Indian Journal of Experimental Biology Vol. 43, March 2005, pp 233-240

Clobetasol propionate solid lipid nanoparticles cream for effective treatment of eczema: Formulation and clinical implications

Mayur Kalariya, Bijay Kumar Padhi , Mahavir Chougule & Ambikanandan Misra*

Pharmacy Department, Faculty of Technology & Engineering, TheM S Uni versity of Baroda, Vadodara, 390 001 , Indi a

Received 15 July 2004; revised 2 December 2004

In the present study clobetasol propionate (Cp) was loaded as solid lipid nanoparticles (SLN), incorporated it in suit­able cream base and evaluated in virro and its performance clinically against equi valent marketed formul ation. Cp was in­corporated into SLN by high-pressure homogeni zat ion technique and characteri zed for mean particle size, surface morphol­ogy and per cent drug entrapment. Drug permeation and sk in uptake studies from Cp creams were carried out in a validated Franz static diffusion cell across human cadaver skin (HCS). Si xteen chronic eczema patients were enro lled in a controlled double blind clinical trial. Optimized Cp-SLN was smooth and spherical under scanning electron microscopy; with average particle size of 177 nm and per cent drug entrapment of 92 .05%. In vitro permeat ion studies revealed lower mean flu x value and higher sk in uptake of Cp from Cp-SLN cream compared to marketed drug cream. Both formulation s were found to be responsive to manifes tations of chronic eczema, while Cp-SLN cream prepared in thi s in vestigation registered significant improvement in therapeutic response (1.9 fold ; inflammation , 1.2 fold; itching) in terms of per cent reduction in degree of inflammation and itching against marketed cream. Further clinical trial s are required to asce rtain the efficiency of the pres­ent formulati on.

Keywords: Clobetasol propionate, Solid lipid nanoparticles , High pressure homogenization technique, Mean flux , Chronic eczema.

IPC Code: Int Cl7 A6 1 P

Eczema is a reaction pattern in which primary lesions clinically manifest as pruritus, erythema, oedema, papules, vesicles, scaling, and lichenification 1. Ae­tiopathogenesis of eczema is not definite, but it may be the common final expression of allergic conditions such as atophic dermatitis, contact dermatitis or sebor­rhoeic dermatitis2. Cp is a potent topical glucocorti­coid; found valuable in the treatment of various der­matological di seases and for controlling exacerbations of pruritic eczema and psoriasis2

. Corticosteroids are thought to act by induction of phospholipase A2 in­hibitory proteins, collectively called lipocortins. It is postulated that these proteins control the biosynthesis of potent mediators of inflammation like prostaglan­dins and leukotrienes by inhibiting release of their common precursor, arachidonic acid, which leads to decreased extravasations of leukocytes to areas of in­jury, and-ultimately-decreased fibrosis3. Topical glu­cocorticoids has sufficient absorption through in­flamed skin to cause sys temic toxicity, including sup­pression of the hypothalamic-pituitary-adrenal axts

*Correspondent author- Phone: + 9 1-265-2434187; Fax:+ 91-265-2418927 E- mai 1-misraan@ hotmai I. com

and growth retardation, particularly in young chil­dren4·5. Topical drug delivery system, which provides higher skin retention of drug on target cells improves therapeutic response and reduces adverse effects of plain drug therapl-9

. Localization of Cp in effected layers of skin is likely to improve the role of topical dosage form of the drug as a supplementary to exist­ing therapy by prolonging the drug release with re­duced systemic uptake. One of the possibilities for increasing rate of healing of lesions is the incorpora­tion of Cp into solid lipid nanoparticles.

In recent years, solid lipid nanoparticles (SLN) have been used in topical drug formulations and are proven clinically as superi.or to pl ain drug topical therapy 10

. Small particle size ensures close contact to the stratum corneum and drug encapsulated in lipid improves selective drug delivery to skin layers1

1.12.

SLN possess a solid matrix, which has the potential to modulate the drug release over a prolonged period of time 13"14 with a reduced rate of systemic absorption. Other benefi t of SLN for topical delivery of active compounds is the time-to-market is very short fo r these products 1 1"15"16. Topical Cp-SLN formulation 17

has been diffusely reported in the literature and the

234 INDIAN J EXP BIOL, MARCH 2005

clinical performance of the formulation has not been studied. Hence, the aim of the present study was to develop Cp loaded SLN topical cream to increase skin uptake of the drug and provide prolonged effect to improve therapeutic index with reduced systemic ab­sorption rate and toxicity.

Materials and Methods Chemical-Ciobetasol propionate and Compritol

888 were generously gifted by M/s Paari Pharma Pvt. Ltd. , Ahmedabad, and Colorcon Asia Pvt. Ltd. , Mumbai respectively. Cetyl alcohol , stearic acid and Tween 80 were purchased from S .D. Fine Chern. Ltd ., Boisar. Poloxamer 188 and human cadaver skin were obtained from BASF, Frankfurt and S.S.G. Hospital, Vadodara respectively. All o ther chemicals were e i­ther of laboratory grade or analytical g rade.

Preparation of Cp-SLN- So!id lipid nanopartic les loaded with Cp were prepared using high-pressure ho mogenization technique as described by Muller et a l18•19 L .. d It d t b . I . . . 1p1 was me e o a ove Its me tmg po111t

(1 0°C) . Cp was di ssolved in the lipid melt by sonica­tion at 120 W for 15 sec using probe sonicator (V33 Vibronics Pvt. Ltd., Mumbai) and Tween 80 was added into the above mixture under continued stirrin a 1:>

until clear di spersion was obtained. Pluronic F68 (2% w/w) was dissolved in aqueous phase and heated to same temperature as that of lipid phase. Lipid phase was dispersed in aqueous phase under constant stir­ri ng fo llowed by sonication. This pre-mix was ho­mogenized for three cycles in a high-pressure homo­genizer (E mul siflex®-C5 , A VESTIN Inc., Ottawa, Canada) kept in a constant temperature bath main­

tained at 90°C. The hot di spersion was cooled quickl y

to 2°-4°C to fo rm Cp-SLN suspension. After additi on of protamine sul phate solution (10 mg/ml), the un­entrapped drug was separated by centrifugation at 15,000 rpm for 10 min using Sigma 3 K 30 refriger­ated laboratory centrifuge (S igma Laborcentrifugen, GmB H). Supernatant was separated and analyzed fo r drug content. Lip id of sediment was adjusted to 50

mg/ml with aqueous phase and stored at 4 °C in amber colour vial s until further use.

Taguchi orthogonal experimental des ign20 was used to optimize the combinatio n of four independent vari­ab les type of lipid, drug: li pid molar rati o, concentra­tion of surfactant, and homogenization pressure (psi) to achieve max imum POE and optimum parti c le size as shown in Table 1. It resulted into optimized com­bin ations of independent variables only in nine ex-

periments (Table 2). Each experiment was repeated thrice and optimized batch was repeated 6 times on 6 different days to ascertain reproducibility as shown in Table 3.

Characterization of Cp-SLN Particle size distribution-Particle size di stribution

of Cp-SLN was carried out by laser light diffracto­metry , using Malvern MasterSizer 2000SM Hydro (Malvern Instruments Inc., Worcestershire , UK) . Samples were prepared by dispersing Cp-SLN with sufficient amount of water to achieve obscuration between 10-20% and kept under stirring using a blade stirrer at 1000 rpm to keep Cp-SLN in suspended form and results a re recorded in Table 2 .

Surface morphology-Surface morphology of Cp­SLN was studied by scanning electron microscopy (JSM-56 10LV, JEOL, Japan) . The scanning electron microphotograph was taken using a double adhesive tape applied on the aluminium dies and Cp-SLN was spread uniformly on it (Fig. 1) .

Table !- Coded units ofTa "'a uchi orthooonal experimental des ion 4 0 b

L9 (3 ) fo r preparation of Cp-SLN

Group/1 ndependent Levels vari ables 2 3

A: Type of Lipid Comprito l Stearic Cetyl 888 acid alcohol

B: Drug : Lipid Molar Ratio I :3 1:5 1:7

C: Concentrati on of 3% 5%

Surfacta nt (Tween 80) 8%

D: Homogeni za ti on Pressure 5,000 8,000 10,000

( si)

Fig. !- Scanning electron microphotograph of Cp-SLN

KALARIYA et al.: CLOBETASOL PROPIONATE NANOPARTICLE CREAM x ECZEMA TREATMENT 235

Encapsulation efficiency-Drug loading and en­trapment efficiency of Cp-SLN was determined col­orimetrically using method as described earlier21

• Ali­quots of stock solution were transfened to volumetric flasks (10 ml) containing 2 ml of tetrazolium blue solution and 6.5 ml of 0.1 N tetrabutylammonium hy­

droxide reagent and heated to 80°C on a water bath for 15 min . The solution was cooled to room tem­perature and volume was made with glacial acetic acid and absorbance was measured at 513 nm against reagent blank. For estimation of Cp in Cp-SLN sus­pension, 0.1 ml of suspension was dissolved in 1 ml of methanol (chloroform in case of compritol 888 SLN) in a volumetric flask (25 ml) and volume (25 ml) was made up with methanol. The resulting solu­tion was suitably diluted and treated as mentioned

above and absorbance was measured at Amax 513 nm against reagent blank. Components of Cp-SLN were not found to be interfere with estimation of Cp at 513 nm at concentrations used in the formulation. Simi­larly, unentrapped drug in supernatant liquid was es­timated by transferri ng supernatant (0.5 ml) into a clean and dry 10 ml volumetric flask and volume was made up to 10 ml with methanol. The resulting solu­tion was further diluted if necessary and treated si mi­larly as menti oned above and measured absorbance at

Amax 513 nm against reagent blank.

Preparation and evaluation of cream-Ingredients listed under oil and aqueous phases (Table 4) were heated to 70°C in separate containers and oil phase gradually was added to aqueous phase under constant stin·ing. The stirring was continued till the mixture solidified and cream base was formed. Prepared cream base was allowed to stand over night. Cp-SLN cream was prepared by incorporating SLN pellets containing 0.05 g of drug by levigation method. Cp contents in cream formulations were estimated by dis­solving specific quantity (200 mg) of cream in metha­nol (10 ml) and analysed by HPLC system with a UV detector as per method described22 in USP 26-NF 21. The HPLC system consisted of a LC-1 0 Avp pump (Shimadzu), 20-1 loop injector (Reodyne), SPD-lOA UV spectrophotometric detector (Shimadzu) at 240 nm and a c-R6A recorder (Shimadzu) . The separation was carried out on a 4.6 mm x 25 em column con­taining packing LJ. The mobile phase was acetonitrile -0.05 M of monobasic sodium phosphate (adjusted with 85% phosphori c acid to a pH of 2.5) -methanol (95:85:20, v/v/v) and was used at flow rate of 1.0 ml min-1

• All form ul ations contained 90-110% (0.045-

0.055% w/w) of the labelled amount of Cp. Samples of clinical studies (CpCl, CpC2) were separately filled in 10 g lacquered aluminium collapsible tubes and sealed securely. The tubes were coded (A, B and C) randomly for clinical evaluation.

Drug permeation studies-Human cadaver skin (HCS) from elbow regions of the bodies of either sex (aged between 25-35 years), was obtained using Humby's knife from autopsy at S.S.G. Hospital (Va­dodara, India) with prior permission of hospital ethi­cal committee. The skin was washed thoroughly with water and subcutaneous fat was removed and stored at -4°C till further use. Full thickness HCS membrane was prepared by shaving the skin, punching out a disc

of approximately 2.5 cm2 area and slicing to 500 11m thickness using a Davis Dermatome 7 . These slices were hydrated with diffusion medium for 24 hr at room temperature ptior to use.

In vitro permeation studies for Cp-SLN and plain Cp creams were carried out across HCS using vali­dated Franz static diffusion cell22

'23 with an effective

diffusion area of 2.271 cm2. The receptor compart­

ment, with an effective volume of 20 ml , was filled with diffusion medium (mixture of PEG 400, DMSO and acetate buffer pH 5.0, 1 :0.5:8.5) and stined con­tinuously at 50 rpm with a Teflon-coated magnetic bar. The test system was equilibrated at 37°± 1 °C with a re-circulating water bath . Cream (0.1g) was spread over dermatomed and prepared HCS and set in such a way that the dermal surface just flushes to the surface of the diffusion medium and the donor surface re­mained unoccluded. Serial samplings from the dermal compartment were carried out at specified time inter­vals (l, 3, 6, 12, 18, 24 and 36 hr) and replaced with fresh medium. The samples were analyzed col­

orimetrically for the drug content at Amax 513 nm against the reagent blank. Each study was continued for period of 36 hr. The mean cumulative amount of drug permeated, Q (1-!g/cm2

) (n=3) and mean flu x val­ues, J (1-!g/min), were calculated for both creams. The results of permeation studies were recorded (Table 5) and shown graphically as Q vs. t (hr) and Q vs. e/2 (sec 112

) in Figs 3 and 4, respect ive ly. Each set of result represents the mean value of three experimental de­termi nations along with its standard error. At the com pletion of the study (i.e. 36 hr) , HCS was re­moved. The amount of drug remaining on the surface of the skin and the amo unt of drug retained in the ski n was determined by washing the surface, three times

236 INDIAN J EXP BIOL, MARCH 2005

with methanol (3 ml for each wash) . The combined washings were diluted suitably and Cp was estimated by measuring absorbance at Amux 513 nm against rea­gent blank. The residual methanol was carefully wiped off from the skin with a cotton swab, digested for overnight at 40°C with 10 ml methanol and Cp deposited in the skin was quantified as above. The procedure used for washing the skin surface was found to be effective in removing > 99% of the resid­ual drug and mass balance data from deposition stud­ies account for high recovery of the applied drug dose.

Clinical evaluation

Double blind clinical study was conducted on six­teen chronic eczema patients in Department of Der­matology and STD, Civil Hospital attached to B.J. Medical College, Ahmedabad, India, for a period of 6 weeks with prior permission of the hospital ethical committee. Written consent was obtained from the patients after explaining the objective and possible consequences of the studies. Out-door patients (5 male/11 female), ranging between 8 and 50 years of age with chronic eczema having not more than 15% body surface area involvement joined the study. Sub­jects were randomized into two parallel groups; the mean duration of the disease at entry was 3.2 years (range 1-5 years). The diagnosis of eczema was rein­forced by clinical presentation of the characteristic pruritus, ery thema, oedema, papules, ves icles, scaling, and lichenification. Before beginning the study, each

patient underwent a general physical examination and standard laboratory tests. All previous treatments were discontinued at least two weeks prior to studies. Each patient was given 10 g identical tubes (no visible differences between CpC 1 and CpC2) with instructions to clean the lesions thoroughly with luke-warm water and to apply twice daily without occlusion. The pro­gresses of treatment were evaluated on a weekly basi s by a group of phys icians in terms of average reduction in score for degree of inflammation and itching (Ta­ble 7). The test sites were scored weekly during ther­apy, for the degree of inflammation and itching on a scale of 0 to 4+ (0, clear; 1 +, mild; 2+, moderate; 3+, severe; and 4+, extreme) the findings were duly fill ed in the patient data-sheet. The results of the studies obtained wi th Cp-SLN cream was compared with those obtai ned with plain drug cream using analysis of variance (ANOV A); differences greater than P < 0.01 were considered significant.

Results and Discussion Nine batches of Cp-SLN were prepared by high

pressure homogenization technique using Taguchi orthogonal experimental design [L9(34

) ] varying four indepenclent variables (Table 1 ), type of lipid (A), drug: lipid molar ratio (B), concentration of surfactant (C) and homogenization pressure (D) at three levels. The particle size and PDE (dependent variables) of prepared batches were determined and recorded in Table 2. A substantial high drug entrapment (92.05 %) and optimal particle size (177 nm) of SLN (batch

Table 2- Experimental design and their corresponding results [Values are mean± SE of three determinations]

Batch Independent variables Dependent variables Cpb (%)

Particle size POE" A B c D (nm)

Cp1 557 (2.58) 61.28 (0.34) 35.12 (0.45)

Cp2 2 2 2 492 (3.32) 63.7 1 (0.84) 34.08 (0.54)

Cp3 I 3 3 3 431 (4.67) 64.93 (0.41) 32.22 (0.25)

Cp4 2 I 2 3 318 (3.59) 79.66 (0.18) 19.33 (0.37)

CpS 2 2 3 399 (5.49) 80.95 (0.67) 18.20 (0.54)

Cp6 2 3 2 343 (1.31) 82.04 (0.54) 12.76 (0.41)

Cp7 3 I 3 2 224 (4.45) 91.21 (0.86) 06.59 (0.59)

CpS 3 2 3 177 (2.33) 92.05 (0.62) 06.38 (0.48)

Cp9 3 3 2 297 (2.39) 92.89 (0.43) 05.94 (0.67)

A-D= Group/Independent variabl es; POE= per cent drug entrapment; a= per cent of added Cp actually entrapped into SLN; and b =per cent of Cp left unentrapped in supernatant liquid

KALARIYA et al.: CLOBETASOL PROPIONATE NANOPARTICLE CREAM x ECZEMA TREATMENT 237

Cp8) achieved at 3 level of A (cetyl alcohol), 2 level of B (1 :5), 1 level of C (3%) and 3 level of D (1 0,000 psi) . The results (dependable variables) of Taguchi orthogonal experimental design revealed that cetyl alcohol influenced PDE of Cp-SLN maximum, fol­lowed by stearic acid and compritol 888, respectively . This was consistent with the findings that chemical and physical structure of solid lipid matrix determines the loading effici ency of drug in SLN24

. The smaller particle size of cetyl alcohol SLN might be due to shorter fatty chain length and bearing of surface ac­tive properties25

'26

. PDE remain unaltered as drug: lipid molar ratio increases, due to high solubility of drug in melted lipids27

. Homogenization pressure was found to have significant impact on the particle size of Cp-SLN. Decrease in particle size during homogeni­zation was associated with pushing liquid with high pressure through a narrow gap, accelerating the fluid on a very short distance to very high velocity. Very high shear stress and cavitation forces disrupt the par­ticles down to the submicron range27

·28

. Reproducibil­ity of optimized batch was established by preparing six batches using the same composition at six differ­ent days. No significant differences (P <0.05) were observed within and among the batches with respect to their particle size and PDE (Table 3). Surface mor­phology of Cp-SLN was found to be smooth and spherical (Fig. 1) under scanning electron microscopy and no free drug crystals were visible in the scanning electron microphotograph of Cp-SLN. Hence, it was concluded that centrifugation was an efficient means of separating the unentrapped drug from dispersions of SLN.

Cumulative amount of drug diffused (Q) and mean flux value (J) across HCS were found to be lower for Cp-SLN cream than plain Cp marketed cream

Table 3-Reproducibility of optimized Cp-SLN (CpS) and their results

Particle Size (nm) PDE" Cpb (%)

179 91.87 6.31 176 92.14 6.07 177 92.22 6.84 174 92.07 7.02 177 91.94 6.96 175 92 .01 6.89

I 76 ± (0.328)c 92.04 ± (0.086) c 6.68 ± (0.053) c

a = Per cent of Cp actually entrapped into SLN; b =Per cent of Cp left unentrapped in supernatant liquid ; and c =Value are mean of six determinations

(Table 5) . This is in agreement with the earlier re­ports 10•

29-34 that entrapment of drug into SLN signifi­

cantly prolongs the drug permeation across HCS. Hi­guchi's diffusion controlled model 35 for release ki­netic behaviour of Cp creams suggests non-linear re­lationship between Q Vs t (Fig. 2). However, the

. ff' . f d Q 1/2 ( 1/2 p· 3) regressiOn coe ICient o ata vs t sec ; 1g. found to be 0 .992 and follows linear relationship, indicating first order release kinetics. Higher deposi­tion of Cp from CpC2 (79.41±0.46%) than CpCl (53.28±0.32%; Table 6) further supports encapsu­lation of Cp into SLN for enhancement of drug depo­sition into HCS 12

.3° and mechanism of formation of cetyl alcohol reservoir containing Cp in the skin layers36

.

'E (,)

& ~ (,)

E .._ 0

0.4

0.3

0.2

0.1

Table 4- Composition of Cp-SLN cream base

Ingredients

Oil phase

Hard paraffin wax

Microcrystalline wax

Heavy liquid paraffin

Cetostearyl alcohol

Sorbitan sesquieoleate

Propylparaben

Aqueous phase

Cetomacrogol 1000

Methy lparaben

Propylene glycol

Glycerine

Purified water

0 10 20

Time (hr)

%w/w

10.0

6.0

8.0

5.0

1.0

0.05

5.0

0.15

12.0

8.0

44.80

--cpc1 ---CpC2

30 40

Fig. 2- /n vitro release profile Q vs . time of Cp from cream

238 INDIAN J EXP BIOL, MARCH 2005

Our earlier investigation has shown that topical SLN formu lation is significantly more effective and reduces adverse effects of therapy when compared to plain drug topical formulations 10

. Clinical efficacy and to lerance of the trial preparations were ascer­tained by physical inspection and overall general re­sponse of patients at each schedu led weekly vis it. At each clinic visit, used tubes were replaced with fresh coded tubes. Patients who showed progressive remis­sion of lesions followed by d~creased inflammation and itching were considered effectively treated. It is evident from the results (Table 7) that manifestations of chronic eczema were responsive to both marketed plain Cp cream and Cp-SLN cream developed in this investigation. Significant improvement in therapeutic response (1.9 fold; inflammation, 1.2 fo ld; itching) in terms of per cent reduction in degree of inflammation and itchi ng was observed with Cp-SLN cream com­pared to marketed plain Cp cream. Moreover, SLN cream of Cp almost cures inflammation (92.5 % re­duction in degree of inflammation) within 6 weeks compared to 57.89% reduction in degree of inflam­mation with the plain Cp cream. Simi larly, the degree of itching drastically reduced at 4111 week (87 .18% reduction) and complete elimination was observed within 5111 week and onwards. Lower (95%) red uction in degree of itching was observed at the end of 6 weeks with plain Cp cream. The resu lts in terms of weighted means are also shown graphically in Figs 4 and 5. Cure of the skin diseases requires longer dura­tion of treatment especially in terminal segment of therapy and in many cases the lesions are not respon­sive beyond certain degree of improvement in skin con-

Table 5-Cumulative amount of Cp permeated (Q) at 37°C across HCS

[Values are mean± SE of three determinations]

Time (hr) Cumulative amount diffused (gg/cm2)

CpC I CpC2

0.016 ± 0.0008 0.004 ± 0.0002

3 0.045 ± 0.0012 0.017 ± 0.0009

6 0.087 ± 0.003) 0.050 ± 0.0011

12 0.168 ± 0.0084 0.106 ± 0 .0064

18 0.206 ± 0.0 I 07 0. 127 ± 0.0086

24 0.240 ± 0.0101 0.146 ± 0.0105

36 0.287 ± 0.0 136 0.169 ± 0.0095

Mean* flux ± SEM 0.041 ± 0.0017 0.0 19 ± 0 .0008 ( o/min)

Table 6- Amount of Cp deposited in to HCS after in vitro drug permeation study

[Values are mean± SE of three determi nations]

Test formulation Human skin(%) Washing medium

'[ 0.30 cT -a, u .§. IJI

~ 0.20 iii > 0

~ IJI IJI

~ 0.10 C) Cl> cr

CpCI

CpC2

0 100

(%)

53.28 ± 0.32 46.27 ± 0.88

79.4 1 ± 0.46 20.05 ± 0.6 1

200 300

t1/2 (sec 1'2 )

Fig. 3- Q vs. t 112 plot for release of Cp from cream

100

90

80 0 Q) 70 ~ g> § 60 '0 . .,

. ~ E so § E 't3 ~ 40

~ .s 30

~ 0

20

10

0

0

o CpC1

•CpC2

2 3 Time in weeks

4 5 6

400

Fig. 4- Per cen t reduction in degree of infl am mati on vs time plot after trea tment of eczema patient with CpC I and CpC2 for six weeks

120

0 100 -(l)

~ Ol

80 (l) - · u Ol c c --

£ c 0 .~ 60 "ti :J u 40 £:> ~ 0

20

0

KALARIYA et al. : CLOBETASOL PROPIONATE NANOPARTICLE CREAM x ECZEMA TREATMENT 239

oCpC1

• CpC2

0 2 3 4 5

Time in weeks

$ ~ f,r

i'&

~. ~ ~\ rg :'!'i ~ .'!i:' .;f! ~

~ Jf

6

ditions. Hence, localization of the drug in specific lay­er of the skin is desired for complete remission of the disease. Photographs of chronic eczema patient skin treated with Cp-SLN cream has been shown in Fig. 6.

The results of present investigation demonstrated localization and reservoir effect of Cp onto the skin accompanied by slower systemic dilution after incor­poration of Cp in hydrophobic carrier (SLN). It helped in complete remission of symptoms of chronic eczema and reduction in duration of therapy . How­ever, the role of Cp SLN cream developed in the pres­ent investigation still requires clinical evaluation in large number of human subjects at different locations .

Acknowledgment

Fig. 5-Per cent reducti on in degree of itching vs time plot after treatment of eczema patient with CpC I and CpC2 for six weeks

We are thankful to the Pharmacy Department, The M.S. University of Baroda, Vadodara, for providing

Pre-treatment Post-treatment

Fig. 6- Photograph of chronic eczema patient skin treated with CP-SLN cream for six weeks

Table 7-Average score and per cent reduction of degree of inflammation and itching of chronic eczema patients treated with Cp creams [Values are mean of eight patients]

Time Average score (±SE) for Percent reduction in Average score (±SE) for Per cent reduction (weeks) inflammation degree of inflammation itching in degree of itching

CpCI CpC2 CpCI CpC2 CpC I CpC2 CpC I CpC2

0 3.8 (0. 106) 4.0 (0.131) 0.00 0.00 4.0 (0.110) 3.9 (0.150) 0.00 0.00

3.5 (0. 1 05) 3.3 (0.099) 7.90 17.50 3.4 (0 082) 3.2 (0.064) 15.00 17.95

2 3.1 (0.092) 2.6 (0. 101 ) 18.42 35.00 2.7 (0.09 1) 2.3 (0.029) 32.50 41.02

3 2.8 (0.046) 1.9 (0.048) 26.3 1 52.50 1.9 (0.043) 1.4 (0.053) 52.50 64.10

4 2.4 (0.077) 0.9 (0.014) 36.84 77.50 1.1 (0.022) 0.5 (0.006) 72.50 87.18

5 1.9 (0.025) 0.7 (0.008) 50.00 82.50 0.8 (0.009) 0.0 (0.000) 80.00 100.00

6 1.6 (0.007) 0.3 (0.002) 57.89 92.50 0.2 (0.008) 0.0 (0.000) 95.00 100.00

240 INDIAN J EXP BIOL, MARCH 2005

research facilities and B.J. Medical College, Ahmed­abad for conducting clinical trials.

References I Halder B, Basic principles of diagnosis and treatment of ec­

zema, Indian J Dermatol, 30 (1985) 19. 2 Satoskar R S & Kale A K, Antiseptics, di sinfectants, insecti­

cides and pharmacotherapy of sk in di seases, Bhandarker's pharmacology and pharmacotherapeutics, 5'11 edition (Popu­lar Prakashan, Mumbai) 1997,809.

3 Cynthi a Guzzo, Gerald S Lazarus & Victoria P Werth, Der­matological pharmacology, in Goodman & Gilman's The pharmacological basis of therapeutics, !O'h edition, edited by Perry B Molinoff and Raynod W Rudan , (McGraw Hill , New York) 2001, 1667 & 1804.

4 Bondi E E & Kligman AM, Adverse effec ts of topical corti­costeroids, Prog Dermatol 14 ( 1980) I.

5 Wester R C & Maibach H I, Percutaneous absorption of topical corti costeroids, Ct,u.,- Probl Dennatol, 21(1993) 45.

6 Patel V B, Misra AN & Marfatia Y S, Topical liposomal gel of tretinoin for the treatment of acne: research and clinical implications, ?harm Dev Tech, 5 (2000) 455.

7 Patel V B, Misra A N & Marfatia Y S, Preparation and com­parative clinical eva luation of liposomal gel of benzoyl per­ox ide for acne, Drug Dev lnd ?harm, 27:8 (1999) 863.

8 Patel V B, Misra A N & Marfatia Y S, Clinical assessment of the combination therapy with liposomal gels of tretinoin and benzoyl peroxide in acne, AAPS ?harm Sci Tech 2:3 (2001) I.

9 Patel V B, Misra A N & Marfati a Y S, Topical dosage form of liposomal clofazimine: research and clini cal implications, Pharmazie, 54 ( 1999) 448.

10 Kalariya M, Padhi B K, Chougu le M & Misra Ambikanan­dan, Methotrexate loaded sol id lipid nanoparticles for topical treatment of psorias is: Formulation and clinical implications, Drug Del Tech, Oct (2004) (in press).

II Jenning V, Feste Lipid Nanoparrikel a is tragersystem for die dennale applikatio11. von reti11.ol , Ph.D. Thesis, Free Univer­sity of Berl in , ! 999.

12 Santos Maia C, Mehnert W & Schafer-Korting M, Solid lipid nanoparticles as drug carriers for topical glucocorticoids, lnt J ?harm, 196 (2000) 165.

13 Mull er R H & Dingler A, Feste Lipid-nanopartikel (Lipo­pear!s™) alsneuartiger carrier fur kosmeti sche und derma­tologische Wirkstoff, Pharmazeut Zeitung Dermopharma, (1998) 11.

14 Muller R H & Dingler A, The next generation after the lipo­somes: Solid lipid nanoparticles (SLN™, Lipopear! s™) as dermal carrier in cosmetics, Euro Cosmetics, 718 (1998)19.

15 Jeffes E W III, McCullough J L, Pittelkow M R, McCormick A, Almanzar J Liu, Dang G, Voss M, Voss K, Scholtzhauer J & Weinstein G D, Methotrexate therapy for psoriasis: differ­enti al sensiti vity of proliferating lymphoid and epithelial cells to the cytotoxic and growth-inhibitory effects of Methotrexate, J In vest Dermatol, !04 (1995)183.

16 Ball M A, McCullough J L & Weinstein G D, Percutaneous absorption of Methotrexate: effect on epidermal DNA syn­thesis in hairless mice, J Invest Dermatol, 79 ( 1982) 7.

17 Hu F Q , Yuan H, Zhang H H & Fang M, Preparation of solid lipid nanoparticles with clobetasol propionate by a novel

solvent diffusion method in a aqueous system and physico­chemical chai acterization, lnt J ?harm, 239 (2002) 121.

18 Muller R H & Lucks J S, Arzneistofftrager aus festen Lipid­teilchen, Feste Lipidnanospharen (SLN), European Patent 0605497, 13'11 July 1994.

19 Muller R H, Schwarz C, Mehnert W & Lucks J S, Production of solid lipid nanoparticles (SLN) for controlled drug deliv­ery, Control Release Bioact Marer (Proc Int Symp) 20 (1993) 480.

20 Taguchi G, Taguchi method: Design of experiment, quality engineering series Vol 4, edited by Kon ishi S (American Supplier Institute Inc, Dearborn) 1993.

21 P D Sethi , Quantitative analysis of pharmaceutical dosage forms, 3'd edition (CBS publi shers, New Delhi) 1997,542.

22 Franz T J, Percutaneous absorption on the relevance of in vitro data, J i11 vesr Dermatol, 67 (1975) 190.

23 Washington C, Drug release from micro di sperse system: a critical review, lnt J ?harm, 58 (1990) I.

24 Mull er R H, Mader K & Gohla S, Solid lipid nanoparticles (SLN) for controlled drug delivery - A review of the state of the art, Eur J ?harm Biopharm , 50 (2000) 161.

25 Siekmann B & Westensen K, Sub-micron sized parenteral carrier systems based on solid lipid , ?harm Pharmacal Lett, I ( 1992) 123.

26 Ahlin P & Kri st! J, Optimization of procedure parameters and physical stability of solid lipid nanoparticles in disper­sions, Acra ?harm, 48 (1998) 257.

27 Mehnert W & Mader K, Solid lipid nanoparticles production, characterization and applications, Adv Drug Del Rev, 47 (2001) 165 .

28 ZurMuhlen A, Fesre Lipid-Nanopartikel mit prolongierter Wirksrojjliberation: Herstellung, I~ngzeitstabilitat,

Charakterisierung, Freisetzungsverhalten und mechanismen, Ph.D. Thesis, Free University of Berlin , 1996.

29 Jenning V, Gysler A, Schafer-Korting M & Gohla S, Vita­min A loaded solid lipid nanoparticles for topical use: occlu­sive properties and drug penetration into porcine skin, Eur J ?harm Biopharm, 49 (2000) 211.

30 Jenning V, Schafer-Korting M & Gohla S, Vitamin A loaded solid lipid nanoparticles for topical application: drug release properties, J Conrrol Rel, 66:2-3 (2000) 115.

31 Dingler A, Feste Lipid-Nanopartikel als kolloidale Wirkstoffiragersysreme zur dermalen Applikation, Ph.D. Thesis, Free University of Berlin,l998.

32 Jenning V & Gohla S, Characterisation of solid lipid nano­partic les (SLN) based on binary mi xtures of liquid and solid lipids, lnt J ?harm, 205 (2000) 15.

33 Dingler A, Hildebrand G, Niehus H & Muller R H, Cosmetic anti-aging formul ation based on vitamin E-loaded solid lipid nanoparticles, Control Release Bioact Mater (Proc Int Symp) 25 ( 1998) 433.

34 Jenning V, Gohla S & Muller R H, Solid lipid nanoparticles (SLN): effect of homogeni zation parameters on drug stabil­ity, Proc 2'"1 World Meeting APGIIA PV, (1998) 619.

35 Higuchi T, Rate of release of medicaments from ointment bases containing drugs in suspension, J ?harm Sci, 50 (1961 ) 874.

36 Wade A & Walker P J, Hand book of pharmaceutical excipi­ents (Pharmaceutical Press, London) 1986, 63.