Formulation and Characterization of Ethosomal Formulation of Ciclopirox olamine

 

Kundlik Girhepunje, U.K Patil*

School of Pharmacy, Peoples University, Bhopal, India

 

ABSTRACT:

The goal of the current investigation is to evaluate the transdermal potential of novel vesicular carrier, ethosomes, bearing Ciclopirox olamine an antifungal having limited transdermal permeation. Ciclopirox olamine loaded ethosomes were prepared, optimized and characterized for vesicular shape and surface morphology, vesicular size, size distribution, vesicles skin interaction and stability. The ethosomal formulation (E9) having 3% phospholipids content and 45% ethanol showing the greatest entrapment (72.81±3.5%) and size range (152±11) was selected for further transdermal permeation studies. Stability study was performed for 120 days, which revealed low aggregation and growth in vesicular size (8.5±0.9%). The formulation retained its permeation power after storage.  Vesicle skin interaction study also showed that there was no interaction between the formulation and rat skin. Furthermore ethosomal delivery system could be considered for the treatment of number of dermal infections with better efficiency.

 

KEYWORDS: Candidiasis, Skin, Enhanced drug delivery, ethosomes, vesicle

 

INTRODUCTION:

Microbial skin infections are very wide spread in population of various ages and it can be classified by wound depth and pathogen species. The most common fungal infections are the Candidiasis, Aspergillosis, Dermatophytes, Pityriasis versicolor, Blastomycosis, and Protothecosis. Infections caused by fungi constitute a major public health problem in many parts of the world, both in developed and developing countries [1]. Microbial skin infections are very wide spread in population of various ages and it can be classified by wound depth and pathogen species. In most cases of dermal and subdermal, primary and secondary skin bacterial infections, the disease treatment by simple topical drug application is not sufficient, a deeper penetration of antifungal drug of choice during therapy is felt very much essential [2,3]. This is due to lack of ability of drug molecules as well as its conventional topical formulations to get self-permeated to deeper sections of the skin viz dermis and epidermis [4].

 

Ciclopirox olamine is the ethanolamine salt of ciclopirox, which is a 6-cyclohexyl-1-hydroxy-4-methyl-2(1H)-pyridone. It has a very broad spectrum of activity and inhibits nearly all clinically relevant dermatophytes, yeasts, and molds, including the frequently azole-resistant Candida species Candida glabrata, Candida krusei, and Candida guilliermondii

 

It acts against a wide range of bacteria including many gram-positive and gram-negative species pathogenic for humans [5,6].

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There are several advantages offered by transdermal route, but only few drug candidates are administered via this route due to formidable barrier nature of stratum corneum [7]. To overcome the constraint, a great number of vesicular approaches are under investigation and the major among them are elastic liposomes and ethosomes. Ethosomes, the high ethanol containing vesicles are able to penetrate the deeper layers of the skin and hence appear to be vesicles of choice for transdermal drug delivery. Ethosomes present interesting features correlated with ability to permeate through the human skin due to their high malleability. The physicochemical characteristics of ethosome allow this vesicular carrier to transport wide variety of active substances more efficiently across the skin barrier [8,11]. With the goal of developing a potential effective treatment for deep dermal and intracellular bacterial infections, ciclopirox ethosomal delivery systems were designed and characterized [12].

 

MATERIALS AND METHODS:

Ciclopirox olamine was purchased from Sigma Lab. New Delhi. Lipoid S PC-3, containing not less than 98% PC was a kind gift from Lipoid GmbH (Ludwigshafen, Germany).  Ethanol was purchased from Sigma Lab. New Delhi. All other chemicals were of analytical grade.

 

Preparation of Ciclopirox olamine loaded vesicles:

The ethosomes was prepared by Jain et al. and Touitou et al. [13]. The ethanolic vesicular system investigated here was composed of 2.0% w/w of Lipoid S PC-3 (PC), 35% w/w of ethanol, drug (Ciclopirox olamine, 0.77% w/w). Lipoid S PC-3 (PC) was dissolved along with drug in ethanol. Triple distilled water was added slowly in a fine stream with constant mixing at 700 rpm with a mechanical stirrer (Remi Equipment, Mumbai, India) in a house built closed container. Mixing was continued for additional 5 min. The system was maintained at 30 ± 10C during the preparation and then left to cool at room temperature for 30 min.

 

Vesicular shape and surface morphology:

Vesicular shape of the ethosome preparations were assessed by using Transmission Electron Microscope (TEM) (Philips CM12 Electron Microscope, Eindhoven, Netherlands. Samples were dried on carbon-coated grid and negatively stained with aqueous solution of phosphotungstic acid. After drying the specimen was viewed under the microscope at 10–100 k-fold enlargements at an accelerating voltage of 100 kV. Scanning electron microscopy (SEM) was also conducted to characterize the surface morphology of the ethosomal vesicle. One drop of ethosomal system was placed on clear glass stub, air dried and coated with Polaron E 5100 Sputter coater (Polaron, UK) and visualized under Scanning Electron Microscope (Leo-435 VP, Cambridge, UK).

 

Size distribution and vesicular size:

The size distribution of ethosomal preparation was measured in two sets of triplicates, in a multimodal mode, by Dynamic Light Scattering (DLS) technique using a computerized Malvern Autosizer 5002 inspection system (Malvern, UK). For vesicle size measurement, ethosomal preparation were mixed with the appropriate medium (PBS, pH 6.5) and the measurements were taken in triplicate.

 

Entrapment efficiency:

Ethosomal preparations were separated from the free (unentrapped) drug by a Sephadex G-50 minicolumn centrifugation technique [14,15]. The method was repeated at least three times with a fresh syringe packed with gel each time until the fraction collected was free from unentrapped drug. The vesicles were lysed by Triton X-100 (0.5% w/w) and entrapped drug were estimated using HPLC.

 

Vesicle skin interaction studies:

To observe the ultrastructural changes in the skin upon exposure to ethosomal formulation, the vesicles were applied on the skin of nude rats (albino rats 5–6 week old, 80–100 g). Preparations were applied topically to the skin for 6 hr., animals were sacrificed, skin were excised and stored in formalin solution (10%) in phosphate buffer saline (pH 7.4) followed by dehydration with alcohol. It was then treated with anti-media and embedded in paraffin wax for fixing. Controls skin section was prepared by similar procedure without application of any preparation. Sections of 5 μm thickness were cut from each piece and stained with heamotoxyline and eosin and histological changes in stratum corneum, epidermis and dermis were examined under optical microscope (Leica, DMLB, Heerbrugg, Switzerland)[16-17].

 

Storage-physical stability of ethosomes:

The ability of ethosomal preparations to retain the drug (i.e., drug-retentive behavior) was checked by keeping the preparations at different temperatures, i.e., 25 ± 2 (room temperature, RT), 37 ± 2 and 45 ± 20C for different periods of time (1, 20, 40, 60, 80 and 120 days). The ethosomal preparations were kept in sealed vials (10 ml capacity) after flushing with nitrogen. The stability of ethosomes was also determined quantitatively by monitoring size and morphology of the vesicles.

 

HPLC assay of ciclopirox olamine:

The quantifications of ciclopirox olamine was done by HPLC using methanol/acetonitrile/pH 5.4 buffer solution (9.0/6.8/85 v/v) solvent system. Flow rate was kept at 0.6 ml min−1, by LC 10-AT vp pump (Shimadzu, Japan). 20 μl of injection volume was eluted in LUNA 54, C18, 4.6Χ150 mm, column (Phenomonex, USA) at room temperature. The column eluant was monitored at 302 nm using SPD-M10A vp diode array UV detector (Shimadzu, Japan), ciclopirox olamine peaks were separated with a retention time of 9.8 min.

 

Statistical analysis:

Data were expressed as the mean ± standard deviation (SD) of the mean and statistical analysis was carried out employing the Student's t test using the software PRISM (Graph Pad). A value of Pb0.05 was considered statistically significant.

 

RESULTS AND DISCUSSION:

Ciclopirox olamine loaded ethosomes were prepared using varying concentration of Lipoid S PC-3 (PC) and ethanol, when examined by Transmission Electron Microscope (TEM) appeared as unilamellar vesicles with a predominant spherical shape (Fig. 1a). Surface morphology and three-dimensional nature of ethosomes were performed by further analysis of the preparation by Scanning Electron Microscopy (SEM), which confirmed the vesicular characteristics possessed by this novel carrier (Fig. 1b).

 

Fig 1a. Visualization of

Ethosomal Vesicles.

(Magnification 315 000)  

Fig 1b.Sem (Magnification (100 000) of  Ethosomal) Vesicles.

 

The two basic parameters on the basis of which the formulations were optimized are vesicular size and entrapment efficiency on increasing the phospholipids concentration. It was observed that the vesicular size was increased, though with increase in ethanol concentration the vesicular size decreased (Table 1). This indicates that at higher ethanol the membrane thickness is reduced considerably probably due to the formation of phase with interpenetrating hydrocarbon chain. 

 

In terms of entrapment efficiency, among all the ciclopirox olamine loaded ethosomal formulations, E9 (45% v/v ethanol, 3.0% w/v Lipoid S PC-3 and drug) showed the greatest entrapment efficiency, thus justifying itself as the optimized  formulation with greatest entrapment efficiency (72.8±3.5%) and optimum size (152±11 nm) thus showing the greatest  opportunity to the ciclopirox olamine loaded ethosomal preparation to attain a better skin penetration, by providing a safe homing to the ciclopirox olamine and optimized vesicular size which has been reported to affect the skin permeation parameters [17]. An optimum  polydispersity index (0.099±0.012) of ciclopirox olamine loaded ethosomal formulation could better justify the homogeneous nature of the prepared ethosomal formulation (Table 1).

 

Vesicle skin interaction studies was performed by skin histopathology on application of ethosomal system, it showed that there was no specific changes in the skin histopathology, though skin lipid fluidization could be observed in the form of some penetration pathway, which could be followed by these ethanolic vesicles, justifying their proposed mechanism of penetration into the skin and further fusing with the skin lipids in the epidermis and dermis.

 

Further, mild swelling of corneocytes could also be observed, which suggesting the retention of fluids and thus providing an insight on sustained drug delivery mechanism of ciclopirox olamine loaded ethosomal preparations shown in fig 2, 3 a and b.

 

Table 1. Composition and characterization of ethosomal formulations.

Formulation

Composition

Vesicle size a

PI b

%Entrapment Efficiency

Ethanol (%v/v)

Phospholipids (%w/v)

E1

25

1

143±8.5

0.049±0.003

35.2±1.7

E2 

25

2

199±9.2

0.052±0.004

41.1±2.5

E3

25

3

262±11.7

0.054±0.007

46.22±2.9

E4

35

1

133±8.0

0.046±0.005

43.1±1.8

E5

35

2

169±12.3

0.064±0.007

49.22±2.9

E6

35

3

194±13.1

0.083±0.008

59.75±3.0

E7

45

1

120±7.3

0.076±0.007

54.3±3.2

E8

45

2

141±10.2

0.096±0.01

63.8±3.1

E9

45

3

152±11.0

0.099±0.012

72.81±3.5

Values represent mean±SD (n=3)

 a Vesicle size just after preparation; b polydispersity index

 

 

Fig. 2 a) The application of the ethosomes on nude skin of rats

 

Fig. 2 b) Histopathology of the normal rat skin

 

Fig.2 c) Histopathology of the rat skin after application of the ethosome

 

Before the proposal of a ciclopirox olamine as a potential carrier for transdermal drug delivery system, an important characteristic to be evaluated is its in vivo skin tolerability/irritancy studies. As, skin non-irritancy of ciclopirox olamine loaded ethosomal formulation is well justified by Kanninkkanan et al. [19] after applying the preparation.

 

The stability study was performed on ciclopirox olamine loaded ethosomal formulations, which was evaluated for substantial loss of drug at various temperatures (refrigeration, room temperature, 37±2 °C and 45±2 °C) which suggested the storage of  ethosomal formulation at refrigerated temperature (4±2 °C), as at elevated temperatures greater drug loss was observed from the formulation (Fig. 3), that might be ascribed to the effect of temperature on the gel-to-liquid transition of lipid bilayers together with possible chemical degradation of the phospholipids, leading to defects in the membrane packing [20]. Still a small amount of drug was loaded out from the ethosomal system at refrigerated temperature (8.5±0.9%), a phenomenon possibly occurring due to hydrosoluble nature of ciclopirox olamine Shown in Table 2.

 

Values represents mean ± SD (n=3).

 

FIG 3. Extent of ciclopirox olamine leakage from ethosomal formulation at different temperatures during storage.

 

Vesicular size of ciclopirox olamine loaded ethosomal formulations were measured over the period of 120 days and result showed in Table 3.

 

Table 2. Drug-retentive behavior of ethosome (E9) at different time and temperatures

No. of days

Refrigeration 4±10C

Room temp. 25 ± 20C

37±2°C

45±2°C

1

0

0

0

0

20

4

8

10

11

40

6

11

13

15

60

7

13

18

21

80

8.1

17

22

25

100

8.4

18

25

29

120

8.5

20

26

31

Vesicular size measurements of ethosomes which stored at room temperature for various time periods showed only 9.2±0.9% size increase (in 120 days) which suggest a stabilizing effect of ethanol in the formulation, in terms of aggregation of vesicles by providing a net negative charge on the surface to the ethosomal formulations thus avoiding aggregation.

 

Table 3. Stability of ethosomes: vesicle size (nm).

Days (after preparation)                                Vesicular size (nm)                                                                 

                                                                       Ethosomes

1                                                              152±07

20                                                            154±11

40                                                            157±12

60                                                            158±09

80                                                            163±11

120                                                          165±08

 

Ethosomal formulation is reported to be non-irritant with the skin, which establishing the potential transdermal drug delivery. The enhanced transdermal efficacy obtained from the ethosomal system could be justified on the basis of function performed by ethanol present in the ethosomal formulations i.e. fluidizing both the vesicular lipid bilayers and Lipoid S PC-3, thus providing a greater malleability to the vesicles[18,21].

 

CONCLUSION:

From the study it was confirmed that ethosomal formulation of ciclopirox olamine showed a enhanced accumulation of ciclopirox olamine via ethosomal carrier within the skin might help to optimize targeting of this drug to the epidermal and dermal sites and better stability profile. Thus it concluded that ethosomes is a very promising carrier for transdermal delivery and creating a new opportunities for topical application of ciclopirox olamine in the fungal infections.

 

ACKNOWLEDGMENT:

The authors are grateful to Lipoid GmbH (Ludwigshafen, Germany) for providing the gift sample.

 

REFERENCES:

1.     http://www.dermnetnz.org/fungal/candida.html.

2.     M.L. Dell,  Skin and wound infections: an overview. Am. Fam. Phys. 1998; 57: 2424– 2432.

3.     G.L. Darmstadt, A.T. Lane, Impetigo: an overview. Pediatr. Dermatol. 1994; 11: 293– 303.

4.     B. Godin, E. Touitou, Mechanism of bacitracin permeation enhancement through the skin and cellular membranes from an ethosomal carrier. J. Controlled Release. 2004; 94: 365– 379.

5.     Martindale, The Complete Drug Reference. 2005; 34: 396.

6.     Markus Niewerth, Donika Kunze, Michael Seibold, Martin Schaller, Hans Christian Korting, and Bernhard Hube, Ciclopirox Olamine Treatment Affects the Expression Pattern of Candida albicans Genes Encoding Virulence Factors, Iron Metabolism Proteins, and Drug Resistance Factors,  Antimicrob Agents Chemother. 2003; 47(6): 1805–1817.

7.     V. Dubey, D. Mishra, T. Dutta, M. Nahar, D.K. Saraf, N.K. Jain, Dermal and transdermal delivery of an anti-psoriatic agent via ethanolic liposomes. J. Controlled Release.  2007; 123: 148–154.

8.     Donatella Paolinoa, Giuseppe Lucaniab, Domenico Mardentea, Franco Alhaiquec, Massimo Fresta, Ethosomes for skin delivery of ammonium glycyrrhizinate: In vitro percutaneous permeation through human skin and in vivo anti-inflammatory activity on human volunteers. J.  Controlled Release. 2005; 106: 99–110.

9.     M.A. Mustafa, Elsayed, O.Y. Abdallah, V.F. Naggar, N.M. Khalafallah, Deformable liposomes and ethosomes: Mechanism of enhanced skin delivery. Int. J. Pharmaceutics. 2006, 322, 60–66.

10.   B.W. Barry, Novel mechanisms and devices to enable successful transdermal drug delivery. Eur. J. Pharma. Sci. 2001; 14: 101–114.

11.   M.A. Mustafa, Elsayed, O.Y. Abdallah, V.F. Naggar, N.M. Khalafallah, Lipid vesicles for skin delivery of drugs: Reviewing three decades of research. Int. J. Pharmaceutics. 2007; 332: 1–16.

12.   B. Godin, E. Touitou, Mechanism of bacitracin permeation enhancement through the skin and cellular membranes from an ethosomal carrier. J. Controlled Release. 2004; 94: 365– 379.

13.   S. Jain, R.B. Umamaheshwari, D. Bhadra, N.K. Jain, Ethosomes: a novel vesicular carrier for enhanced transdermal delivery of an anti- HIV agent. Indian J. Pharm. Sci. 2004; 66(1):  72–81.

14.   D.W. Fry, J.C. White, I.D. Goldman, Rapid separation of low molecular weight solutes from liposomes without dilution. J. Anal. Biochem. 1978; 90: 809–815.

15.   E.N. Sorensen, G. Weisman, G.A. Vidaver, A Sephadex column for measuring uptake and loss of low molecular weight solutes from small vesicles. Anal. Biochem. 1977, 82, 376–384.

16.   Dubey Vaibhav, Mishra Dinesh, Dutta Tathagata, Nahar Manoj, Saraf DK, Jain NK. Dermal and transdermal delivery of an anti-psoriatic agent via ethanolic liposomes. J Control Release. 2007; 123: 148–154.

17.   D.D. Verma, S. Verma, G. Blume, A. Fahr, Particle size of liposomes influences dermal delivery of substances into skin. Int. J. Pharm. 2003; 258: 141–151.

18.   E. Touitou, N. Dayan, L. Bergelson, B. Godin, M. Eliaz, Ethosomes-novel vesicular carriers for enhanced delivery: characterization and skin penetration properties. J. Controlled Release. 2000; 65: 403–418.

19.   N. Kanninkkanan, T. Jackson, M.S. Shaik, M. Singh, Evaluation of skin sensitization potential of melatonin and nimesulide by murine local lymph node assay. Eur. J.  Pharm. Sci. 2001; 14: 217–220.

20.   V. Dubey, D. Mishra, N.K. Jain, Melatonin loaded ethanolic liposomes: physico-chemical characterization and enhanced transdermal delivery. Eur. J. Pharm. Biopharm. 2007; 67: 2, 398–405.

21.   B. Godin, E. Touitou, Erythromycin ethosomal systems: physicochemical characterization and enhanced antibacterial activity. Curr. Drug Deliv. 2005; 2: 269–275.

 

Received on 09.09.2012

Modified on 20.09.2012

Accepted on 29.09.2012        

© A&V Publication all right reserved

Research Journal of Pharmaceutical Dosage Forms and Technology. 4(5): September –October, 2012, 285-289