Formulation Characterization Optimization of Duloxetine loaded Ethosome Patch for Transdermal Application

 

A. Eswar Kumar¹, Ch. Niranjan Patra²

¹Department of Pharmaceutics, University College of Pharmaceutical Sciences, Palamuru University, Mahabubnagar, 509001, Telangana, India.

²Professor, Department of Pharmaceutics, Roland Institute of Pharmaceutical Sciences, Orissa, India.

 

ABSTRACT:

The present investigation deals with the development of duloxetine ethosome then loading into transdermal patch to impart lower drug side effect, enhanced bioavailability, avoid first pass metabolismt.3² factorial design was applied to optimize the formulation. Materials and Methods, Ethanol (X1) and Phospholipid (X2) were taken as independent variable, Responses are vesicle size (Y2), entrapment efficiency (Y1). Optimum desirability was identified and, an optimized formulation was prepared, characterized and loaded into transdermal films. Ex-vivo permeation study for the prepared films was conducted and, the permeation parameters and drug permeation mechanism were identified. Results The percent of alcohol was significantly affecting all the studied responses while the other factors and their interaction effects were varied on their effects on each response. The optimized ethosomes formulation showed observed values for Y2, Y1 a of 161nm and 98.79% respectively. Ex-vivo permeation of films loaded with optimized ethosomal formulation was superior to that of the corresponding pure drug transdermal films.

 

 

INTRODUCTION:

Flexible vesicular carriers are one of the approach to enhance skin permeability of essential drugs, through the stratum corneum barrier. Liposomes, transfersomes and ethosomes are three common types of these flexible particles although niosomes, non-ionic surfactant based vesicles, possess similar properties and uses¹ Conventional liposomes are the least among them in their transdermal application since they are confined to the upper layer of the skin stratum corneum and therefore, are topical delivery. Ethosomes are lipid based vesicular particles containing ethanol in a relatively high concentration and water. These particles have been emerged due to the unique characteristic features and advantages they possess.

 

High deformability, high drug entrapment efficiency, and enhanced transdermal permeation rate in the drug- delivery system make them suitable for transdermal administration of different therapeutic agents^2,3. Deeper penetration and enhanced permeation to the skin layers have been reported to be attributed to the presence of phospholipid and high concentration of ethanol^4,5. Ethosomes have showed extensively higher drug transdermal flux in comparison to liposomes and 24-fold higher in vitro skin permeation when compared with aqueous drug solution⁶. Duloxetine inhibits the reuptake of serotonin and norepinephrine (NE) in the central nervous system.The analgesic properties of duloxetine in the treatment of diabetic neuropathy and central pain syndromes such as fibromyalgia are believed to be due to sodium ion channel blockade

 

The aim of this study was to develop duloxetine transdermal films loaded with an optimized drug ethosomes formulation to enhance the skin drug permeability, decrease dosing frequency, extend the drug release, and avoid drug per-oral side effects.

 

Fig:1 Structure of Duloxetine

 

MATERIAL AND METHODS:

Materials:

Duloxetine was a kind gift from Dr.Reddy Labrotories (Hyderabad) Soya lecithin was kindly supplied by Research lab Fine chem (Mumbai), Phosphate buffer, Ethanol and Propylene glycol were procured from MSN lab (Hyderabad). Dimethyl sulfoxide (DMSO) from Techno Pharmchem (Bahadurgarh, India). Hydroxypropyl methyl cellulose (HPMC) MW 86,000, viscosity 4000 cp (2% solution) from Acros Organics (New Jersey, USA).

 

Drug–excipient compatibility study:

Drug-excipient compatibility was carried out by FTIR spectroscopy. Individual sample of PC-90G, RXL, physical mixture of PC-90G and RXL and the final optimised formulation of ethosomes in a freeze-dried form were scanned for spectral observations. A total no. of sixteen scans within a scanning range of 400 cm−1 to 4000 cm−1 were performed .

 

Design of Experiment:

3² full factorial design:

Based on our preliminary investigation and information from the literature two independent formulation parameter are selected A 3² factorial design was applied^11,12. According to this design, 9 runs were conducted in total The independent variables selected for this study were X1, Ethanol; X2, Phospholipid. The dependent variables were Y1, Entrapment Efficiency (Y1); and Vesicle size (Y2). The levels of independent and dependent variables were listed in Table 1

 

 

Preparation of the formulations:

Table 2 illustrates the composition of the Duloxetine ethosomal formulations proposed by the 3² full factorial design. Preparation of the formulations was achieved according to the method previously published^5,6. except for slight modification. Briefly, the calculated amount of cholesterol, phospholipids (X2), and drug (1%) were dissolved in a mixture of the specified amount of ethanol (X1) and propylene glycol in a covered glass bottle vessel at room temperature by vigorous stirring. The mixture was heated to 30⁰C in water bath. Distilled water, quantity sufficient to prepare 10ml formulation, was added slowly at a constant rate of 1ml/min in a fine stream with a constant mixing using a magnetic stirrer at 1500rpm in a closed vessel. The temperature was maintained at 30⁰C during the experiment. Mixing was continued for additional 5 min while the temperature was kept at 30⁰C. The preparation was stored at 4⁰C. Ethosome prepared by the above procedure were subjected to sonication at 4⁰C using probe sonication in 3 cycles of 5 min each with 5 min rest between each cycle.

 

Characterization of the formulations:

Vesicle size and zeta potential. Dynamic light scattering technique was utilized to measure the particle size and zeta potential of the prepared vesicles using Zetatrac (Microtrac Inc., York, PA). An aliquot from each formulation was taken, loaded into the sample chamber and measured. Measurement was done in triplicate for each formulation

 

Entrapment efficiency:

An aliquots from each formulation were placed in eppendorf tubes (n = 3) and subjected to centrifugation at 16000rpm for 30 min at 4⁰C using (Sigma Laboratory centrifuge, 3K30, Ostrode, Germany). The supernatant was separated, filtered and the amount of free drug was determined using UV method at 289nm.

 

Table 1: Duloxetine ethosomal formulation independent variables level and studied responses

X1: Volume of alcohol, X2: amount of phospholipid, Y1: entrapment efficiency,Y2: vesicle size

 

Table 2: Formulation Table for Duloxetine Ethosome

Ingredients	F1	F2	F3	F4	F5	F6	F7	F8	F9
Duloxetine	30	30	30	30	30	30	30	30	30
Ethanol	3	5	7	3	5	7	3	5	7
Phospholipid	10	10	10	20	20	20	30	30	30
PEG	0.2	0.2	0.2	0.2	0.2	0.2	0.2	0.2	0.2
Ethanol: Chloroform(5:5)	10	10	10	10	10	10	10	10	10

 

Ethosome morphological study:

Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to investigate the morphology of the optimized ethosomal formulation. For SEM, the formulation was lyophilized using (alpha 1-2 LD plus, Christ lyophilizer, Germany). The freeze dried sample was sputter coated with gold then was investigated using scanning electron microscope (Philips XL30, Eindhoven, Netherlands). For TEM, few drops of the prepared formulation was mounted on a carbon coated grid, left for about 3 min to permit better adsorption of the vesicles on the carbon film, then a filter paper was used to remove the excess liquid. A drop of phosphotungstic acid of 1% concentration was added and the sample was examined under TEM (Model JEM-1230, JOEL, Tokyo, Japan). The optimized ethosomal drug formulation was also examined under an optical microscope (Olympus System Microscope Model CX41, Tokyo, Japan). Images were taken under 100 and 400_ magnification power to illustrate the structure of the prepared vesicles^7,8.

 

Preparation of duloxtine ethosomal based transdermal films:

In order to prepare a medicated transdermal films, known weight of HPMC was added to a specified volume of the optimized drug ethosomal formulation to prepare 1% polymeric solution. DMSO and propylene glycol, in a concentration of 2% each, were added as a skin permeation enhancer and plasticizer, respectively. The dispersion was thoroughly mixed on a magnetic stirrer at 1000rpm then was left for 24 h in the refrigerator at 5⁰C in order to obtain a clear solution. The prepared medicated polymeric solution was poured into a glass petri dish covered with a silicone-coated liner (Scotchpak TM, 3M, St Paul, USA) and kept in an oven at 40 _C until complete evaporation of the solvent. The dried semi-solid formulation was cut into films of 1.76cm2. The prepared films were finally stored in a tightly closed container maintained at room temperature. For comparative study, pure drug films, containing pure duloxetine instead of the drug ethosomal formulation, were prepared using the same concentrations of DMSO and propylene glycol in 1% HPMC solution using distilled water as a solvent utilizing the same procedure.^9,10

 

In vitro release studies:

Transdermal patches can be in vitro evaluated by Franz diffusion cell the cell using synthetic membrane. The Franz diffusion cell composed of two compartments: donor and receptor. The receptor compartment has a volume of 5-12ml and effective surface area of 1-5 cm². The diffusion buffer is continuously stirred at 600rpm by a magnetic bar. At appropriate intervals (1, 2, 3, 4, 5, 6, 7, 8, 12, 14, 24 hr), 1ml of sample was withdrawn from receptor medium and immediately equal volume of fresh receptor solution was added to maintain sink condition of receptor medium. The temperature in the bulk of the solution is maintained by circulating thermos stated water through a water jacket that surrounds the receptor compartment.^11-12 The drug content is analyzed using suitable UV method, at 289nm .

 

Ex-vivo Permeability Study:

Franz diffusion cell was used for skin permeation study with an effective diffusion area of 2.668 cm2. The experiment was carried out using freshly killed goat dorsal skin acquired from the local slaughter house and stored at 18°C in the ringer solution. Skin was first flushed with ringer solution at room temperature for 2 hrs with aeration. Then further fat content was removed from subcutaneous tissue of skin by using hand razor. The moisten skin was further wiped with distilled water. A circular piece of skin (about 3.2cm diameters) was sandwiched between donor and receptor compartment of the vertical diffusion chamber and 30 mg drug loaded Transdermal patch was added to the mucosal side^13,14. The receptor compartment was filled with phosphate buffer pH 7.4, kept at constant temperature of 37±05ºC and stirred using magnetic stirrer. At appropriate intervals (1, 2, 3, 4, 5, 6, 7, 8 to12 hr), 1 ml of sample was withdrawn from receptor medium and immediately equal volume of fresh receptor solution was added to maintain sink condition of receptor medium. Periodically samples were collected and analyzed by UV spectrophotometer (Shimadzu-1800, Japan) at 282nm using phosphate buffer (pH 6.8) as blank. Each experiment was performed in triplicate. The cumulative amount of duloxetine permeated per unit area from the transdermal system through the skin into the receptor compartment medium was plotted as a function of time, and the slope of the linear portion of the plot was estimated as steady state flux^15-16.

 

RESULTS AND DISCUSSION:

The concept behind the improved transdermal flux of ethosomes relies on the concentration of ethanol and its penetration enhancing ability by disrupting the stratum corneum.

 

Effect on the vesicle size:

From the results of the statistical analysis illustrated in Table 3, it was concluded that; the main effect of X1 and X2, the interaction effect of X1X2, and the quadratic effect of (X1) were significantly affecting the vesicle size (Y1) of the prepared ethosomal formulations¹⁷. All of which showed a positive, synergistic, effect on Vesicle size, Size ranging from. 161nm to 1062nm

 

Entrapment efficiency:

It was noticed that the main effect of X1 and X2, and the interaction effect of X1X2 were significantly affecting the entrapment efficiency of the prepared drug ethosomal formulations^18,19. All these variables had a synergistic, positive, effect on EE%,it ranges from 98.79% to 94.55%.

 

Table 3: Composition of the Duloxetine ethosomal formulations and observed values of the studied responses

Run	Independent variable X1 (ml) X 2(mg)		Responses Y1(%) Y2(nm)
1	3	10	98.56	862
2	5	10	98.79	161
3	7	10	98.69	1062
4	3	20	94.55	673
5	5	20	97.95	891
6	7	20	98.01	1013
7	3	30	98.04	786
8	5	30	97.76	972
9	7	30	97.77	321

 

Figure2: a. Duloxetine ethosome b. SEM image of Duloxetine ethosome

 

 

Figure 3: FTIR of pure Duloxetine

 

Table 4: Physical characterization of transdermal film containing Duloxetine

Formulation	Thickness(mm)	Folding endurance	Drug content(%)	Moisture uptake (%)	Moisture content (%)
P1	0.4569±0.10	206	95±0.11	7.98±0.12	3.77±0.21
P2	0.4510±0.22	210	92±0.21	25.05±0.31	9.2±0.24
P3	0.3470±0.31	281	97±0.31	13.09±0.34	5.16±0.31
P4	0.4496±0.42	296	90±0.44	15.63±0.41	5.66±0.34
P5	0.4460±0.52	201	97±0.32	11.73±0.49	4.87±0.44
P6	0.4517±0.51	324	92±0.54	19.65±0.51	12.67±0.53
P7	0.4478±0.62	210	100±0.61	9.42±0.62	3.43±0.65
P8	0.4437±0.71	227	93±0.69	10.87±0.71	4.72±0.77
P9	0.4503±0.79	207	95±0.74	16.44±0.81	6.62±0.79

 

Physical evaluation of Transdermal formulation:

Total nine patches were developed and evaluated in triplicate, the drug content for all the formulation are more than 90%, Folding endurance for all formulation ranges between 201 to 296 ,thickness of all formulation ranges from 0.3470mm to 0.4569mm

 

In-vitro release Results:

In-vitro drug release is excellent in patches containing ethosome PE3 showing drug release 94.7% in 24 hr ,PP6 is showing 64.7% release in 24hr.

 

 

Table 5: Cumulative Drug release % at 24 hr from transdermal patch

Formulation	%Cumilative Drug Release at 24 hr SD±Mean n=3
PE1	84.2± 7.3
PE2	85.8± 1.2
PE3	94.7± 7.8
PE4	89.4± 6.3
PE5	75.4± 4.3
PP6	64.7± 6.3
PP7	81.9± 7.3
PP8	78.7±1.3
PP9	79.1±2.3

Table 6: Permeation flux(µg/cm²/hr) at 24 hr from transdermal patch

Formulation	Permeation flux (µg/cm2 /hr)
PE1	79.12± 0.3
PE2	82.24± 1.3
PE3	86.34± 2.3
PE4	72.44± 0.3
PE5	29.14± 1.3
PP6	62.33± 2.6
PP7	52.34± 1.9
PP8	22.14± 1.6
PP9	39.54± 2.6

 

 

 

Figure 4: Transdermal formulation containing Duloxetine ethosome

 

 

Ex vivo permeation studies through goat abdominal skin:

The results of ex-vivo drug permeation studies of ethosomal formulations (PE1, PE2, PE3, PE4, PE5) were compare with patches containing only pure drug²⁰. The results indicated that drug penetration was increased with ethosome incorporation and the percent drug permeated from PE3 was found to be up to 86.34% and in PP8 it was found to be 22.14%.

 

CONCLUSION:

The optimization design has been successfully implemented to prepare an optimized ethosomal formulation characterized by enhanced drug percutaneous absorption. Moreover, transdermal films loaded with ethosomal drug formulation showed extended drug release profile Finally, this drug delivery system could avoid the hepatic first pass metabolism and considered an alternative administration route for acid sensitive drugs

 

REFERENCES:

1.     Tarek A. Ahmed, Khalid M. El-Saya,b, Bader M. Aljaeida, Usama A. Fahmya, Fathy I. Abd-Allah Transdermal duloxetine delivery system based on optimized ethosomal nano-vesicles: Preparation, characterization, in vitro,ex vivo and clinical evaluation, Pharmaceutical Nanotechnology. International Journal of Pharmaceutics. 2016, (500) 245-254

2.     Ah, Y.-C., Choi, J.-K., Choi, Y.-K., Ki, H.-M., Bae, J.-H.et al . A novel transdermal patch incorporating meloxicam: in vitro and in vivo characterization. Int. J. Pharm. 385 (1-2), 12–19 https://doi.org/10.1016/j.ijpharm.2009.10.013

3.     Dubey, V., Mishra, D., Dutta, T., Nahar, M., Saraf, D.K., Jain, N.K, Dermal and transdermal delivery of an anti-psoriatic agent via ethanolic liposomes. J. Control. Release. 2007https://doi.org/10.1016/j.jconrel.2007.08.005

4.     Abdel Messih, H.A., Ishak, R.A., Geneidi, A.S., Mansour, S., 2017. Nanoethosomes for transdermal delivery of tropisetron HCl: multi-factorial predictive modeling, characterization, and ex vivo skin permeation. Drug Dev. Ind. Pharm. 43, 958–971. https://doi.org/10.1080/03639045.2017.1287717

5.     Touitou, E., Godin, B., Dayan, N., Weiss, C., Piliponsky, A., Levi-Schaffer, F, Intracellular delivery mediated by an ethosomal carrier. Biomaterials. 2001, 22 (22), 3053–3059 https://doi.org/10.1016/s0142-9612(01)00052-7

6.     Davis, S.N., 2004. The role of duloxetine in the effective management of Type 2 diabetes. J. Diabetes Complications. 18 (6), 367–376

7.     Dibyalochan Mohanty, A.Mounika, Vasudha Bakshi, Chinmayee kumar sahoo ,Ethosome: A Novel Approach in Transdermal Drug Delivery, International Journal of Chem Tech Research. 2018, Volume-11(8) ,Page no:219-226

8.     Liu, X., Liu, H., Liu, J., He, Z., Ding, C., Huang, G., et al., Preparation of a ligustrazine ethosome patch and its evaluation in vitro and in vivo. Int. J. Nanomed. 2011,6, 241–247 https://doi.org/10.2147/IJN.S16044

9.     Hamaguchi, T., Hirose, T., Asakawa, H., Itoh, Y., Kamado, K., Tokunaga, K., et al., 2004. Efficacy of duloxetine in type 2 diabetic patients treated with glibenclamide. Diabetes Res. Clin. Pract. 66 (Suppl 1), S129–S132

10.  Karande, P., Mitragotri, S., Enhancement of transdermal drug delivery via synergistic action of chemicals. Biochim. Biophys. Acta. 2009, 1788 (11), 2362–2373, https://doi.org/10.1016/j.bbamem.2009.08.015

11.  Jobin Jose, Shweta Shetty, Sandeep D S, Prashanth Nayak. Ethosomes as a Promising Delivery Carrier for Herbal Drugs: Recent Developments. Research J. Pharm. and Tech. 2018; 11(7): 3197-3200 https://doi.org/10.5958/0974-360X.2018.00587.5

12.  Sneh Priya, Divya Jyothi, Jainey P James, Amala Maxwell. Formulation and Optimization of Ethosomes loaded with Ropinirole Hydrochloride: Application of quality by Design Approach. Research J. Pharm. and Tech. 2020; 13(9):4339-4345. https://doi.org/10.5958/0974-360X.2020.00767.2

13.  Singh Shikha Y., Aher Smita S., Saudagar Ravindra B.. Ethosomes –Novel Drug Delivery System. Research J. Topical and Cosmetic Sci. 6(1):Jan.–June 2015 page 7-14. https://dx.doi.org/10.15171%2Fapb.2015.074

14.  Tejashree A. Ghadge, A.S. Kulkarni, S.H. Majumdar, S.D. Chavare. Ethosome: A Promising Approach for Antifungal Drug Delivery. Research J. Topical and Cosmetic Sci. 6(1):Jan.–June 2015 page 32-37. https://www.doi.org/10.5958/2321-5844.2015.00001.1

15.  Shraddha Pandey, Shashi Kiran Misra, Nisha Sharma. Ethosomes- A Novelize Vesicular Drug Delivery System. Research J. Pharm. and Tech. 2017; 10(9): 3223-3232, https://doi.org/10.5958/0974-360X.2017.00572.8

16.  T. M. Jondhalekar, S. S. Aher, R. B. Saudagar. Transethosome: Novel Vesicular Carrier for Enhanced Transdermal Drug Delivery System. Research J. Pharm. and Tech. 2017; 10(6): 1816-1819. https://doi.org/10.5958/0974-360X.2017.00320.1

17.  Kundlik Girhepunje et al Formulation and Characterization of Ethosomal Formulation of Ciclopirox olamine. Research J. Pharma. Dosage Forms and Tech. 2012; 4(5): 285-289.

18.  S. J. Daharwal, Veena Devi Thakur, Shikha Shrivastava et al . Designing and Optimization of medicated chewing gum of Ambroxol HCl by using 3² Factorial design. Asian J. Pharm. Res. 3(3): July-Sept. 2013; Page 118-120.

19.  Pradnya M Khandagale, Manisha M. Rokade et al, Formulation Development and Evaluation of Nasal In- Situ Gel of Hydrocortisone. Asian J. Pharm. Tech. 2018; 8 (2):92-102 . https://doi.org/10.5958/2231-5659.2021.00010.2

20.  Monisha C, GNK. Ganesh, Mythili L, Kayalvizhi Radhakrishnan et al A Review on Ethosomes for Transdermal Application. Research J. Pharm. and Tech. 2019; 12(7): 3133-3143.

 

 

Accepted on 29.03.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2022; 15(12):5565-5570.