INTRODUCTION:

Zidovudine (AZT), the first anti-HIV compound approved for clinical use, still widely used for the treatment of AIDS and AIDS related complex, either alone or in combination with other antiviral agents.¹ However, the main limitation to the therapeutic effectiveness of AZT is its dose dependant hematological toxicity. This virustatic drug has a very short half-life of 1h and undergoes considerable first-pass metabolism thus necessitating frequent administration of large doses (200mg for every 4h) to maintain therapeutic drug levels thus side effects occurs frequently.² After per oral administration AZT is completely and rapidly absorbed thus leading to very high initial plasma concentrations and consequently high incidence of toxicity. As AZT undergoes considerable first-pass metabolism, transdermal route is a better alternative to per oral administration, which additionally provides better patient compliance¹. The transdermal route has vied with oral treatment as the most successful innovative research area in drug delivery. The negatives of oral route can be overcome, and benefits of intravenous drug infusion such as to by-pass hepatic “first-pass” elimination to maintain constant prolong and therapeutic effective drug level in the body can be closely duplicated, without its potential hazards, by transdermal drug administration through intact skin.³

Since, AZT is relatively polar molecule with a log P-0.09, its transdermal permeability is poor and below the level necessary to achieve a therapeutic effect².

Attempts have been made to circumvent the skin barrier by several means such as hydrogels and emulgels are promising vehicles for successful delivery of AZT via transdermal route.⁴ Hydrogels are three-dimensional, cross-linked networks of water-soluble polymers. Hydrogels can be made from virtually any water-soluble polymer, encompassing a wide range of chemical compositions and bulk physical properties. The unique physical properties of hydrogels have sparked particular interest in their use in drug delivery applications. Their highly porous structure can easily be tuned by controlling the density of cross-links in the gel matrix and the affinity of the hydrogels for the aqueous environment in which they are swollen. Their porosity also permits loading of drugs into the gel matrix and subsequent drug release rate dependent on the diffusion coefficient of the small molecule or macromolecule through the gel network.⁵Emulgel is emulsion, either of the oil‐in‐water or water-in-oil type, which are gelled by mixing with a gelling agent. Emulgels are stable and better vehicles for hydrophilic as well as hydrophobic drugs. Oil-in-water emulsions are most useful as drug delivery water washable bases.⁶

In the present study, attempts had been made to develop hydrogels as well as emulgels to compare the in vitro permeation performance of AZT.

MATERIALS AND METHODS:

AZT gifted from Aurobindo Pharma, Hyderabad. Tween-20 and Span-20 was purchased from NR Chem. Pvt. Ltd, Mumbai. Carbopol 940, methyl paraben, propyl paraben, liquid paraffin and propylene glycol (PG) was supplied by Loba chemie, Mumbai. Ethanol and Triethanolamine (TEA) was obtained from SD Fine chemical Ltd., Mumbai. All other chemicals and solvents were of analytical grade.

Preparation of AZT hydrogels:

Appropriate quantity of carbopol 940 was soaked in water for a period of 2 hours. Carbopol was then neutralized with TEA with stirring. Then specified amount of AZT was dissolved in appropriate and preweighted amounts of propylene glycol and ethanol. Solvent blend was transferred to carbopol container and agitated for additional 20 min. The dispersion was then allowed to hydrate and swell for 60 min and pH was adjusted between 6.8-7 with TEA. During pH adjustment, the mixture was stirred gently with a spatula until homogeneous gel was formed.⁷

Figure 1: In vitro release of AZT from hydrogels

Figure 2: In vitro release of AZT from emulgels.

Preparation of AZT emulgels:

AZT emulgel was prepared by dispersing Carbopol 940 in distilled water with constant stirring at a moderate speed then the pH are adjusted to 6.8 to 7 using TEA. The oil phase of the emulsion was prepared by dissolving Span 20 in light liquid paraffin while the aqueous phase was prepared by dissolving Tween 20 in distilled water. Methyl and Propyl paraben was dissolved in PG whereas AZT was dissolved in ethanol and both solutions were mixed with the aqueous phase. Both the oily and aqueous phases were separately heated to 70° to 80°C; then the oily phase were added to the aqueous phase with continuous stirring until cooled to room temperature. The obtained emulsion was mixed with the gel in 1:1 ratio with gentle stirring to obtain the emulgel.⁸

Table 1: Formulation of AZT hydrogels and emulgels.

Ingredients (% w/w)	H1	H2	H3	E1	E2	E3
AZT	1	1	1	1	1	1
Carbopol 940	0.5	1	1.5	1	1	1
Liquid paraffin	-	-	-	5	6.25	7.5
Tween 20	-	-	-	0.6	0.8	1
Span 20	-	-	-	0.9	1.2	1.5
PG	5	5	5	5	5	5
Ethanol	2.5	2.5	2.5	2.5	2.5	2.5
Methyl paraben	0.03	0.03	0.03	0.03	0.03	0.03
Propyl paraben	0.01	0.01	0.01	0.01	0.01	0.01
Distilled water q.s.	100	100	100	100	100	100

Evaluation of AZT gels:

Organoleptic properties:

The prepared hydrogels as well as emulgels containing AZT were inspected visually for their color, homogeneity, consistency and phase separation.⁹

Determination of pH:

The pH of the formulated hydrogels as well as emulgels was determined using pH meter (Model: 7007, Digisun electronics, Hyderabad). The electrode was immersed in gel and readings were recorded on pH meter.¹⁰

Drug content analysis:

A specified quantity (1.0 g) of gel was extracted with 5 mL of ethanol and then volume was made upto 50 mL with water, the 5 ml of the above solution was further diluted to 50 ml with distilled water. The absorbance of the solution was measured spectrophotometrically at 266.5 nm against blank and drug content was calculated.¹¹

Determination of viscosity:

Viscosity studies of formulated gels were carried out using cone and plate programmable Brookﬁeld Rheometer (Model: DV-ІІІ ULTRA, Brookfield Engineering Lab; Inc; Middleboro. USA) ﬁtted with spindle Cp-52 at 100 rpm and at temperature 25 ◦C.¹²

Spreadability studies:

Spreadability of AZT gels was determined by a wooden block and glass slide apparatus, which was provided by a pulley at one end. A ground glass slide was fixed on the block and an excess of formulated gel (about 2 gm) was placed on it. The gel was then sandwiched by using another glass slide having the dimensions as that of fixed ground slide and provided with the hook. Weight of 1 kg was placed on the top of the two slides for few minutes to remove entrapped air and to form a uniform gel film between the slides. The excess of the gel was scrapped off from the edges. Pulley was attached to the hook and 80 g weight was incorporated to it. The time taken by the top slide to travel a distance of 7.5 cm was noted.¹³

In vitro drug release studies:

AZT release rates from the gels were measured through cellophane membrane using a modified Keishery chein cell. Cellophane membrane allowed to equilibrating with the diffusion medium for 15 minutes by immersing in diffusion medium for 30 minutes. It was then placed on the support screen of the diffusion cell assembly. All the joints were properly sealed with adhesive tape to avoid the penetration of diffusion medium. Saline phosphate buffer solution was used as the receptor medium and 1 g of the test gel was placed on the donor side. The receptor medium was kept at 32^o C. At predetermined time intervals, 5 mL samples were taken from the receptor compartment, for 12 h period and replaced by the same medium to maintain a constant volume. Absorbance of these solutions was measured at 266.5 nm using UV/VIS double beam spectrophotometer. Cumulative percent release of AZT was calculated.¹⁴

Kinetics of drug release:

In order to investigate the mode of drug release from the developed gels, the release rate were analysed according to zero (equation І) and first order (equation ІІ) kinetics as well as diffusion controlled mechanism (equation ІІІ-Higuchi equation)using linear regression analysis.

І) Q = k_ot

ІІ) In (100-Q) = In Q_o – k₁t

ІІІ) Q = k_H t_1/2

In the above equations, Q is the percentage of drug released at time t and k_o, k₁t and k_H are coefficients of the equations. The statistical analysis was performed by calculating the correlation (r) existing between the in vitro release and the proposed at different n-values.¹⁵

Skin compatibility studies:

Skin compatibility studies were carried out with the permission of Institutional Animal Ethical Committee, N.E.T. Pharmacy College Raichur, Karnataka, (IAEC No. 576/2002/bc/IAEC/CPCSEA). Albino rats (150-200 g) of either sex were used for testing of skin irritation. The animals were acclimatized under standard conditions with free access to water Ad libitum. Hair was shaved from back of rats and area of 4 cm² was marked on both the sides, one side served as control while the other side was test. AZT gel was applied (500 mg/rat) twice a day for 7 days and the site was observed for any sensitivity and the reaction if any.¹⁶

Accelerated stability studies:

All the formulations were subjected to a stability testing for three months as per ICH norms at a temperature of 40º ± 2º. All selected formulations were analyzed for the change in viscosity, pH, drug content and phase separation by procedure stated earlier.¹⁷

RESULTS:

Table 2: Evaluation parameters of AZT hydrogels and emulgels.

Formulation Code	Colour	Homogeneity	Consistency	Phase Separation	Skin Compatibility
H1	Transparent	Homogenous	Excellent	Nill	Compatible
H2	Transparent	Homogenous	Excellent	Nill	Compatible
H3	Transparent	Homogenous	Excellent	Nill	Compatible
E1	Opalescent	Homogenous	Good	Nill	Compatible
E2	Opalescent	Homogenous	Good	Nill	Compatible
E3	Opalescent	Homogenous	Good	Nill	Compatible

Table 3: Evaluation parameters and drug release kinetics of AZT hydrogels and emulgels.

Formulation code	Drug content (%) (±SD), n=3	Viscosity (cps) (±SD), n=3	pH (±SD), n=3	Spreadability (gcm/sec) (±SD), n=3	% drug released in 12 h (Q) (±SD), n=3	Cumulative % Drug released in 12 h (Q/A) (±SD), n=3	First order (R²)	Zero order (R²)	Higuchi Mode (R²)
H1	99.32 ± 0.22	1854.2 ± 0.07	6.83 ± 0.11	28.33 ± 0.09	72.91 ± 0.11	3.72 ± 0.01	0.9504	0.9958	0.9713
H2	98.52 ± 0.42	2932.6 ± 0.02	6.91 ± 0.03	33.48 ± 0.10	67.05 ± 0.12	3.42 ± 0.03	0.9628	0.9967	0.9837
H3	99.49 ± 0.34	4382.2 ± 0.04	6.88 ± 0.13	34.73 ±0.03	62.24 ± 0.23	3.18 ± 0.04	0.9398	0.9789	0.9695
E1	99.55 ± 0.63	2988.7 ± 0.06	6.84 ± 0.12	33.82 ± 0.74	77.20 ± 0.19	3.94 ± 0.05	0.9493	0.9983	0.9724
E2	98.84 ± 0.24	3319.3 ± 0.04	6.93 ± 0.18	37.69 ± 0.32	74.92 ± 0.13	3.83 ± 0.03	0.9507	0.9974	0.9868
E3	98.92 ± 0.82	4738.8 ± 0.09	6.96 ± 0.09	38.64 ±0.21	71.47 ± 0.16	3.65 ± 0.02	0.9499	0.9945	0.9667

Table 4: Stability study data of AZT hydrogels and emulgels.

Formulation Code	Drug content (%) (±SD), n=3	Viscosity (cps) (±SD), n=3	pH (±SD), n=3	Spreadability (gcm/sec) (±SD), n=3	Phase Separation
H1	99.92 ± 0.53	1834.4 ± 0.03	6.93 ± 0.13	24.24 ± 0.43	Nil
H2	98.14 ± 0.84	2183.3 ± 0.04	6.97 ± 0.09	29.84 ± 0.19	Nill
H3	98.23 ± 0.12	4324.3 ± 0.09	6.79 ± 0.05	31.47 ±0.12	Nill
E1	98.21 ± 0.32	2734.3 ± 0.05	6.73 ± 0.08	30.74 ± 0.46	Nill
E2	99.38 ± 0.24	3264.2 ± 0.02	6.87 ± 0.13	34.84 ± 0.64	Nill
E3	98.53 ± 0.45	4738.4 ± 0.06	6.82 ± 0.07	27.38 ±0.83	Nill

Figure 3: Comparative in vitro drug release between H1 and E1

DISCUSSION:

AZT transdermal gels were formulated in order to bypass the side effects associated with oral therapy. Hydrogels (H1, H2 and H3) were formulated by using Carbopol 940 in varying concentrations (0.5, 1 and 1.5 g) to provide the adequate consistency and elegancy also it is free from the toxicological effects on skin. In order to solublize the drug in the formulation, PG was used as a solvent as well as humectants to avoid the drying of gels.

Ethanol enhances the drug solubility in water because it acts as a co-solvent. Excess amount of ethanol reduces the viscosity of the formulated gels, this may be probably due to break down of cross linking between the polymer hence its concentration is optimum in the preparations i.e. 2.5%w/w. One more advantage of using ethanol is that it enhances the drug permeation through the skin.

In another batch of formulation emulgels were developed (E1, E2 and E3). Light liquid paraffin containing Span 20 acts as oil phase and water containing Tween 20 constitutes aqueous phase. Along with the oil as well as water phases; PG and ethanol also included in the formulation to enhance solubility and permeability of the drug. Methyl and propyl paraben are well known preservatives used to avoid the microbial growth in both hydrogels as well as emulgels. (Table 1)

Formulated gels were tested for physical appearance, homogeneity and consistency. All the hydrogels were found to be transparent and homogenous with excellent consistency where as emulgels were found to be opaque, homogenous with good consistency and no phase separation were observed. (Table 2) Drug content uniformity is the indicative of uniform dispersion of drug throughout the gels. The drug content of the formulated hydrogels and emulgels were found in between 98.52 ± 0.42 to 99.55 ± 0.63% (Table 3).

A flow characteristic of the topical gels depends upon viscosity of formulation. Since viscosity is inversely proportional to the in vitro drug release hence a change in viscosity reduces effectiveness of the product.Viscosity of the hydrogel formulations depends upon the concentration of Carbopol 940, it was found that gels H1, H2 and H3 having viscosities 1854.2 ± 0.07, 2932.6 ± 0.02 and 4382.2 ± 0.04 cps in the concentration of 0.5, 1 and 1.5 g respectively, whereas emulgels E1, E2 and E3 bear viscosities as 2988.7 ± 0.06, 3319.3 ± 0.04 and 4738.8 ± 0.09 cps respectively. In the emulgels the gelling agent concentration was kept constant (1%w/w) and the increased viscosities from E1 to E2 may be due to increased concentration of emulsifying agents (Span 20 : Tween 20). (Table 3)

The pH of the formulated gels not only influences the solubility of drug in the formulation, but may also affect the skin compatibility. Stability of the gels may affect, if the pH changes throughout the shelf life. Hence, pH of the gels must be even and in accordance with skin pH (5.4 to 6.9). All the formulated gels have pH in the range of 6.83 ± 0.11 to 6.96 ± 0.09. (Table 3)

In order to give uniform applicability on the skin, Spreadability of the gel is the essential consideration which depends upon viscosity of the gels and these were in the range of 28.33 ± 0.09 to 38.64 ± 0.21gcm/sec for all the formulations. The in vitro drug release studies helpful to optimize the gel formulations. Drug release studies were carried out for 12 h from both hydrogels as well as emulgels. It was observed that in vitro drug release from hydrogels is inversely depends upon the Carbopol 940 concentration, as polymer concentration increases viscosity of the gels increases and thus release rate decreases from H1 to H3. (Table 3 and Figure 1)

In case of emulgels higher release rate was found in case of formulation E1 and it decreases further with formulation E2 and E3. (Table 3 and Figure 2) It may be due to higher viscosities of E2 and E3 which is the result of increased concentrations of emulsifying agents (Span 20: Tween 20).

Comparison between hydrogels and emulgels with respect to in vitro drug release profile revealed that emulgels are capable to deliver AZT with higher release rates; it may be due to partition of the drug in both oil as well as aqueous phases. Figure 3 depicts that E1 is the better formulation than H1 and among all the other formulations with respect to all the properties.

Kinetics of drug release from the gels was studied by using various mathematical models like first order, zero order and Higuchi. (Table 3) The study revealed that all the gels followed the zero order kinetics as their R² values ranges between 0.9789 to 0.9983 and the mechanism of the drug release was found to be diffusion controlled (Higuchi data), which is the rate limiting step in the drug permeation.

All the gels are composed of pharmaceutically approved (non-immunogenic and biocompatible) excipients in desired amounts. But still then there may be chances of some allergic manifestations after applying on skin. Hence, the skin compatibility study was carried out by using albino rats. It was observed that all the gels showed some behavioral changes in rats after first application, it may be due to the cooling effect by the ethanol. But on further application they showed the tolerability to that action. No allergic manifestation was observed during study (Table 2).

With the purpose to deliver safe and effective formulation throughout its shelf life to the patient it is very essential to study the stability. When all the formulation were exposed to exaggerated temperature condition (40º ± 2ºC), no changes were found in their physical stability. Emulgels are the o/w emulsions may undergo phase separation at higher temperature. But the developed emulgels were found to be stable without phase separation throughout the study period. Also no significant changes were found in drug content, viscosity, pH and spreadability of all the formulations. (Table 4)

CONCLUSION:

The study concludes that transdermal delivery of AZT is one of the better alternatives over the oral route which is associated with serious side effects and emulgels are the effective transdermal vehicles with attractive properties.

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Received on 15.10.2011 Modified on 30.10.2011

Research J. Pharm. and Tech. 5(1): Jan. 2012; Page 41-45