Development and Evaluation of Topical Drug Delivery System for Terbinafine Hydrochloride using Niosomes

 

P.S. Salve*

Department of Pharmaceutical Sciences, Rashtrasant Tukadoji Maharaj Nagpur University Campus,

Mahatma Fuley Shaikshanik Parisar, Amravati Road, Nagpur – 440 033 (MS)

*Corresponding Author E-mail: pramodsalve77@yahoo.com

 

ABSTRACT

Fungal infection caused by fungus called dermatophyte infects top layer of skin, hair or nails. An allylamine antifungal agent terbinafine hydrochloride is used topically and orally. Its topical administration is preferred but barrier properties of stratum corneum decreases absorption and requires frequent application. It has low oral bioavailability due to hepatic first pass metabolism and many systemic adverse effects. Niosomes have been reported to enhance  residence time of drugs in stratum corneum and epidermis, while reducing  systemic absorption and improve penetration of entrapped drug across skin.  Niosomes of terbinafine hydrochloride were prepared by film hydration method in size range of 0.24 to 9.4 μm. Maximum entrapment efficiency was observed in formulation containing span 60 at 1:1 molar ratio of cholesterol and surfactant.  Zeta potential values of niosomes containing span 60 (1:1) were more stable than other niosomal formulations. Niosomes were incorporated in 1.5 %w/v carbopol gel at pH 6.8-7.0. In in vitro antifungal study against candida albicans, vesicular systems were found to be more effective than conventional gel. The formulations containing tween 60 (1.5:1) and span 80 (1:1) were found to have maximum zone of inhibition. In ex vivo percutaneous permeation studies, niosomal formulations have shown superior skin penetration and drug deposition as compared to conventional formulation. The formulation containing tween 80 has shown higher drug deposition in rat skin as compared to other formulations. The niosomal vesicles can be used to enhance penetration and deposition of terbinafine hydrochloride in skin.

 

 


INTRODUCTION:

A fungal infection is caused by type of fungus called dermatophyte that infects top layer of skin, hair or nails. Fungal infection of skin is called ring worm (Tinia). Types of ring worm are body ring worm (Tinia corporis), jock itch(tinia cruris), Athlete’s foot (Tinia pedis), scalp ring worm (Tinia capitis), nail ring worm (Tinia unguium)35,36.

 

The mainstay of management of skin and nail fungal infection has been oral and topical antifungal agents. Terbinafine hydrochloride is an allylamine antifungal used topically and orally. Although its topical administration is most preferred route for treatment but barrier properties of  stratum corneum decreases absorption and requires frequent application. Also it has low oral bioavailability due to hepatic first pass metabolism and many systemic adverse effects.

 

Niosomes have been reported to enhance residence time of drugs in stratum corneum and epidermis, while reducing systemic absorption and improve penetration of entrapped drug across skin. Niosomes are microscopic lamellar structure of cholesterol and nonionic surfactants such as alkyl ether, alkyl ester or alkyl amide. The vesicle has an infrastructure consisting of hydrophilic, amphiphilic and lipophilic moieties together and as a result can accommodate drug molecules with a wide range of solubility.

 

The vesicles prepared from cholesterol and polyoxyethylene alkyl ether surfactants were studied with isolated human stratum corneum incubated for 48 hours and for vesicle skin interactions. The fusion of liquid as well as gel state vesicles on the superficial layer of stratum corneum takes place, but liquid state vesicles induced perturbations in liquid organization, so water pool formation within stratum corneum was observed.16 The stacks of lamellae and irregular structures were formed on skin with fusion and adsorption of vesicles onto stratum corneum surface. These structures and interactions strongly depend on vesicle composition and physiological properties.17The available data suggests that the tested surfactant damaged the epidermis membranes.18 Surfactant causes modification of physicochemical characteristics of natural membrane and can also disrupt artificial membranes. The nonionic surfactants have ability to increase permeability of sarcoplasmic reticulum. This phenomenon has been frequently exploited to extract and solubilize sparingly soluble proteins such as membraneproteins.15Vesicles are prepared by film hydration method, ether injection method, sonication, reverse phase evaporation, aqueous dispersion, microfluidization, multiple membrane extraction method, transmembrane pH gradient drug uptake process, the bubble method and formation of vesicles from provesicles11. Ether injection method and sonication method used for water soluble drug which give unilamellar vesicles hence film hydration method is used for lipid soluble drugs which provides  multilamellar vesicles.

 

Various factor affecting niosomal physical chemistry as drug, amount and type of surfactant, cholesterol content and charge, method of preparation, temperature and general characteristics of nonionic surfactants. Vesicles are characterized by vesicle size, its shape and morphology, entrapment efficiency, in-vitro drug release and vesicle surface charge. The niosomal dispersion in an aqueous phase can be emulsified in a non-aqueous phase to regulate the delivery rate of drug and administer normal vesicle in external non-aqueous phase.

 

Terbinafine hydrochloride has more than 70% absorption on oral administration and around 47% of absorbed drug is metabolized by first pass metabolism. Its topical absorption is less than 5% from intact skin.

 

Hence, it was envisaged to develop topical delivery of terbinafine Hydrochloride in treatment of fungal infection.

 

MATERIALS AND METHODS:

Materials

Terbinafine hydrochloride was obtained as a gratis sample from Dr. Reddy’s Laboratories (India), cholesterol, isopropyl alcohol were obtained from SRL Chemicals (India), citric acid, tri-sodium citrate, sodium hydroxide were obtained from Loba Chemicals, (India), Tween 60, Tween 80 were obtained from Sisco Chemicals, (India). Span 60, span 80, potassium dihydrogen orthophosphate, chloroform, methanol were obtained from SD.Fine Chemicals, (India). The dialysis membrane was obtained from HiMedia Laboratories, (India) and membrane filter was obtained from Pall Corporation, (India)

 

Method:

Pre-formulation studies were performed by organoleptic evaluation, spectroscopical analysis performed by UV visible spectroscopy at 400 to200 nm and FT-IR spectroscopy.

 

Dialysis of drug

The amount of terbinafine hydrochloride equal to 20 mg was solubilized in 2 mL of pH 3 citrate buffer and was placed in dialysis bag (Hi Media Laboratories) to dialyze against 400 mL of pH 3 citrate buffer and magnetically stirred at 37±0.5oC. From it, 5 mL sample was withdrawn at regular time intervals. The drug content was estimated at 283 nm against pH 3 citrate buffer. Dialysis was performed for 5 hours to separate un-entrapped drug.

 

Preparation of terbinafine hydrochloride loaded noisome by film hydration method

Weighed quantities of surfactants (tween-60, tween-80, span-60 or span-80) and cholesterol in different molar ratios, 1:0, 1:1 and 1:1.5 (table 1), were dissolved in 10 mL of chloroform: methanol mixture (2:1, v/v) along with terbinafine hydrochloride in round bottom flask. The organic solvent were removed under vacuum in a rotary evaporator at 50oC for 1 hour to form a thin film on wall of flask and kept under vacuum for 2 hours to ensure total removal of trace solvents. Hydration of surfactant film was carried out using 10 mL pH 7.4 phosphate buffer at 60ºC for one hour which is above the gel–liquid transition temperature (Tc) of sorbitan monoesters and polyoxyethylene alkyl ether surfactants. The resulting suspension was mechanically shaken for 1 hour using the horizontal mechanical shaking water bath. The dispersion was left for 4 hours at room temperature for complete hydration and stored at 4ºC overnight before use, and it was sonicated in 3 cycles of 1 min “on” and 1 min “off” leading to formation of multilamellar niosomes.6

 


Table 1  Composition of niosomes

Compositions

Terbinafine HCl (mg)

Tween 60 (mL)

Tween 80 

(mL)

Span 60 (mg)

Span 80 (mL)

Molar quantity of cholesterol (mg)

Batches

 

 

 

 

 

0.5

1

1.5

F1

50

1.312

---

---

---

193.8

---

---

F2

50

1.312

---

---

---

---

386.68

---

F3

50

1.312

----

---

---

---

---

579.9

F4

50

---

1.3

---

---

193.8

---

---

F5

50

---

1.3

---

---

---

386.68

---

F6

50

---

1.3

---

---

---

---

579.9

F7

50

---

---

430.0

---

193.8

---

---

F8

50

---

---

430.0

---

---

386.68

---

F9

50

---

---

430.0

---

---

---

579.9

F10

50

---

---

---

0.429

193.8

---

---

F11

50

---

---

---

0.429

---

386.68

---

F12

50

---

---

---

0.429

---

---

579.9


Characterization

Niosomes were characterized for microscopic characteristics, entrapment efficiency, vesicle size and zeta potential.

 

Microscopic examination51

Niosomal suspension was examined for structure and lamellarity under motic plus 2.0 microscope at magnification power of 10X and 45X and photomicrographs were recorded.

 

Entrapment efficiency

Niosomes encapsulation efficiency was determined by dialysis technique for separating un-entrapped drug. 52 Quantity of drug loaded niosomal dispersion equal to 2 mL was placed into dialysis bag and exhaustively dialyzed against 200 mL of pH 3 citrate buffer, magnetically stirred at 150 rpm and 37±0.5ºC for 5 hours. Vesicles were disrupted with 4 mL of isopropyl alcohol and diluted to 10 mL with pH 3 citrate buffer and sonicated for 15 min. Quantity of resultant dispersion equal to 1 mL was diluted up to 25 mL with pH 3 citrate buffer and drug content was estimated by the UV spectroscopy.40Amount of terbinafine hydrochloride entrapped in vesicles was determined using following equation.                                                               

 

EE (%) = [(Ct-Cf)/Ct] 100

 

Where, Ct is concentration of total terbinafine hydrochloride, Cf is concentration of free terbinafine hydrochloride

 

Measurement of vesicle size53

The mean particle size diameter and size distribution (polydispersity index, PI) was determined by Malvern zetasizer nano. Each sample was run 3 times and analysis was carried out at 25ºC with an angle of detection 173°.

 

Measurement of zeta potential53

The zeta potential was determined by Malvern zetasizer nano after suitable dilutions. Each sample was run 3 times and the analysis was carried out at 25ºC with an angle of detection 173°.

 

Stability studies56

The physical stability studies were carried out to investigate the leaching of drug from niosomes during storage. The niosomal formulations were sealed in 20 mL glass vials. The stability studies of vesicular dispersions were carried out at refrigerator temperature (4-8ºC) and at room temperature for 45 days. The effects of temperature on the % EE were monitored at 0, 15, 30, 45 and 60 days for vesicular dispersion.

 

Preparation of gels

Vesicular gel

Aqueous dispersion of 1.5 %w/w carbopol-934P was prepared and niosomal suspension was incorporated with gentle shaking to prevent breakage of vesicles. To it, 0.2g and 0.02 g methyl paraben and propyl paraben respectively were incorporated and pH was adjusted to 6.8 to 7 by addition of triethnolamine.

 

Conventional gel

Conventional gel was prepared by dispersing micronised drug equal to 100 mg in aqueous dispersion of carbopol-934P.

              

Evaluation of vesicular gel

The appearance, drug content, pH, viscosity and spreadability of vesicular gel were determined.

 

Study of in vitro antifungal activity

Sabourd dextrose agar (SDA) media was used for the cultivation of pathogenic fungi. In it candida albicans was inoculated and incubated at 25ºC for 3 days.

 

The in vitro antifungal activity of niosomal gel and conventional gel were carried out against candida albicans using cup plate method. The antifungal assay agar was used for the assay of antifungal activity. The quantity of fungal culture equal to 1 mL was inoculated to antifungal assay agar media and uniformly poured into a sterile petridish. The agar medium was left to solidify to and 5 holes were made by cork-borer having diameter of 1 cm and agar disk was removed. The quantity of gel formulations equal to 30 mg were sampled in cup. It was allowed to diffuse at room temperature for 1 hour. The plates were placed in incubator at 25ºC. The diameter of growth inhibition zones were measured after every 24 hours for 3 days.

 

Ex vivo percutaneous permeation and skin uptake behavior

Ex vivo permeation studies were carried out using the abdominal skin of Sprague Dawley rat mounted on static vertical Franz diffusion cells with dermal-side in contact with receptor phase. The diffusion cell with an effective diffusion area of 3.14 cm2 and diffusion medium pH 7.4 phosphate buffer.The test system was equilibrated at 37ºC. Terbinafine hydrochloride accumulation in skin was assessed after 24 hours treatment with vesicular gels (tween 60, tween 80, span 60 and span 80), and conventional gel.

 

The skin content of drug was determined using methanol by sonication which was analyzed by UV visible spectroscopy at 282 nm.

 

RESULTS:

Preformulation studies

Fourier transform Infrared (FT-IR) spectroscopy

 The FT-IR spectrum of terbinafine hydrochloride is shown in figure 1.

 

Figure 1 FT-IR spectrum of terbinafine hydrochloride in methanol

 

Drug-Excipients interaction

FT-IR spectras of terbinafine hydrochloride in combination with excipients (figure 2,3,4 and 5) were compared with FT-IR spectrum of the terbinafine hydrochloride (figure 1).

 

Figure 2 FT-IR spectras of terbinafine hydrochloride, cholesterol and tween 60

 

Figure 3 FT-IRspectras of terbinafine hydrochloride, cholesterol and span 60

 

 Figure 4 FT-IR spectras of terbinafine hydrochloride, cholesterol and span 80

Figure 5 FT-IR spectras of terbinafine hydrochloride, cholesterol and tween 80

 

Cumulative drug dialyzed

The cumulative % amount of dialysed terbinafine hydrochloride is shown in the table 2.

 

Table 2 Cumulative % drug dialyzed with respect to time

Time (Hours)

Cumulative % drug released  (Mean± SD)

0.5

34.777 ± 0.052

1

57.542 ± 0.223

2

63.851 ± 0.524

3

76.111 ± 0.263

4

81.147 ± 0.994

5

86.296 ± 0.313

6

82.962 ± 0.209

7

81.517 ± 0.156

SD-Standard deviation

 

From the table 2 it can be observed that more than 85% drug was dialyzed at the end of 5 hours.

 

Formulation development

Preparation of terbinafine hydrochloride loaded niosomes

Vesicle forming ability of surfactant

It has been reported that niosomes prepared without cholesterol forms a gel and only on addition of cholesterol, homogenous niosomal dispersion obtained.27 Thus, in this study, cholesterol was added at 0.5, 1 and 1.5 molar ratios, and amount of surfactant and drug was kept constant. As shown in table I, surfactants were able to form vesicles at cholesterol ratios studied.

 

 Characterization of vesicles

Microscopy

The morphological characteristics of terbinafine hydrochloride loaded niosomes are shown in figures 6, 7, 8, 9, 10 and 11.

  

Figure 6 Photomicrograph of niosomes containing tween at 45X magnification 

 

 Figure 7 Photomicrograph of niosomes containing tween at 10X magnification

     

 Figure 8 Photomicrograph of niosomes containing span at 45X magnification .

 

Figure 9 Photomicragraph of niosomes containing span at 10X magnification.

 

Figure 10 Photomicrograph of niosomes containing span at 45X magnification

 

Figure 11 Photomicrograph of niosomes containing span at 45X magnification

 

 

As observed from photomicrographs at various magnifications (figure 7, 8, 9, 10, 11 and 12) terbinafine hydrochloride loaded niosomal vesicles were found to be well identified perfect spherical structures in  size range of 0.24 to 9.4 μm and multilamellar in nature formulated by classic thin film hydration method and no aggregation of vesicles was observed.

 

Entrapment efficiency

 Effect of cholesterol on niosomal formulation

The effect of cholesterol on terbinafine hydrochloride entrapment was found to vary according to nonionic surfactant used and cholesterol was found to have an insignificant effect on drug entrapment into sorbitan esters (span) niosomes (figure 12). For tweens, entrapment of drug was found to increase with increase of cholesterol ratio from 0 to 1.5. As HLB value of surfactant was increased above 10, minimum amount of cholesterol necessary to form vesicles was increased.13  

              

Figure 12 Effect of cholesterol molar ratio on the entrapment efficiency of terbinafine      hydrochloride into niosomes (n = 3)

 

In formulations prepared with sorbitan monoesters, span 60 showed maximum entrapment efficiency at 1 molar ratio of cholesterol.

 

Vesicle size

The mean vesicle diameters along with polydispersity index of the optimized formulations are shown in table 3.

 

All vesicles formed were in mean area-number diameters (dAN) ranging from 0.24 μm to 9.4 μm. While comparing formulations that had shown maximum entrapment efficiencies, larger size was observed of those vesicles that had surfactants with a saturated alkyl chain. As observed from table III, in case of vesicles containing surfactants with saturated alkyl chain,  size of niosomes containing span 60 (1754 nm) were larger than tween 60 containing niosomes (1332 nm) and for vesicles containing surfactants with unsaturated alkyl chain, niosomes containing span 80 (807.23 nm) were larger in size than those containing tween 80 (756.2 nm).

 

Table 3  Particle diameter (nm) and polydispersity index (PDI) of optimized formulations (n=3)

 

SD- Standard deviation; PDI- Polydispersity index

                

Zeta potential

As shown in table 4, the formulations containing the sorbitan esters, zeta potential value of formulation containing span 60 (HLB 4.7) was found to be higher than, span 80 (HLB 4.3).Similarly zeta potential value of the formulation containing tween 80 (HLB 15) was found to be higher than formulation containing tween 60 (HLB 14.9).

 

Table 4  Zeta potential of optimized formulations (n=3)

SD-Standard deviation

 

Stability study

A direct relationship between percentage drug leaching out of vesicles and ageing was observed. With increase in storage period, the degree of leaching was also increased

 

Figure 13 Effect of storage time and temperature on the entrapment efficiency of vesicle

 

From figure 13, it was observed that span 60 and tween 80 niosomal suspensions has shown higher stability in terms of entrapment efficiency over other niosomal suspensions. It was observed that niosomal suspension containing tween 60 showed low stability. Vesicular dispersions were found to be more stable at low temperature and should be stored at low temperature.

 

In-vitro antifungal activity

The results of in vitro antifungal activity are shown in figure 15 and table 5.

   

Figure 14 Zone of inhibition of terbinafine hydrochloride containing niosomal gels and conventional gel against candida albicans (n=3)

Table 5 In vitro antifungal activity of terbinafine hydrochloride containing     niosomal gels and conventional gel against candida albicans (n=3).

 

Terbinafine hydrochloride was found to be active against Candida albicans. The vesicular formulations of terbinafine hydrochloride were found to be more effective than conventional formulation, as they were found to have larger zone of inhibition. The vesicular formulations containing tween 60 and span 80 were found to have maximum zone of inhibition followed by span 60 and tween 80.

 

Ex vivo percutaneous permeation and skin uptake behaviors

From figure 15, better ex vivo permeation and skin partitioning of drug by niosomal gel was observed as compared to conventional gel.

 

Figure 15.Ex vivo permeation studies of niosomal formulations

 

The superior skin permeation was observed with formulations containing tween 80 and span 80 as compared to formulations containing tween 60 and span 60. From figure16, it was clearly indicated that drug deposition in excised rat skin from vesicular gels were greater than conventional gels.

 Figure 16 Drug Deposition in rat skin from different formulations, (Mean±SD) (n=3).

 

DISCUSSION:

Drug excipients interaction

From FT-IR spectrum of physical mixture of drug and excipients, excipients were found to be compatible with terbinafine hydrochloride.

 

Characterization of vesicles

Entrapment efficiency

Effect of cholesterol on niosomal formulation

The effect of cholesterol on terbinafine hydrochloride entrapment was found to be varying according to nonionic surfactant used. Cholesterol was found to have insignificant effect on terbinafine hydrochloride entrapment into sorbitan esters (span) niosomes (figure 13). For tweens, entrapment of terbinafine hydrochloride was found to increase with increase of cholesterol ratio from 0 to 1.5. As HLB value of surfactant was increased above 10, minimum amount of cholesterol necessary to form vesicles was increased.13 More amount of cholesterol was necessary to compensate for larger head group. In present study, tweens have highest HLB value indicating low hydrocarbon chain volume in comparison with hydrophilic surface area. Thus, increased cholesterol content might have increased the lipophilic behavior and crystallinity of lipid bilayer of niosomes containing tweens.13 .Hence, higher drug entrapment of niosomes containing tween was observed in presence of higher content of cholesterol. In niosomal formulations prepared with sorbitan monoesters, span 60 showed maximum entrapment efficiency at 1 molar ratio of cholesterol. Increasing cholesterol content from 0 to 1 molar ratio leads to an increase in entrapment efficiency of sorbitan ester niosomes. The improvements in drug entrapment with increased cholesterol content (0–1) and major reduction in drug entrapment when the cholesterol content was further increased (1–1.5) may be due to two conflicting factors: (1) with increased cholesterol, bilayer hydrophobicity and stability was found to increase135 and permeability was decreased136 which may lead to efficiently trapping of hydrophobic drug into bilayers as the vesicles were formed. (2) In contrast, the higher amounts of cholesterol may compete with drug for packing space within the bilayer, hence excluding the drug as amphiphiles assemble into vesicles.28, 30

 

Span 60 was found to have significantly higher entrapment efficiency than span 80. This could be due to surfactant chemical structure. All spans have same head group and different alkyl chain. Increasing alkyl chain length leads to higher entrapment efficiency.41 The span 60 and span 80 have same head groups but span 80 have an unsaturated alkyl chain, 137 demonstrated that  introduction of a double bonds into the paraffin chains caused a marked enhancement in permeability of niosomes, possibly explaining lower entrapment efficiency of span 80 formulation. In addition, span 80 have lowest transition temperature (Tc =−12ºC) amongst all spans.138 Span 60 having highest phase transition temperature provided highest entrapment of drug and vice versa.27

 

Vesicle size

It had been widely known that diameter of vesicles depends on length of alkyl chain of surfactants. Surfactants with longer alkyl chains generally gave the larger vesicles.13

 

The size distribution was observed from polydispersity index shown in table III, a polydispersity index of 1 indicates large variations in particle size and a value of 0 means size variation is absent. It indicates that all niosomal formulations were multidispersed. While comparing formulations that had shown the maximum entrapment efficiencies, polydispersity index was found to be 1 for niosomal formulations containing span 60 and tween 80 surfactants and niosomal formulations containing tween 60 (PI 0.994) was found to be more polydispersed as compared to span 80 (PI 0.744). Among all formulations, span 80 (1.5:1) showed least degree of variation in particle size (PI 0.203).

 

Zeta potential

Zeta potential of niosomal formulations containing span was increased with increase in hydrophilicity of surfactants. This could be due increased surface free energy of span surfactants with increased HLB value.32 Similar kind of results were observed in case of polyoxyethylene sorbitan esters, zeta potential value of formulation containing tween 80 (HLB 15) was found to be higher than that of formulation containing tween 60 (HLB 14.9).

 

Evaluation of antifungal gel

Ex vivo percutaneous permeation and skin uptake behaviors

Surfactant in formulations acts as a permeation enhancer, which might partly contribute to enhancement of terbinafine hydrochloride permeation from niosomes.1140 The niosomes fused at interface of stratum cornium, and high local drug concentration in bilayers generates a high thermodynamic activity of drug in upper part of the stratum cornium.141, 142

 

 

The superior skin permeation was observed with formulations containing tween 80 and span 80 as compared to formulations containing tween 60 and span 60. This could be due to better release of drugs from tween 80 and span 80 niosomes due to low phase transition temperature of these surfactants and smaller size of niosomes.27 The packing nature of unsaturated fatty acids changed the fluidity of the stratum corneum lipid structure and facilitated skin permeation of drug.143 Moreover, it was reported that sorbitan ester niosomes exhibit an alkyl chain length dependent drug release; the higher the chain length, the lower the release rate.106, 100

 

CONCLUSION:

Niosomes entrapped with terbinafine hydrochloride were prepared by film hydration method for topical skin fungal infection. The niosomal gel was found to improve the antifungal activity as compared to conventional gels. This study provided the evidence that niosomal vesicles are valuable as topical delivery carrier to enhance the penetration and deposition of terbinafine hydrochloride in skin.

 

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Received on 20.08.2011                    Accepted on 02.10.2011        

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Res. J. Topical and Cosmetic Sci. 2(2): July – Dec. 2011 page 52-63