Formulation and Evaluation of Film Forming Solution of Tavaborole for Treatment of Skin Infections

 

Pravin D. Harak*, Amar G. Zalte, Vishal S. Gulecha

School of Pharmaceutical Sciences, Sandip University, Nashik.

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

 

ABSTRACT:

Film forming solutions (FFS) as a novel approach attracted the interest of pharmaceutical scientist for skin drug delivery system development and characterization concerning with their mechanical properties and water vapor permeability.  FFS were developed by varying concentration of the film forming polymers also nature and content of the plasticizer. The formulations were prepared using 32 full factorial design. They were tested for drying time, drug release, antifungal activity, skin irritation and stability studies. The FFS was characterised for pH, viscosity, drug content, effective dosage volume and mechanical properties of the film formed after application; bioadhesion and water vapour permeability were also tested. All the formulations showed results within acceptable range for various tests. The optimized formulation showed drug release of 97.2% and antifungal activity in terms of efficacy as 99.2%. Such a formulation can be claimed to decrease duration of therapy, will be more accepted by the patients and be a breakthrough in treating skin infections.

 

KEYWORDS: FFS, Filmforming solutions, Tavaborole, Eudragit, HPC

 

 

 


INTRODUCTION: 

Fungal diseases can be classified into 3 groups: the superficial, subcutaneous, and deep or systemic mycoses. Superficial infections are confined to skin, hair, nails or mucous membranes. The most common fungal skin infections are the dermatophytoses, pityriasis versicolor, and candidiasis. Approximately 90% of fungal skin infections are caused by ‘dermatophytes’, which are parasitic fungi affecting the skin, hair, nails. 1 One of the leading antifungal agents for topical treatment of fungal infections is terbinafine. It has been approved by the US Food and Drug Administration in cream, gel, solution and spray dosage forms.2

 

Tavaborole is a small, boron-based molecule capable of effectively penetrating the nail unit as described below. It is the first molecule in this class of drugs to achieve FDA approval in July 2014 as tavaborole (formerly AN2690). It is formulated for the treatment of toenail

 

onychomycosis as 5% topical solution. Tavaborole is a novel broad-spectrum antifungal pharmaceutical agent of the oxaborole class of boron-containing compounds. These antifungal compounds were designed from a previous class of borinic acid quinolone ester compounds having antibacterial properties and they were specifically selected based on their antifungal properties. The broad-spectrum antifungal activity of these compounds was optimized by the addition of a 5-fluoro group and their hydrophilicity which was optimized by the substitution of a 1-phenyl group for a 1-hydroxy group. 1-hydroxy-5-fluoro-1,3-dihydro-2,1 benzoxaborole is an optimal compound that has also been shown to maintain its activity in presence of keratin3 and also claim to have better in-vitro nail penetration than other oxaboroles at a 10% concentration in ethanol and ciclopirox 8% nail lacquer4.

 

Tavaborole has low molecular weight (approximately half of the most antifungal medications such as terbinafine and efinaconazole) which permits optimal nail plate penetration, higher to that of an existing topical antifungal medication5. The amount of 5% solution tavaborole penetrating an ex vivo cadaver fingernail plate was 250-fold higher than ciclopirox 8% solution in a head-to-head study with ciclopirox, for daily application after 14 days (524.7 µg/cm2 versus 13.0 µg/cm2 , respectively) 6, 7. In vitro nail penetration studies, it also shows that tavaborole is able to penetrate human nails better compared with ciclopirox 8% and amorolfine 5% nail lacquers. Tavaborole acts by inhibiting synthesis of protein in the fungus. It inhibits an enzyme known as cytosolic aminoacyl transfer RNA synthetase (AARS) with 1000 times’ greater selectivity than for mammalian AARS (Also known as leucyl-transfer RNA synthetase, or LeuRS) which is the enzyme that plays a key role in fungal essential protein synthesis. Termination of protein synthesis leads to inhibition of fungal cell growth which ultimately leads to death of fungus8,9. Tavaborole exhibits broad-spectrum activity against a variety of fungi, including non-dermatophytes, dermatophytes and yeasts. It was also tested for an in-vitro activity against a panel of different fungal strains including dermatophytes T. rubrum, T. mentagrophytes, Cryptococcus neoformans, C. albicans and Aspergillusfumigatus. Tavaborole exhibited an inhibitory activity to all of the fungal strains tested (1 mg/mL) 9.Tavaborole safety have been studied, results found no evidence of teratogenicity in pregnant rabbit and rabbits10. 5% tavaborole solutions have demonstrated a good safety profile across Phase II, Phase III studies, with most adverse events considered mild and unrelated to study drug. None of the serious adverse events reported were considered treatment-related11.The FDA in 2014 approved a 5% nail solution of tavaborole which is to be applied daily for the period of 48 weeks and should completely cover the entire surface of the toenail and underneath the toenail tip.

 

MATERIALS AND METHODS:

Materials

Tavaborole was received as gift sample from M/S Enaltec Pharma Research Pvt. Ltd. Ambernath Thane. Eudragit was received as gift sample from Evonik Degussa India Pvt. Ltd., Mumbai. HPC and TEC were purchased from SD Fine Chemicals, Mumbai. Wistar albino rabbit and were purchased from National Institute of Biosciences, Pune. All other chemicals were of analytical grade and were obtained commercially.

 

Methods

Preparation of FFS:

The polymeric solutions of Eudragit RS PO and Hydroxypropyl cellulose were prepared in ethanol using dispersion method. Eudragit S-100 was sprinkled over 10 mL of ethanol containing triethyl citrate (7.5 % w/w of S-100). Hydroxypropyl cellulose was sprinkled over 10 mL of ethanol separately. Both solutions were allowed to swell for 24 hours to produce clear solutions. The polymeric solutions were mixed properly with continuous stirring. Accurately weighed quantity (0.25g) of the Tavaborole was dissolved in 5 mL ethanol. The drug solution and polymeric dispersion were mixed properly with continuous stirring and volume was made up to the mark using ethanol. 12-16 32 factorial design was followed for the development of the formulations. In this design, 2 factors were evaluated each at 3 levels and experimental trials were performed at all 9 possible combinations as reflected in table 1.

 

Physicochemical evaluation17

Formulations were evaluated based on physicochemical properties of film formed upon casting the FFSs on a plane glass slide. Formulations were evaluated based on 5 parameters, appearance and viscosity of solution, drying time, outward stickiness and nature of formed film. Based on these physicochemical properties, suitable formulations were selected for further development.

 

Appearance and microscopy17

Both appearance and microscopy were evaluated based on visual examination. The drug loaded films, cast on plane slide were observed under electrical optical microscope (Labomed, Vision 2000) to detect possible crystallization of the drug, if any, on standing.

 

Viscosity17

Viscosities of the solutions were measured using Brookfield viscometer (Brookfield Ametek, BRK instruments) to determine the flow property of the solution. An ideal film forming solution should have low viscosity.

 

Drying time17

Formulations were cast onto a plane slide to determine drying time. After a set time span, another glass slide was mounted onto the film without pressure. Film is considered dry, if after removal there was no liquid visible on glass slide. Experiment was repeated with a rise in drying time if remains of liquid were visible on glass slide. A good FFS should have a minimum drying time to prevent long waiting for patients.


 

Table No. 1: Composition of formulations as per 32 factorial design

Batches

F1

F2

F3

F4

F5

F6

F7

F8

F9

Tavaborole (gm)

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

0.5

Eudragit S-100 (gm)

0.5

0.5

0.5

0.625

0.625

0.625

0.75

0.75

0.75

HPC  (gm)

2

5

8

2

5

8

2

5

8

Triethyl citrate (ml)

0.4

0.4

0.4

0.4

0.4

0.4

0.4

0.4

0.4

Methanol (ml)

10

10

10

10

10

10

10

10

10


Outward Stickiness17

The outward film stickiness was measured by pressing a fixed weight of cotton wool onto dry film with low pressure. Stickiness was graded ‘high’ when dense fibre accumulation was seen on film, ‘medium’ when thin fiber layer was noted on film and ‘low’ when fibre adherence was seen occasionally or not seen, depending on the amount of cotton fibres retained by film. The details of all the physicochemical properties evaluated are captured in Table .

 

In vitro release studies18

The in vitro diffusion studies were carried out in vertical Franz diffusion cells using dialysis membrane (12,000 MWCO) as a barrier. The membrane was sandwiched between donor and receiver chamber and firmly secured between the two chambers using a stainless steel clamp. A tiny magnetic bead is used in the receiver chamber to stir the PBS (pH 7.4) during the course of the experiment. About 0.5 mL of FFS solution was dispensed into the donor compartment and allowed to form a film. The receiver chamber was filled with buffer and the receiver was kept at 37.0 ± 0.5°C. About 1 mL of samples were withdrawn at predetermined time points from the receptor compartment and were replaced with fresh buffer equivalent to the volume withdrawn, before the samples were analyzed using UV spectrophotometer. To inspect the repeatability, studies were run in triplicate (n=3).

 

Antifungal Activity: 19

An agar diffusion method was used for the determination of antifungal activity of formulations. Standard Petri dishes (7.5 cm diameter) containing medium to a depth of 0.5 cm were used. The sterility of the lots was controlled before use. Inoculum was prepared by suspending 1-2 colonies of Candida albicans (NCIM no. 3102) from 24 hr. cultures in Sabouraud's medium into tubes containing 10 mL of sterile saline. The tubes were diluted with saline. The inoculum (0.5 mL) was spread over the surface of agar and the plates were dried at 35°C for 15 min prior to placing the formulation. Bores of 0.5 cm diameter were prepared and 20μl samples of formulation (1% w/v) were added in the bores. After incubation at 35°C for 2 days, the zone of inhibition around the bores was measured.

 

Skin irritation study:20-24

The protocol was approved by Institutional Animal Ethics Committee with approval no- “RP. No. 22/2122” approved by IAEC dated 21/August/2021. The rabbit (n=3) were randomly divided into 3 equal groups for application of standard irritant, optimized formulation or test and control (no application). Hairs were removed by hair removal cream (Anne French) from an area (2 cm2) on the dorsal side of the rabbit to make a hairless area. A 0.8% v/v aqueous solution of formalin was applied as a standard irritant to rabbit chosen randomly for standard irritant application (n=3) on the following day. The optimized formulation was applied to group 2 of rabbit (n = 3) for assessing any kind of irritation at specified sites. Formulation was removed after 24 h and skin was examined for any sign of erythema and oedema. The administration sites were assessed for signs of skin irritation, and this test procedure was repeated for another 6 days. The resulting reactions were compared against control group (n=3) and scored according to table no. 2.

 

Table No. 2: Score rating for skin irritation study

Sr. no.

Score

Rating

1

0

Nil

2

0-2

Mild

3

2-4

Moderate

4

4-6

Severe

5

6 and above

Very Severe

 

RESULTS AND DISCUSSION:

Appearance

All formulations were found to be clear on visual inspection.

 

Formulation pH

The pH of the formulations was found to be between 5.26  and 5.87. Ideally, the dermal gel should possess pH in the range of 5-6, so as to minimize discomfort or irritation due to acidic pH and microbial growth due to basic pH. Hence, the formulations displayed pH values within acceptable range.

 

Table No. 3: pH  of Formulation F1-F9

Sr. no.

Formulation code

Observed pH (±SD)

1.

F1

5.66±0.01

2.

F2

5.87±0.01

3.

F3

5.56±0.01

4.

F4

5.77±0.01

5.

F5

5.67±0.02

6.

F6

5.83±0.01

7.

F7

5.85±0.01

8.

F8

5.26±0.01

9.

F9

5.62±0.02

 

Viscosity

The viscosity profile of formulations F1 to F9 has been shown in Table 4 .


 

Table No. 4: Viscosity of Formulation  F1-F9

Rpm

Viscosity (cP) at Room Temperature

Formulation code

F1

F2

F3

F4

F5

F6

F7

F8

F9

0.3

2030.43

2563.67

3174.65

4664.54

5647.63

5763.14

5612.12

5875.74

7638.47

0.6

1573.74

2153.75

2965.13

4153.63

5587.74

5367.67

5634.74

5676.44

7167.78

1.5

1264.64

1834.85

2645.82

4067.83

4845.96

5165.94

5268.17

5136.53

6534.85

3

1035.59

1567.34

2134.84

3885.13

4246.18

5084.85

4914.74

5086.12

6258.10

 


Drying time

The drying time of formulations F1 to F9 has been shown in Table 5. Formulation batch F8 shows least drying time of 1 min 50 sec.

 

Table No. 5: Drying time of Formulation  F1-F9

Sr. no.

Formulation code

Drying time

1.

F1

2 min 10 sec ±5 sec

2.

F2

2 min 50 sec ± 5 sec

3.

F3

2 min 30 sec  ± 5 sec

4.

F4

2 min 10 sec ± 5 sec

5.

F5

2 min 10 sec ± 5 sec

6.

F6

2 min 20 sec ± 5 sec

7.

F7

2 min 30 sec ± 5 sec

8.

F8

1 min 50 sec ± 5 sec

9.

F9

2 min 30 sec ± 5 sec

 

Outward Stickiness

The outward stickiness of formulations F1 to F9 has been shown in Table 6. Formulation batch F1-F3 shows medium stickiness while formulation batch  F4-F9 shows low stickiness.

 

In-vitro release studies

The In-vitro drug release study of formulations is shown in table-7.

 

 

Of the nine formulations, maximum release was found to be for formulation F8 after 24 hours. 97.2% of the drug in the formulation was available for antifungal activity. The composite film had hydrophobic and hydrophilic portions which provide competition for drug release as both the polymers have different release properties. Therefore, as the polymer ratio varies, competition to release drug also varies. Formulation F8 showed steady state release up to 24 hours which also indicates that this formulation would show better contact with biological membrane.

 

Table No. 6: Outward Stickiness of Formulation  F1-F9

Sr. no.

Formulation code

Observation

1.

F1

Medium

2.

F2

Medium

3.

F3

Medium

4.

F4

Low

5.

F5

Low

6.

F6

Low

7.

F7

Low

8.

F8

Low

9.

F9

Low

 

Antifungal Activity

The results of antifungal activity of formulations have been shown in table-8.

 

 


Table No. 7: In- vitro release studies of Formulation  F1-F9

Time (hr.)               Cumulative Drug Release (%) (±S.D.)

            F1

F2

F3

F4

F5

F6

F7

F8

F9

0

0

0

0

0

0

0

0

0

0

  0.6

11.7 ±0.01

13.23±0.02

26.43±0.02

29.34±0.22

26.24±0.05

27.43±0.04

31.43±0.02

34.33 ±0.02

31.43±0.01

1

17.17±0.06

25.53±0.02

33.45±0.01

37.53±0.15

30.44±0.54

34.53± 0.53

35.43±0.02

43.22 ±0.02

38.33±0.01

2

26.47±0.02

34.63±0.02

41.52±0.02

45.42±0.92

46.54±0.35

41.24±0.53

41.42±0.02

51.42 ±0.01

43.35±0.03

3

31.09±0.01

41.68±0.03

44.55±0.03

51.42±0.42

56.35±0.55

45.54±0.54

46.22±0.04

55.33 ±0.01

51.74±0.04

4

42.88±0.04

50.26±0.02

57.65±0.04

54.53±0.43

65.35±0.53

54.73±0.05

50.83±0.01

60.4±0.02

54.61±0.05

5

48.17±0.01

56.75±0.04

63.24±0.05

65.63±0.25

74.55±0.43

61.24±0.22

55.33 ±0.02

64.33± 0.05

60.54±0.06

6

60.13±0.01

65.83±0.05

71.64±0.05

73.65±0.25

81.73±0.53

65.44±0.52

60.42±0.01

71.35±0.02

65.74±0.09

12

76.98±0.02

74.43±0.04

81.67±0.03

82.53±0.25

88.56±0.03

73.53±0.57

78.33±0.01

73.42±0.02

75.84±0.07

18

86.84±0.02

85.25±0.02

86.74±0.09

90.54±0.02

91.45±0.34

83.62±0.54

84.33±0.01

89.12±0.01

87.22±0.02

24

90.20±0.02

91.60±0.04

93.43±0.12

94.60±0.24

95.4±0.05

96.3±0.1

96.8±0.01

97.2±0.004

98.9±0.03

 


Table No. 8: Antifungal Activity  of Formulation  F1-F9

Sr. no.

Formulation

Code

Candida albicans

Zone of Inhibition (mm) ±SD

% Efficacy

1.

Standard value

24

100

2.

F1

21.74±0.2

90.6

3.

F2

22.12±0.2

92.2

4.

F3

22.60±0.3

94.2

5.

F4

22.75±0.3

94.8

6.

F5

22.87±0.3

95.3

7.

F6

23.47±0.3

97.8

8.

F7

23.68±0.3

98.7

9.

F8

23.80±0. 3

99.2

10.

F9

24±0.3

100

11.

Ethanol (control)

1.07±0.03

4.45

12.

Drug suspension

22.54±0.02

93.91

13.

Commercial cream

17.13±0.03

71.37

The standard value of Tavaborole against Candida albicans for zone of inhibition is 24 mm. The study indicates that Tavaborole retained its antifungal efficacy when formulated as a film forming dermal gel and drug was active against selected strain of micro-organism. F8 formulation showed a zone of inhibition of 23.80 mm and 99.2%.


Rat No.

Control group

Formulation group

Standard group

Erythema

Oedema

Erythema

Oedema

Erythema

Oedema

1.

0

0

0

0

0

0

2.

0

0

0

0

0

0

3.

0

0

0

0

0

0

Erythema scale: 0- none; 1-slight; 2- well defined; 3-moderate; and 4- scar formation

Oedema scale: 0- none; 1- slight; 2- well defined; 3- moderate; and 4- severe.

             

     A: Control group                                             B: Formulation Group                                              C. Standard Group

Figure no. 1: Photographs of skin irritation test

 

 


Skin irritation study

Skin irritation study on rabbit showed that after application of the optimized formulation there was no evidence of irritation (erythema and oedema). Hence, the optimized formulation F8 was found to be safe.

 

CONCLUSION:

FFS of Tavaborole were successfully prepared using hydroxypropyl cellulose, Eudragit S 100, Triethyl citrate as plasticizer and ethyl alcohol as solvent. The films formed were found to display low viscosity, low outward stickiness and short drying time. The films formed of formulation batch F8 display relatively better release compared to other batches. The concentrations of both the polymers were optimized by 32 full factorial design to obtain optimum drug release and antifungal activity. Thus, desirable goals could be achieved by systematic formulation approach. Antifungal study showed that developed film forming solution can reduce the fungal burden and thus, is more effective as compared to commercial product. The film forming solution prepared in this study fulfills all necessary parameters required for treatment of skin infections.

 

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Received on 21.01.2023             Modified on 19.02.2023

Accepted on 23.03.2023           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(3):1342-1346.

DOI: 10.52711/0974-360X.2023.00220