Formulation and Optimization of a Niosomal Gel for Transdermal Drug Delivery of Baclofen for treating alcohol dependence

WHO reports that over 400 million adults worldwide live with Alcohol Use Disorder (AUD). About 2.6 million deaths per year are attributed to overconsumption of alcohol, with a higher percentage of men affected than women¹. Major health problems associated with alcohol abuse include liver and gastrointestinal disorders, neurological complications and cancers, psychological problems such as depression, erectile dysfunction etc. Alcohol abuse also has a serious social impact through cases of domestic violence, marital dispute, reduced workplace performance, debt and poverty, drunk driving accidents, increase in crime rate etc.^{2 3 4 5}. AUD is a chronic condition that can be treated by pharmacotherapy and counselling. Disulfiram has been used conventionally for the treatment of alcohol dependence which causes unpleasant reactions if alcohol is consumed, thus leading to abstinence. However, it requires voluntary patient involvement and poor patient compliance⁶. Naltrexone reduces alcohol cravings but is contraindicated in hepatitis which may accompany AUD⁷. Acamprosate is also an anticraving agent but requires multiple daily dosing⁸.Baclofen is a selective GABA B agonist which works by blocking withdrawal symptoms associated with alcohol dependence. A very small amount is metabolized by the liver and hence it is suitable particularly for liver compromised patients. It helps to reduce anxiety and restlessness strongly associated with AUD patients^{9 10}.Baclofen is available as oral tablets which require frequent administration as it has a short half life of about 4 hours. This makes it inconvenient for chronic treatment and results in low patient compliance^{11 12 13}. A sustained release transdermal drug delivery system could overcome this limitation thus reducing the dosing frequency.Niosomes are non-ionic surfactant containing vesicles which can incorporate hydrophilic as well as lipophilic drugs for transdermal delivery with better permeability, sustained release and drug stability^{14 15 16 17}. The current research work aims to formulate a sustained release niosomal gel of baclofen for treating alcohol dependence by reducing the dosing frequency.

MATERIALS AND METHODS:

Materials

Baclofen was a gift sample from Piramal Pharma and Carbopol^® 980 from Lubrizol. Cholesterol was purchased from Sigma Aldrich and Span^® 20 from SD Fine Chemicals, Chloroform and Methanol from Merck & Co.

Methods

Solubility studies

10ml of phosphate buffer 7.4 was taken in a test tube. Baclofen was added to the solvent in increments of 1mg at a time. The solution was mixed after each addition using a cyclomixer, ultrasonicated for 10 minutes and visually observed for any precipitate or residual solid. This was continued till the drug formed a precipitate and was no longer soluble in the solvent. The supernatant was separated by filtration using 0.45µ membrane filter. The filtrate was analyzed using UV spectrophotometer (Jasco V630 with Borwin software) at 225nm. The unknown concentration was extrapolated from a standard curve of the drug made previously using the same solvent^{18 19}.

Selection of non-ionic surfactant

Preliminary batches of Niosomes were formulated using Cholesterol and three different non-ionic surfactants viz. Span 20^®, Span^® 60 and Span^® 80 ^{20 21 22 23}. The concentration of all the ingredients was kept constant, only varying the type of surfactant. Thin film hydration method was used and the stability was observed for one week in order to select the suitable surfactant for formulation.

Preparation of Baclofen Niosomes

Thin film hydration method was used for the formulation of Baclofen Niosomes (methodology in Figure1). Cholesterol (1-2%) and Span^® 20 (2-4%) were dissolved in chloroform and methanol mixture (1:2 ratio) and placed in a round bottom flask. The solvent was evaporated on a vacuum rotary evaporator at 50ºC for a period of 15 minutes for obtaining a thin film which was further dried for 30 minutes. This thin film was then hydrated with desired dose of Baclofen (dissolved in phosphate buffer 7.4 for a period of 1 hour). The resultant solution was then allowed to stand overnight for the liposomes to anneal and subjected to sonication on the consecutive day to obtain the niosomal suspension^{24 25 26} . One Factor at a Time (OFAT) approach was used for the various niosomal batches represented in Table 1.

Characterization

a. Particle size determination

Zetasizer Malvern Nano ZS90 (UK) was used to determine the average particle size and polydispersity index of the various niosomal batches that were formulated. Each sample was diluted suitably using phosphate buffer 7.4, placed in the sample cuvette and the particle size was measured²⁷.

b. Entrapment efficiency

4ml of the formulation was used to carry out ultracentrifugation (Eltek high speed centrifugation RC4100D) at 17,000rpm at -4ºC for one hour. The resultant pellet was separated and the supernatant solution was analysed for the free drug using HPLC (Jasco UV-2077 with Rheodyne manual sample injector with 20 µL capacity and UV detector at 225 nm. Data processing was done by using Borwin software). The following formula was used to determine the drug that was entrapped in the niosomal vesicles ^{28 29}.

% Entrapment efficiency=Amount of drug loaded-Amount of drug in supernatantAmount of drug loaded x 100

c. Transmission Electron Microscopy (TEM)

The particle size of the formulation N14 was confirmed using TEM. The sample was diluted as required and then phosphotungstic acid was used to spray the solution onto a carbon grid. After drying the grid was observed under an electron microscope for noting the particle size³⁰.

d. Zeta potential measurement

The zeta potential of the optimized formulation N2 was also determined using Malvern zetasizer³¹ .

Differential Scanning Colorimetry studies

Differential scanning calorimetry (DSC) was carried out in order to determine the compatibility of Baclofen with the excipients. A physical mixture of the drug and excipients was prepared by mixing in the same ratio as required for formulation. Also the niosomal formulation was subjected to DSC in order to confirm the drug encapsulation in the niosomal vesicles. 3 mg of each sample was weighed and sealed in DSC pans keeping an empty pan as a reference. The samples were subjected to heating between 30- 300°C under a nitrogen purge. The thermograms were recorded and analyzed^{32 33 34}.

Formulation of Niosomal gel

Formulation N2 was selected for incorporation in a Carbopol gel. Transcutol P was used as a permeation enhancer. The concentration of polymer and permeation enhancer was varied in order to obtain six different batches of Niosomal gel represented in Table 2. Carbopol 980 was allowed to swell for 60 minutes. The niosomal suspension was incorporated slowly in parts by constant stirring on a magnetic stirrer. Triethanolamine was used to adjust the pH to physiological values³⁵.

In vitro drug diffusion

Cellophane membranes (Himedia laboratories with molecular weight 12,000-16,000) were used for the study. Franz diffusion cell was used to mount the membrane. 2g of niosomal gel was applied on the donor side, while the receptor compartment was filled with phosphate buffer 7.4. At various time intervals viz. 0.5, 1, 2, 3, 4, 5, 6 and 24 hours aliquots were withdrawn from the receptor compartment and analyzed using HPLC (Jasco with UV detector) at 225nm for quantifying the amount of drug released^{36 37 38 39}.

Evaluation of the Niosomal gel

a. pH determination

1gm of gel was used to determine the pH using a digital pH meter (Electrolabs).

b. Viscosity determination

Sufficient amount of gel was placed in a beaker in order to dip the spindle of the Brookfield viscometer USA (spindle 5 was used). The viscosity was measured by gradually increasing the speed of the spindle thus increasing the shear stress and then gradually decreasing the stress values. The dial reading was noted, multiplied by the factor in order to determine the viscosity. A graph was plotted of shear stress v/s viscosity⁴⁰.

c. Spreadability

On a glass tile, a small quantity of gel was applied in a circle of 0.5cm radius. Another glass tile was placed over the first one thus enclosing the gel between the two slides. A weight of 500gm was placed over the upper tile for 1 minute. The increased gel diameter was measured and compared with a standard marketed topical baclofen gel⁴¹.

RESULTS AND DISCUSSION:

Solubility studies

The solubility of Baclofen in Phosphate buffer 7.4 was found to be 10mg/ml. This data was used to determine the amount of drug to be used for loading into the niosomes using thin film hydration method.

Selection of non-ionic surfactant

The batch consisting of Span^® 20 as the surfactant yielded a batch that remained stable for the test period of one week. Hence, all future batches were formulating using variable concentrations of Span^® 20.

Preparation of Baclofen Niosomes

Formulation of Baclofen Niosomes was successfully done using Thin film hydration method. Span^® 20 was majorly responsible for formation of the bilayer vesicles. The hydrophilic core thus formed was suitable for encapsulation of Baclofen a hydrophilic drug. The bilayer thus formed is quite fluid and hence to prevent leakage of the drug from the core, cholesterol was added to impart rigidity. Span^® 20 yields vesicles with good stability and lower irritation potential. This is suitable for a sustained release formulation that would remain in contact over 24 hours. The various niosomal batches are indicated in Table 1 each containing 160mg of drug in 10ml:

Table 1: Niosomal batches with their characterization data

Figure1: Methodology to formulate Niosomes

Characterization

a. Particle size determination

The particle sizes of the various batches are indicated in Table 1. N1, N7, N10, N14 were found to have lower particle sizes as compared to others. Low particle size is desirable for better transdermal drug delivery.

Figure2: Particle size analysis of batch N14

b. Entrapment efficiency

The entrapment efficiency of the various batches is indicated in Table 1. N4, N14, N15, N18 were found to have higher entrapment efficiencies as compared to others. Higher entrapment indicates that the drug is well enclosed into the niosomal vesicles, thus providing better therapeutic effect, sustained release and reduced systemic toxicity. The balance between the fluidity of the vesicles and the rigidity from cholesterol is essential for its delivery through the layers of the skin⁴².

c. Transmission Electron Microscopy (TEM)

Batch N14 was found to have low particle size and high entrapment efficiency and was selected for further characterization. Transmission electron microscopy (Figure 2) revealed spherical vesicles. They had well defined boundaries, homogeneity in size and shape. The dark region within indicates presence of encapsulated drug.

Figure3: Transmission Electron Microscopy image of Baclofen Niosomes

d. Zeta potential measurement

The zeta potential of the selected batch N14 was found to be -20mV. Although Span 20 is non-ionic and cholesterol is neutral, there is surface adsorption of hydroxyl ions which imparts a slight negative charge to the formulation.

Figure4: Zeta potential of Niosomes N14

Differential Scanning Colorimetry studies

DSC thermogram of pure drug showed a prominent endothermic peak at 216?, the narrow and sharp nature indicative of crystalline nature. In the DSC thermogram of baclofen niosomes, the sharp peak disappears. This indicates entrapment of the drug into the niosomal vesicles.

Figure5a, 5b: DSC thermogram of pure drug and drug containing niosomes respectively

Formulation of Niosomal gel

Optimized batch N14 was selected in order to formulate a gel. Carbopol^® 980 was soaked in water for an hour. The niosomes and Transcutol^® P were slowly incorporated in the polymer matrix by manual stirring. The pH was adjusted to 6.5 using triethanolamine⁴³. Table 2 represents the variable concentrations of polymer and permeation enhancer in order to yield six batches. The gel contained 1.5%w/w of Baclofen.

Table2: Niosomal gel batches and their composition