed by CPF5 (95.99 ± 0.13 %). When content of Eudragit RS

282

100 was varied with fixed amount of PVA, encapsulation efficiency was found increased (Table

283

3). Increasing the content of Eudragit RS 100 may result in increased number of pores and

284

availability of larger space to accommodate CP, hence enhanced encapsulation was obtained.

285

These results are in agreement with Pandit et al. (2016), who reported that increasing drug to

286

polymer ratio resulted in increased entrapment efficiency in microsponges. This group fabricated

287

Eudragit RS 100 microsponges for entrapment of nebivolol [23].

288

In the present research work, the particle size of fabricated microsponges ranged from 12.20 ±

289

8.2- 45.80 ± 12.30 µm (Table 2). Formulation CPF1 presented minimum particle size of 12.2 ±

290

8.2 µm, when Eudragit RS 100 was at low levels and conversely at high level of Eudragit RS

291

100, maximum particle size was obtained (CPF4). These results may be attributed to the fact that

292

owing to increase in viscosity of dispersed phase at higher quantity of Eudragit RS 100, larger

293

globules were formed, which were difficult to be divided into small particles. Therefore, these

294

larger droplets resulted in bigger porous particles. These results were in consistency with the

295

report of Obiedallah et al., who worked on acetazolamide microsponges [16]. At fixed amount of

296

PVA, when quantity of Eudragit RS 100 is increased, enhancement in particle size was also

297

observed.

298

4.1.2. Influence of internal phase (DCM)

10

299

It is well documented that drug polymer ratio, amount of emulsifier (PVA) has potential affect

300

on production yield, encapsulation efficiency and particle size. The influence of other leading

301

formulation variable, volume of internal phase (DCM) on these parameters was also checked in

302

present investigation. On varying DCM, the order of particle size was observed as 34.6±7.3 µm,

303

44.2±8.4 µm and 43.5±9.2 µm, corresponding to CPF3, CPF5 and CPF6, respectively. Similar

304

trend in production yield and encapsulation efficiency was observed (Table 4). According to

305

Deshmukh and Poddar, particle size of microsponges directly depends on the viscosity of the

306

internal phase [38]. Therefore, with higher volume of solvent, consequently less viscous

307

dispersed phase is obtained resulting in emulsion globules. These emulsion globules definitely

308

divide into smaller droplets leading to small particle size [39]. The pores must have been formed

309

by rapid evaporation of DCM from microsponge surface during stirring. Hence, the

310

dichloromethane has a key role to play in crafting of microsponges [19,33,40]. These results

311

were in accordance with eberconazole nitrate loaded microsponges [39]. Similarly, the more

312

viscous dispersed phase (due to low solvent volume) hampered the solvent diffusion, hence,

313

bigger droplets with higher entrapment efficiency of CP were obtained [39]. The optimum

314

volume of the solvent (DCM) selected was 10 ml, as it results in high production yield, high

315

encapsulation efficiency and appropriate size, suitable for topical delivery of CP.

316

4.1.3. Influence of amount of emulsifier (PVA)

317

Keeping drug polymer ratio fixed (1:7), the production yield was found increased with increasing

318

PVA amount from 50 mg to 100 mg. The production yield was obtained maximum 96.87 ±0.15

319

% in CPF8, where amount of PVA is 100 mg. Higher amount of emulsifier leads to abridged

320

DCM diffusion from the concentrated solutions to the aqueous medium (at higher CP: Eudragit

321

RS 100 concentrations) providing additional time for droplet generation, subsequently resulting

322

in enhanced production yield. Similar results were reported in case of diclofenac diethylamine

323

[30].

324

Batch CPF5, CPF7 and CPF8 fabricated with fixed amount of Eudragit RS 100 and variable

325

PVA displayed encapsulation efficiencies of 95.99 ± 0.13 %, 95.84 ± 0.01 % and 96.37 ± 0.04

326

%, respectively (Table 5). Using variable PVA concentration, a little variation in encapsulation

327

efficiency was seen. Higher values of encapsulation efficiency in all batches were obtained

328

owing to porous nature of microsponges. 11

329

With increase in concentration of PVA at fixed polymer concentration, particle size were found

330

44.2 ±8.4 µm, 34.1±7.8 µm and 37.2±9.2 µm for CPF5, CPF7 and CPF8, respectively. This

331

could be accounted for decrease in surface tension of aqueous (continuous) phase by increasing

332

surfactant content. As a result, tinier droplets were formed, leading to formation of smaller sized

333

particles which reverses, on further raising the surfactant amount [41,42]. Further, as amount of

334

PVA was increased from 75 mg to 100 mg; the bigger emulsion droplets obtained cannot be

335

splitted into smaller droplets and as a result, finally large sized microsponges were produced

336

[43].

337

On the basis of highest production yield and encapsulation efficiency most sustained drug release

338

pattern, the CPF8 was selected as optimized formulation.

339

4.2. Solid state characterization of clobetasol propionate microsponges

340

A few analytical tools (FTIR, DSC and SEM) were employed to investigate drug and polymer

341

interactions, thermal properties and surface structures of the CPMS. These facts are vital in

342

engineering of rigorous and reliable sustained release formulations.

343 344

4.2.1. Differential scanning calorimetry

345

DSC studies were employed to indicate compatibility between active moiety and polymer. The

346

thermal behavior of CP and CPMS (CPF8) are displayed in Fig. 1. The thermal graphs presented

347

a sharp endothermic peak at 196 °C corresponding to melting point of CP, reflecting its

348

crystalline nature and purity. CPMS exhibited no characteristic peak of CP, indicating its

349

encapsulation inside microsponges [44]. Further, absence of melting endotherm of CP in CPMS

350

advocated transformation of CP from crystalline to amorphous state or disordered crystalline

351

phase inside microsponge cavities, as noted in Fig. 1. Similar results have been reported for

352

diltiazem hydrochloride loaded Eudragit RS 100 microsponges by Ivanova et al., 2019 [45].

353

4.2.2. Fourier transforms Infrared spectroscopy

354

To examine feasiblility of clobetasol propionate and Eudragit RS 100 (polymer) interactions and

355

to assess the degradation of CP during fabrication process, FTIR spectroscopy was performed.

356

The FTIR spectra of CP, CPMS, Eudragit RS 100 and blank MS are recorder and illustrated in

357

Fig. 2a and 2b. Clobetasol propionate is a crystalline active moiety having three prominent 12

358

peaks, one appearing at 1734 cm-1 for C=O stretching vibration of ester, 1662 cm-1 for C-F

359

stretching of the ester and one more appearing at 3309 cm-1 for OH stretching of alcohol group.

360

The results collected in study are in accordance with previous published data [11,46]. Eudragit

361

RS100 displayed an ester C=O stretching peak at1734 cm-1 as like with the given data [47]. All

362

characteristic peaks of CP were also recognized in physical mixture and microsponge

363

formulation (CPF8) spectrum (Fig 2a and 2b). Thus, IR results advocated that CP was

364

compatible with selected polymer, excipients. Further, all retained peaks of CP indicated that

365

original form of drug was maintained without any modification while it’s entrapment in Eudragit

366

RS 100 microsponges and possess good stability in this carrier system [44]. Additionally, the

367

attenuation of peak intensity in CPMS also points out towards pore confinement of CP, where it

368

is available in an amorphous form, confirmed by previous thermal investigation. FTIR showed

369

disappearance of existing peaks or no new peak, discarding chemical interaction possibility in

370

between CP and polymer used. Hence, IR results showed that CP was compatible with Eudragit

371

RS 100 (selected polymer), excipients and have good stability in all MS formulations [44].

372

4.2.3. Scanning electron microscopy

373

For morphological analysis, engineered CP microsponges were subjected to SEM analysis. The

374

SEM images are illustrated in Fig. 3. SEM observations revealed that fabricated MS were highly

375

porous and mostly spherical. Further, no aggregation of microsponges was observed which might

376

be due to negligible surface charge on them. Hence, the prepared microformulations were found

377

to possess desirable physical stability. PVA used as emulsifier during fabrication of

378

microsponges might be responsible for reducing particle surface charge and kept

379

microformulations free from aggregates [38]. The pores must have been formed by rapid

380

evaporation of DCM from microsponge surface during stirring. Hence, the dichloromethane has

381

a key role to play in crafting of microsponges [19,33,40].

382 383

4.3. In vitro drug release and kinetics of drug release

384

Drug release was assessed with an aim to elucidate release pattern of CP from microsponges.

385

The CP release was seemed to decrease from 91.4 to 63.40 % with increase in CP-Eudragit ratio

386

(1:1 to 1:9) (Table 6). The results exhibited that increased drug to polymer ratio led to thicken

387

the microsponge wall and increase its size, resulting in the decrease in surface area and

388

consequently, reduction of drug release from MS [16]. The findings are in accordance with the 13

389

release profile of nebivolol loaded microsponges crafted using Eudragit RS 100 which

390

contributed for delayed release of drug [23]. The highest CP release (91.4 %) was observed for

391

batch CPF1, while minimum was 63.40 % for CPF4. Graphical representation for cumulative CP

392

release of all batches is illustrated in Fig. 4a for CPF1-CPF4 and Fig. 4b for CPF5-CPF8,

393

respectively. The increased amount of DCM also resulted in the precipitation of the drug at the

394

periphery of the microsponge. This might have led to increase in drug release. Hence,

395

enhancement in release rate with increase in amount of DCM for formulations CPF6 was

396

observed (92.82±0.15 %) [48]. However, CPF5 was not found to follow this release pattern. It is

397

noteworthy that voids present in microsponges may acted as reservoir for drug and responsible

398

for delaying its release [16]. The decline in release rate was demonstrated with increase in

399

amount of PVA for formulations CPF7 and CPF8 (61.05 % to 60.06 %).

400

formulation exhibited maximum delayed release in comparison to other CP microsponge

401

batches.

402

In order to understand CP release mechanism from fabricated microsponges, the data obtained

403

from in vitro release was fitted into various release models namely, zero order, first order,

404

Higuchi and Korsmeyer-Peppas. From r2 (Regression co-efficient) value, best fit model was

405

chosen. The in vitro drug release presented highest regression value for the Peppas model (0.988

406

for CPF2 and CPF3) with the release exponent (n) values below 0.43, ascertaining drug release

407

through Fickian diffusion controlled mechanism. Further, it was supported from SEM results

408

(Fig. 3), as the prepared microsponges were found to possess porous structures. [33]. Higuchi

409

model was also best fit for some of the formulations (CPF1, CPF5, CPF6 and CPF7 with

410

regression values as 0.990, 0.914, 0.946 and 0.990, respectively) confirming drug release

411

through diffusion process (Table 7), whereas formulations CPF4 and CPF8 followed the zero

412

order kinetics demonstrating that drug release was independent of drug concentration [30]. This

413

helps in extending the drug efficacy and maintained constant drug levels with maximum

414

therapeutic activity [49]. Salah et al., also investigated kinetics of all batches of miconazole

415

Eudragit RS100 microsponges using these models [33].

416

4.4. Preparation of CP microsponge loaded gel

417

The topical carrier allows the targeting of active moiety directly to the affected skin in the

418

present investigation. Since, prepared CP microsponges were particulate in nature, these were 14

Hence, CPF8

419

less suitable for direct topical application. Hence, the optimized batch (CPF8) was enriched in

420

Carbopol gel. Gel was chosen due to its better aesthetic nature, cosmetic appeal, non-irritant and

421

convenient to apply.

422

4.5. Evaluation of CP microsponge gel/ Semisolid state characterization

423

4.5.1. Visual inspection and pH measurements

424

The prepared gel formulations loaded with CP microsponges were observed visually for

425

appearance, color and texture. All MS formulations were transparent and viscous with smooth

426

texture as well as good homogeneity. The pH values of gel formulations were found in the range

427

of 6.9-7.3, thus, minimizing irritation after application on psoriatic skin (Table 8).

428

CP microsponge gel maintains appropriate moisture, providing an additional benefit to psoriatic

429

patients and overruling psoriasis complications.

430 431

4.5.2. Spreadability investigations

432

Potency of topical formulations depends on their spreading in the form of even layer to

433

administer a standard dose. Viscous nature of fabricated gel proved limited spreadability,

434

therefore reservation of gel on psoriatic skin for longer time. Spreadability of CPMS gel and CP

435

gel was found to be 13.52 g cm/s and 11.18 g cm/s, respectively indicating that spreadability of

436

CPMS gel was slightly better than that of plain CP gel (Table. 8). Psoriatic patients already

437

suffer from discomfortness due to itching, dryness, peeling, redness and consequently, more

438

discomfort due to grittiness and rough consistency of gel during its application is no more

439

tolerable. The absence of course particles exhibited marvelous relevance of gel on psoriatic skin.

440 441

4.5.3. Viscosity investigations

442

Carbopol polymers resulted in gels with maximum viscosity at a pH of 6-7 [50]. The pH of the

443

gel was found between 6.9 to 7.3. To prolong its retention over the skin and to allow complete

444

release of CP from the prepared formulation, gels were prepared [33]. Carbopol gels are known

445

for their compatibility with variety of active moieties, high viscosity at low concentration,

446

stability and patient compliance [51,52]. The viscosity of Carbopol gels generally affects drug

447

release behavior and its retention at application site. At fixed shear rate values, because shearing

448

stress was increased, normally disorganised molecules start to line up their long axes in direction 15

449

of flow. These orientations have internal resistance and permitted better shear rate on every

450

consecutive shearing stress [53]. In the present study, the viscosity was reliant on polymeric

451

content of novel formulation (Table 8). The viscosity of the CPMS gel was investigated at

452

various shear rates. The CPMS gel showed pseudoplastic pattern, which is a necessary feature to

453

break microgel framework of topical gel [50]. As the shear rate was increased, the viscosity of

454

gel was found decreased. The curved rheograms indicated shearing action onto long chain

455

polymer molecules (Fig. 5). Similar results were reported in the previous investigation by Ghosh

456

and Kumar, 2017 [40].

457

As per literature for topical delivery, at optimum viscosity, the gel neither flow instantly after its

458

application on skin, nor it resisted application [33]. CPMS gel was found to possess appropriate

459

viscosity in the acceptable range for topical application. Hence, this provided an additional

460

benefit of prolonged retention of the gel to psoriatic patients and improved patient compliance

461

[33].

462 463

4.6. In vitro release profile of CP microsponge gels

464

The release profile of CP microsponge (CPF8) carbopol gel (G2) produced a profound

465

improvement in the release rate, which was remarkably higher than CP plain gel (G1) (Fig. 6). It

466

was observed that plain CP gel got exhausted by releasing 98.66 % of the drug, at the end of 6

467

hrs only. In contrast, microsponge-based gel exhibited sustained release pattern up to 9 hrs,

468

which may help in minimizing the side effects like, skin irritation and hypersensitivity reactions

469

associated with of clobetasol propionate. Further, the results showed that loading of CP

470

microsponges in Carbopol gel imparted delay in its release. This may be because of extra time

471

taken for dissolution of the gel, before drug release. Hence, the present results advocated the

472

microsponges were efficacious in retarding drug release in comparison to uncapsulated drug. As

473

the CPF8 formulation exhibited zero order drug release kinetics indicating, that drug release was

474

not dependent on drug’s concentration [30]. It was found superior in terms of entrapment

475

efficiency and production yield, it has been considered as most efficacious among the all

476

prepared batches.

477 478

4.7. Photostability and stability studies

16

479

Clobetasol propinate gets absorbed in the UV region displaying a peak around 237 nm, whose

480

intensity retarded upon UVA irradiation. The reduction in intensity indicated photolysis of CP.

481

The % CP content of the CP gel was found 92.15±0.32% whereas 97.12±0.10 % CP content was

482

observed in case of CPMS gel. From the degradation study, it was ascertained that CP

483

microsponge gel was more photostable than plain CP gel. This might be because of strong

484

encapsulation of drug with in microsponge cavities (Fig. 7A). Microsponges provided CP with a

485

physical barrier against UV- induced degradation. Moving to possible pharmaceutical

486

applications of this investigation, it could be speculated that microsponges may protect the

487

bioactives entrapped from degradation via UVA radiations.

488

For stability investigations, CP gel and CPMS gel were examined at 5±2ºC, 25±2ºC and 40±2ºC

489

for 40 days. The both gels were found white, homogenous and smooth. Further, no significant

490

change in pH was observed. The CP and CPMS gel stored at 5±2ºC and 25±2ºC showed non

491

significant drug degradation. It was elucidated from results that negligible changes in drug

492

content were observed (data not shown). The % CP degradation in CP and CPMS gel stored at

493

40±2ºC was found 95. 15 ± 0.29 % and 97.05 ± 0.07%, respectively (Fig. 7B). For comparative

494

assessment of CPMS (CPF8), drug release pattern was assessed initially and after 40 days (Fig.

495

8). From FTIR analysis of stored samples, all characteristic peaks were observed in all spectra

496

after 40 days storage. All prominent peaks of CP were also present in physical mixture

497

advocating its acceptable stability in MS formulations.

498 499

4.8. In vivo antipsoriatic activity

500

The extent of CP antipsoriatic activity of CP loaded gels (treatment-induced orthokeratosis) is

501

illustrated in Fig. 9. In vivo results indicated that CPMS gel (CP microsponge gel equivalent to

502

0.05 %w/v CP) presented significantly higher efficiency than plain CP gel (0.05 %w/v). It was

503

observed that microsponge loaded gel has increased the orthokeratotic regions by 65.14±1.63 %

504

in comparison to control group (untreated). However, the CP gel showed the increase in the

505

orthokeratotic regions by 57.35 ± 2.08 %, (Fig. 10B). Further, microsponge loaded gel has

506

decreased the epidermal thickness as 62.01 ± 0.96 % while the plain CP gel drug decreased the

507

epidermal thickness as 55.85 ± 1.24 % (Fig. 10A). Percent drug activity of microsponge loaded

508

gel was found to be 61.31 ± 1.37 % showing significant antipsoriatic effect in comparison to CP 17

509

gel (53.75 ± 1.81 %) (Fig. 10C). The findings in the present study are in accordance with those

510

of previous reports [11,54], as hyper keratinization is the main issue in psoriasis management.

511

The formulations which potentially help in building microreservoirs in skin, are efficient in

512

orthokeratosis induction in mouse. In the similar fashion, the CPMS gel presented potential

513

enhancement in orthokeratosis vis-à-vis plain CP gel. By and large, fabricated nanocarrier gel

514

improved the anti-psoriatic potential of CP with respect to CP gel, supposedly due to improved

515

interaction of the CP-loaded system with the substrate (skin layers).

516

5. Conclusion

517

Microsponge based novel gel for topical delivery has been proposed in the present study for

518

psoriasis management. CP loaded microsponges were successfully fabricated employing

519

Eudragit RS 100 as matrix polymer and PVA as emulsifier. The crafted microsponges were

520

efficiently characterized and in vitro drug release pattern was determined. Manipulating the

521

quantity of Eudragit RS 100 (polymers) and PVA (surfactant) had potential momentous impact

522

on efficacy of MS formulations. The outcomes demonstrated that microsponge engineered with

523

Eudragit RS 100 and PVA along with 1% triethylcitrate (CPF8) resulted in maximum drug

524

payload with delayed release (following zero order kinetics) from porous MS. Further, MS

525

microsponge embedded gel catered moist climate for psoriatic patients for longer time period.

526

Additionally, this microformulation reduced the probable permeation of CP in systemic

527

circulation. Thus, potential approach of clobetasol propionate, MS and gel accomplished

528

significant orthokeratosis and healing process in psoriasis. Consequently, this approach would

529

offer satisfactory as well as selective safety profile to CP. Such a developed system might

530

enhance patient compliance by optimizing therapeutic performance of CP while reducing side

531

effects associated with it.

532

Declaration of interest

533

The authors have no declaration of interest.

534 535

Acknowledgement

536

The authors are grateful to Department of Pharmaceutical Sciences and Dr. A. P. J. Abdul Kalam

537

Central Instrumentation Laboratory, Guru Jambheshwar University of Science and Technology,

538

Hisar for providing all laboratory facilities to carry out this work. The author Mr. Sunil Kumar, 18

539

is thankful to Indian Council of Medical Research, New Delhi for providing Senior Research

540

Fellowship [Letter No: 45/44/2018-Nan/BMS on dated 14/05/2018].

541

References

542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582

[1] J.C. Chamcheu, I.A. Siddiqui, V.M. Adhami, S. Esnault, D.J. Bharali, A.S. Babatunde, S. Adame, R.J. Massey, G.S. Wood, B.J. Longley, Chitosan-based nanoformulated (-)epigallocatechin-3-gallate (EGCG) modulates human keratinocyte-induced responses and alleviates imiquimod-induced murine psoriasiform dermatitis, Int. J. Nanomedicine. 13 (2018) 4189. [2] M. Pradhan, A. Alexander, M.R. Singh, D. Singh, S. Saraf, S. Saraf, Understanding the prospective of nano-formulations towards the treatment of psoriasis, Biomed. Pharmacother. 107 (2018) 447–463. [3] S. Wiedersberg, C.S. Leopold, R.H. Guy, Bioavailability and bioequivalence of topical glucocorticoids, Eur. J. Pharm. Biopharm. 68 (2008) 453–466. [4] https://www.medicines.org.uk/emc/product/939/smpc Google Search, (n.d.). https://www.google.com/search?q=%5B4%5D+https%3A%2F%2Fwww.medicines.org.uk %2Femc%2Fproduct%2F939%2Fsmpc&rlz=1C1CHBD_enIN862IN862&oq=%5B4%5D+ https%3A%2F%2Fwww.medicines.org.uk%2Femc%2Fproduct%2F939%2Fsmpc&aqs=ch rome..69i57.427j0j4&sourceid=chrome&ie=UTF-8 (accessed September 24, 2019). [5] Clobetasol propionate, (n.d.). https://www.drugbank.ca/drugs/DB01013 (accessed September 24, 2019). [6] Clobex Spray (Clobetasol Propionate Spray): Side Effects, Interactions, Warning, Dosage & Uses, (n.d.). https://www.rxlist.com/clobex-drug.htm (accessed September 24, 2019). [7] U. Nagaich, N. Gulati, Nanostructured lipid carriers (NLC) based controlled release topical gel of clobetasol propionate: design and in vivo characterization, Drug Deliv. Transl. Res. 6 (2016) 289–298. [8] L.A.D. Silva, L.M. Andrade, F.A.P. de Sá, R.N. Marreto, E.M. Lima, T. Gratieri, S.F. Taveira, Clobetasol-loaded nanostructured lipid carriers for epidermal targeting, J. Pharm. Pharmacol. 68 (2016) 742–750. [9] E. Desmet, M. Van Gele, J. Lambert, Topically applied lipid-and surfactant-based nanoparticles in the treatment of skin disorders, Expert Opin. Drug Deliv. 14 (2017) 109– 122. [10] H.K. Patel, B.S. Barot, P.B. Parejiya, P.K. Shelat, A. Shukla, Topical delivery of clobetasol propionate loaded microemulsion based gel for effective treatment of vitiligo: ex vivo permeation and skin irritation studies, Colloids Surf. B Biointerfaces. 102 (2013) 86–94. [11] R. Panonnummal, R. Jayakumar, M. Sabitha, Comparative anti-psoriatic efficacy studies of clobetasol loaded chitin nanogel and marketed cream, Eur. J. Pharm. Sci. 96 (2017) 193– 206. [12] M.C. Fontana, J.F.P. Rezer, K. Coradini, D.B.R. Leal, R.C.R. Beck, Improved efficacy in the treatment of contact dermatitis in rats by a dermatological nanomedicine containing clobetasol propionate, Eur. J. Pharm. Biopharm. 79 (2011) 241–249. [13] C. Mathes, A. Melero, P. Conrad, T. Vogt, L. Rigo, D. Selzer, W.A. Prado, C. De Rossi, T.M. Garrigues, S. Hansen, Nanocarriers for optimizing the balance between interfollicular permeation and follicular uptake of topically applied clobetasol to minimize adverse effects, J. Controlled Release. 223 (2016) 207–214. 19

583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628

[14] T. Şenyiğit, F. Sonvico, A. Rossi, I. Tekmen, P. Santi, P. Colombo, S. Nicoli, Ö. Özer, In vivo assessment of clobetasol propionate-loaded lecithin-chitosan nanoparticles for skin delivery, Int. J. Mol. Sci. 18 (2017) 32. [15] A. Kaur, S.S. Katiyar, V. Kushwah, S. Jain, Nanoemulsion loaded gel for topical codelivery of clobitasol propionate and calcipotriol in psoriasis, Nanomedicine Nanotechnol. Biol. Med. 13 (2017) 1473–1482. [16] M.M. Obiedallah, A.M. Abdel-Mageed, T.H. Elfaham, Ocular administration of acetazolamide microsponges in situ gel formulations, Saudi Pharm. J. 26 (2018) 909–920. [17] H.V. Gangadharappa, N. Vishal Gupta, S. Chandra Prasad M, H.G. Shivakumar, Current trends in microsponge drug delivery system, Curr. Drug Deliv. 10 (2013) 453–465. [18] G. Singhvi, P. Manchanda, N. Hans, S.K. Dubey, G. Gupta, Microsponge: An emerging drug delivery strategy, Drug Dev. Res. 80 (2019) 200–208. [19] S.-S. Li, G.-F. Li, L. Liu, X. Jiang, B. Zhang, Z.-G. Liu, X.-L. Li, L.-D. Weng, T. Zuo, Q. Liu, Evaluation of paeonol skin-target delivery from its microsponge formulation: in vitro skin permeation and in vivo microdialysis, PloS One. 8 (2013) e79881. [20] A. Kumari, A. Jain, P. Hurkat, A. Tiwari, S.K. Jain, Eudragit S100 coated microsponges for Colon targeting of prednisolone, Drug Dev. Ind. Pharm. 44 (2018) 902–913. [21] N. Amrutiya, A. Bajaj, M. Madan, Development of microsponges for topical delivery of mupirocin, AAPS PharmSciTech. 10 (2009) 402–409. [22] M.V. Junqueira, M.L. Bruschi, A review about the drug delivery from microsponges, AAPS PharmSciTech. 19 (2018) 1501–1511. [23] A.P. Pandit, S.A. Patel, V.P. Bhanushali, V.S. Kulkarni, V.D. Kakad, Nebivolol-loaded microsponge gel for healing of diabetic wound, AAPS PharmSciTech. 18 (2017) 846–854. [24] Y. Shahzad, S. Saeed, M.U. Ghori, T. Mahmood, A.M. Yousaf, M. Jamshaid, R. Sheikh, S.A. Rizvi, Influence of polymer ratio and surfactants on controlled drug release from cellulosic microsponges, Int. J. Biol. Macromol. 109 (2018) 963–970. [25] V. Jain, R. Singh, Dicyclomine-loaded Eudragit®-based microsponge with potential for colonic delivery: preparation and characterization, Trop. J. Pharm. Res. 9 (2010). [26] V. Jain, D. Jain, R. Singh, Factors effecting the morphology of eudragit S-100 based microsponges bearing dicyclomine for colonic delivery, J. Pharm. Sci. 100 (2011) 1545– 1552. [27] S. Maiti, S. Kaity, S. Ray, B. Sa, Development and evaluation of xanthan gum-facilitated ethyl cellulose microsponges for controlled percutaneous delivery of diclofenac sodium, Acta Pharm. 61 (2011) 257–270. [28] V.V. Pande, N.A. Kadnor, R.N. Kadam, S.A. Upadhye, Fabrication and characterization of sertaconazole nitrate microsponge as a topical drug delivery system, Indian J. Pharm. Sci. 77 (2015) 675. [29] G. Wadhwa, S. Kumar, V. Mittal, R. Rao, Encapsulation of babchi essential oil into microsponges: Physicochemical properties, cytotoxic evaluation and anti-microbial activity, J. Food Drug Anal. 27 (2019) 60–70. doi:10.1016/j.jfda.2018.07.006. [30] R.A.M. Osmani, N.H. Aloorkar, D.J. Ingale, P.K. Kulkarni, U. Hani, R.R. Bhosale, D.J. Dev, Microsponges based novel drug delivery system for augmented arthritis therapy, Saudi Pharm. J. 23 (2015) 562–572. [31] N. Devi, S. Kumar, S. Rajan, J. Gegoria, S. Mahant, R. Rao, Development and Validation of UV Spectrophotometric Method for Quantitative Estimation of Clobetasol 17Propionate, Asian J. Chem. Pharm. Sci. 1 (2016) 36–40. 20

629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672

[32] E. Yadav, R. Rao, S. Kumar, S. Mahant, V. Prakriti, Microsponge Based Gel of Tea Tree Oil for Dermatological Microbial Infections, Nat. Prod. J. 8 (2018) 1–12. [33] S. Salah, G.E. Awad, A.I. Makhlouf, Improved vaginal retention and enhanced antifungal activity of miconazole microsponges gel: Formulation development and in vivo therapeutic efficacy in rats, Eur. J. Pharm. Sci. 114 (2018) 255–266. [34] R. Ravi, S.K. Sk, Standarization of process parameters involved erythormycin microsponges by Quassi emulsion solvent diffusion method, Int J Pharm Dev Technol. 3 (2013) 28–34. [35] Y. Chandramouli, S. Firoz, R. Rajalakshmi, A. Vikram, B.R. Yasmeen, R.N. Chakravarthi, Preparation and evaluation of microsponge loaded controlled release topical gel of acyclovir sodium, Int J Biopharm. 3 (2012) 96–102. [36] R. VG, A.B. Tambe, V.K. Deshmukh, Topical Anti-Inflammatory Gels of Naproxen Entrapped in Eudragit Based Microsponge Delivery System, (2015). [37] K. Raza, O.P. Katare, A. Setia, A. Bhatia, B. Singh, Improved therapeutic performance of dithranol against psoriasis employing systematically optimized nanoemulsomes, J. Microencapsul. 30 (2013) 225–236. [38] K. Deshmukh, S.S. Poddar, Tyrosinase inhibitor-loaded microsponge drug delivery system: new approach for hyperpigmentation disorders, J. Microencapsul. 29 (2012) 559–568. [39] C. Bothiraja, A.D. Gholap, K.S. Shaikh, A.P. Pawar, Investigation of ethyl cellulose microsponge gel for topical delivery of eberconazole nitrate for fungal therapy, Ther. Deliv. 5 (2014) 781–794. [40] P.M. Kumar, A. Ghosh, Development and evaluation of silver sulfadiazine loaded microsponge based gel for partial thickness (second degree) burn wounds, Eur. J. Pharm. Sci. 96 (2017) 243–254. [41] R. Dinarvand, S. Mirfattahi, F. Atyabi, Preparation, characterization and in vitro drug release of isosorbide dinitrate microspheres, J. Microencapsul. 19 (2002) 73–81. [42] R.M. Mainardes, R.C. Evangelista, PLGA nanoparticles containing praziquantel: effect of formulation variables on size distribution, Int. J. Pharm. 290 (2005) 137–144. [43] M. Jelvehgari, M.R. Siahi-Shadbad, S. Azarmi, G.P. Martin, A. Nokhodchi, The microsponge delivery system of benzoyl peroxide: Preparation, characterization and release studies, Int. J. Pharm. 308 (2006) 124–132. [44] A.J. Shinde, M.B. Paithane, S.S. Sawant, Development and evaluatio n of fenoprofen microsponges and its colonic delivery using natura l polysaccharides, Ame J Pharma Sci Nanotech. 1 (2014) 27–42. [45] N.A. Ivanova, A. Trapani, C. Di Franco, D. Mandracchia, G. Trapani, C. Franchini, F. Corbo, G. Tripodo, I.N. Kolev, G.S. Stoyanov, In vitro and ex vivo studies on diltiazem hydrochloride-loaded microsponges in rectal gels for chronic anal fissures treatment, Int. J. Pharm. 557 (2019) 53–65. [46] Y.F. Bassuoni, E.S. Elzanfaly, H.M. Essam, H.E.-S. Zaazaa, Development and validation of stability indicating TLC densitometric and spectrophotometric methods for determination of clobetasol propionate, Bull. Fac. Pharm. Cairo Univ. 54 (2016) 165–174. [47] R. Pignatello, C. Bucolo, P. Ferrara, A. Maltese, A. Puleo, G. Puglisi, Eudragit RS100® nanosuspensions for the ophthalmic controlled delivery of ibuprofen, Eur. J. Pharm. Sci. 16 (2002) 53–61.

21

673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691

[48] A.P. Pawar, A.P. Gholap, A.B. Kuchekar, C. Bothiraja, A.J. Mali, Formulation and evaluation of optimized oxybenzone microsponge gel for topical delivery, J. Drug Deliv. 2015 (2015). [49] Y.-N. Zhao, X. Xu, N. Wen, R. Song, Q. Meng, Y. Guan, S. Cheng, D. Cao, Y. Dong, J. Qie, A drug carrier for sustained zero-order release of peptide therapeutics, Sci. Rep. 7 (2017) 5524. [50] M.T. Islam, N. Rodriguez-Hornedo, S. Ciotti, C. Ackermann, Rheological characterization of topical carbomer gels neutralized to different pH, Pharm. Res. 21 (2004) 1192–1199. [51] W. Liu, M. Hu, W. Liu, C. Xue, H. Xu, X. Yang, Investigation of the carbopol gel of solid lipid nanoparticles for the transdermal iontophoretic delivery of triamcinolone acetonide acetate, Int. J. Pharm. 364 (2008) 135–141. [52] D.D. Pelot, N. Klep, A.L. Yarin, Spreading of Carbopol gels, Rheol. Acta. 55 (2016) 279– 291. [53] A. Nokhodchi, M. Jelveghari, M.-R. Siahi, S. Dastmalchi, The effect of formulation type on the release of benzoyl peroxide from microsponges, Iran. J. Pharm. Sci. 1 (2005) 131–142. [54] S. Kumar, K.K. Singh, R. Rao, Enhanced anti-psoriatic efficacy and regulation of oxidative stress of a novel topical babchi oil (Psoralea corylifolia) cyclodextrin-based nanogel in a mouse tail model, J. Microencapsul. 36 (2019) 140–155.

22

Table 1 Composition of different microsponges formulations prepared by quasi emulsion solvent diffusion method. Ingredients

CPF1 CPF2 CPF3 CPF4 CPF5 CPF6 CPF7 CPF8

Clobetasol propionate 1:3 (CP):Eudragit RS100 (mg)

1:5

1:7

1:9

1:7

1:7

1:7

1:7

Dichloromethane (ml)

5

5

5

5

10

15

10

10

Triethylcitrate (% v/v)

1

1

1

1

1

1

1

1

Polyvinyl alcohol (mg)

50

50

50

50

50

50

75

100

Water (ml)

100

100

100

100

100

100

100

100

Table 2 Encapsulation efficiency, production yield and particle size of prepared CP microsponge all batches [obtained values are mean±SD (N=3)]. Batch code

Drug : polymer ratio

% Encapsulation efficiency ± SD

% Production yield ± SD

Particle size (µm) ±SD

CPF1 CPF2 CPF3 CPF4 CPF5 CPF6 CPF7 CPF8

1:3 1:5 1:7 1:9 1:7 1:7 1:7 1:7

60.00±0.06 75.68±0.07 92.03±0.12 95.81±0.05 95.99±0.13 95.06±0.11 95.84±0.01 96.37±0.04

73.57±0.23 80.70±0.35 93.25±0.21 94.47±0.42 94.66±0.41 93.93±0.31 95.22±0.22 96.87±0.15

12.2±8.2 18.4±9.1 34.6±7.3 45.8±12.3 44.2±8.4 43.5±9.2 34.1±7.8 37.2±.9.2

Table 3 Effect of drug-polymer ratio of microsponge formulation [obtained values are mean±SD (N=3)] Batch Code Drug-polymer Production yield Encapsulation Particle size ratio (%)±SD efficiency (%)±SD (µm) ±SD CPF1 1:3 73.57±0.23 60.00±0.06 12.2±8.2 CPF2 1:5 80.70±0.35 75.68±0.07 18.4±9.1 CPF3 1:7 93.25±0.21 92.03±0.12 34.6±7.3 CPF4 1:9 94.47±0.42 95.81±0.05 45.8±12.3 Table 4 Effect of composition of internal phase (DCM) of microsponge formulation [obtained values are mean±SD (N=3)] Batches CPF3

Dichloromethane Concentration (ml) 5

Production yield (%) ±SD 93.25±0.21

Particle size (µm Encapsulation efficiency (%)±SD ±SD) 92.03±0.12 34.6±7.3

CPF5

10

95.99±0.13

94.66±0.41

44.2±8.4

CPF6

15

95.06±0.11

93.93±0.31

43.5±9.2

Table 5 Effect of composition of external phase (PVA) of microsponge formulation [obtained values are mean±SD (N=3)] Encapsulation Particle size (µm Batches PVA Amount (mg) Production Yield (%) ±SD efficiency (%) ±SD ±SD) 50 94.66±0.41 95.99±0.13 44.2±8.4 CPF5 34.1±7.8 75 95.22±0.22 95.84±0.01 CPF7 37.2±.9.2 100 96.87±0.15 96.37±0.04 CPF8

Table 6 In vitro release from prepared microsponge formulations (CPF1-CPF8). Batches code % CDR±SD 91.40±0.16 CPF1 89.15±0.09 CPF2 87.40±0.10 CPF3 63.40±.022 CPF4 79.33±0.14 CPF5 92.82±0.15 CPF6 61.05±0.08 CPF7 60.06±0.13 CPF8 Table 7 Release kinetics of CP microsponge formulations (CPF1 to CPF8). Kinetic Models Zero order First order Higuchi Korsemeyer peppas Best fit model

r2 value for microsponge formulations CPF1 CPF2 CPF3 CPF4 CPF5 CPF6 CPF7 0.964 0.970 0.982 0.978 0.823 0.896 0.969 0.851 0.879 0.861 0.955 0.654 0.773 0.883 0.990 0.982 0.990 0.947 0.914 0.946 0.990 0.984 0.988 0.987 0.987 0.889 0.940 0.978 Higuchi Peppas Peppas Zero Higuchi Higuchi Higuchi order

Table 8 Evaluation parameters of CP gel formulations. Formulation Plain CP Gel CP microsponge Gel

Viscosity (CPs) 8980-172060 9280-182000

pH ±SD

% CDR ±SD 6.9±0.02 98.66±1.08 7.3±0.03 66.06±0.98

Drug content (%)±SD 99.52±0.25 98.97±0.41

Spreadibility (g cm/s)±SD 11.18±0.39 13.52±0.27

CPF8 0.985 0.914 0.968 0.977 Zero order

6 . 0 4 . 0 2 . 0 0 . 0 6 . 0 -

) (

4 . 0 8 . 0 0 . 1 -

w o l F t a e H

2 . 0 -

g / W

2 . 1 4 . 1 -

CP CPMS

6 . 1 -

0

50

100

150

200

250

ο

Temperature ( C)

Figure 1 DSC thermograms of optimized microsponge formulation (CPMS) and pure CP (CP).

CP

%T

CPF8

%T

CP

CP+Eudragit RS 100

BMS

Eudragit RS 100 4000

4000

3500

3000

2500

2000

1500 -1

1000

500

0

3500

3000

2500

2000

1500

1000

500

-1

Wavenumber (cm )

Wavenumber (cm )

a

b

Figure 2 FTIR spectrum of (a) CP, blank microsponges (BMS) and optimized CP microsponge formulation (CPF8) (b) FTIR spectrum of CP, physical mixture (CP+Eudragit RS100) and Eudragit RS 100.

Figure 3 SEM images of selected microsponge formulation (CPF8).

120

120

a

b 100

80

F1

60

F2

40

F3

20

F4

0

% Cumulative dru release

% Cumulative drug release

100

80 F5

60

F6 F7

40

F8 20

0 0

2

4 Time (hrs)

6

8

10

0

2

4

6

8

10

Time (hrs)

Figure 4 In vitro release profile of formulations CPF1-CPF4 (4a) and CPF5-CPF8 (4b).

200000 180000

Viscosity (cps)

160000 140000 120000 100000 80000

CP gel

60000

CPMS gel

40000 20000 0 0

50

100

150

Rate of shear (1/sec)

Figure 5 Rheogram of CP gel and CPMS gel. 120 100 % CDR

80 60 G2

40

G1

20 0 0

2

4 6 Time (hrs)

8

10

Figure 6 In vitro release profile of plain CP gel (G1) & optimized microsponge (CPF8) gel (G2).

B 100

a

a

a

CP gel CPMS gel

CP gel

b Drug content (%)

a

CPMS gel

b

98

b

b

96

40

30

20

10

0

40

30

20

10

94 0

Drug content (%)

A 100 99 98 97 96 95 94 93 92 91 90

Time (days)

Time (minutes)

Figure 7. Photostability and stability analysis of the CP and CPMS gel. All data are shown as mean±SD; n= 3, Statistical data analysis from the two-way ANOVA followed by Bonferroni posttests for multiple comparisons. (Photo stability analysis (A) and stability analysis (B): a p < 0.001 versus CP gel with respect to their time period in photo stability analysis, b p < 0.001 versus CP gel with respect to their time period in stability analysis, CP: Clobetasol propionate gel (0.05%w/v), CPMS gel: Clobetasol propionate microsponge loaded gel (0.05%w/v).

90 80 70

%CDR

60 50 40

CPMS after 40 days

30

CPMS initially

20 10 0 0

2

4

6

8

10

Time (hrs)

Figure 8. Drug release profile of CP microsponge gel initially and after 40 days of stability study.

Figure 9. Histopathology of mouse tail with plain gel (a), CP gel (b) and CPF8 (c) gel group treatment. Calibration bar ═100 µm.

100

a

a,b

50

B 60

a

a,c

40 20

0

0

Plain gel Plain CP

CPMS

Plain gel

CP gel

CPMS gel

80

Drug activity (%)

80

A

Orthokeratotic activity (%)

Relative epidermal thickness (%)

150

C

60

a

a,d

40 20 0

Plain gel

CP gel

CPMS gel

Figure 10. In vivo evaluation parameters of the antipsoriatic potential of various groups. All data are shown as mean±SEM; n= 6 per group, Statistical data analysis from the one-way ANOVA followed by Tukey’s test for multiple comparisons. (Relative epidermal thickness (A), % Orthokeratosis (B), Drug activity (C): a p < 0.001 versus plain gel, b p < 0.001 versus CP gel, c p < 0.05 versus CP gel, d p < 0.01 versus CP gel, CP: Clobetasol propionate gel (0.05%w/v), CPMS gel: Clobetasol propionate microsponge loaded gel (0.05%w/v).

Highlights • • •

Common side effects of Clobetasol propionate (CP), limits its use for psoriasis. CP loaded microsponges minimized the adverse effects while controlling its release. MS is the potential carrier of CP for topical delivery in psoriasis management.