Effect of Permeation Enhancers on the Release and Permeation Kinetics of Oxytetracycline Hydrochloride Organogel Formulations

Oxytetracycline HCl is an antibacterial drug used in the treatment of acne vulgaris and dermatitis. Its oral bioavailability is intermediate i.e., 60% and it is rapidly excreted through renal route. This drug has many side effects such as epigastric pain, nausea, vomiting, diarrhea, hypersensitivity, and tooth discoloration when taken orally. To overcome the side effects and to increase the bioavailability of Oxytetracycline HCl, topical formulations were prepared. Organogels were prepared using sorbitan monostearate as gelling agent and isopropyl myristate as vehicle with different permeation enhancers. The prepared formulations were evaluated for drug content, viscosity, extrudability, homogeneity, skin irritation test, in vitro drug release, and antimicrobial activity. A formulation containing eucalyptus oil with isopropyl myristate, showed better in vitro permeation and higher antimicrobial activity as compared to other formulations with different penetration enhancers.


INTRODUCTION
For a drug to be an effective topical agent, it should penetrate the skin as readily as possible.Drug delivery across the skin is a great challenge.The greatest obstacle to this is the stratum corneum, which is the outermost layer of skin and is considered as the primary rate limiting barrier to the permeation of the drugs across the skin.It consists of dead, ß attened cells, Þ lled with keratin embedded in a lipid matrix.Stratum corneum has been described as hydrophilic protein 'bricks' in a hydrophobic mortar.This barrier function of stratum corneum creates difÞ culties for the formulation of the scientists aiming to deliver drugs via the skin.The search for a solution to this problem led the investigators to employ several penetration enhancement polyethylene glycol, ethanol, isopropanol, n-octanol, and eucalyptus oil by applying them concomitantly with drug (Oxytetracycline HCl) to investigate the contribution of vehicle (isopropyl myristate) and accelerant to the observed enhancement.

Preparation of organogels
Organogel formulation was prepared by a two-step method.First, organogel base was formed by melting the sobitan monostearate on a water bath at the temperature range of 60-70°C, adding isopropyl myristate to it with continuous stirring, and then allowing it to cool at room temperature; thus, forming an organogel base.Then drug (Oxytetracycline HCl) along with different penetration enhancers was added to this base to form the desired organogel.The ratios for the different formulation ingredients have been given in Table 1. [3]

Viscosity
BrookÞ eld viscometer DV II model attached with T-bar spindle (no.94) was used for determination of viscosity.Organogel was Þ lled in a beaker of suitable size and spindle was lowered perpendicularly taking care in such a way that spindle does not touch the bottom of beaker.The spindle was rotated at such a speed so as to generate the torque >30%.The viscosity of organogel was then obtained by multiplying the viscometer reading with the multiplication factor given in BrookÞ eld viscometer catalogue. [4]ug content determination A speciÞ ed quantity (100 mg) of gel was shaken with 10 ml mixture of ethanol: PBS (pH 7.4) in 30:70 ratio.The resulting solution was Þ ltered using Whatman Þ lter paper.The Þ ltrate was appropriately diluted and the drug content was determined by measuring the ultra violet (UV) absorbance of Þ ltrate at 356 nm. [4]trudability Extrudability was determined in terms of weight in grams required to extrude a 0.5 cm ribbon of gel in 10 sec from collapsible tube. [5]mogeneity Homogeneity of various formulations was tested by visual observation and was ranked as follows: [4] +++ = Excellent, ++ = Very Good, + = Good, -= Poor.

Skin Irritation Test
The organogel containing drug was applied on the skin.The test gel and cotton swab covering it were secured Þ rmly on the applied surface with the help of adhesive tapes.Then observations were made for any sign of erythema and ranked as follows as per the state of the applied site [5] : +++ = Severe erythema, ++ = Moderate erythema, + = Slight erythema, -= No irritation.

In vitro permeation studies through dialysis membrane
The in vitro diffusion study of the organogels was performed using semipermeable dialysis membrane (dry,  1.The recipient chamber was Þ lled with 22 ml of ethanol: IPBS (pH 7.4) 30:70 solution.
Ethanol was added to maintain sink condition and temperature was maintained at 37 ± 0.5ûC by circulating water in surrounding jacket. 1 g of gel was evenly spread on semipermeable dialysis membrane, which was clamped between donor and receptor compartment.Air bubbles, if any, were removed carefully and medium was stirred by external driven Teß on coated magnetic bar.At predetermined time intervals, 2 ml of solution was pipetted out from the receptor compartment and immediately replaced with 2 ml fresh phosphate buffer.
The drug concentration of the receptor fluid was  Preparation and enhancement study of organogel of Oxytetracycline HCl determined spectrophotometrically (Shimadzu, Tokyo, Japan) against appropriate blank solution. [6]

Antimicrobial activity
Antimicrobial activity was by ditch plate technique.This technique has been used for evaluation of bacteriostatic and fungistatic activity of compounds especially in semisolid preparations.Agar plates were prepared and sterilized as per standard procedure.A cup (2.5 X 0.5 cm 2 ) was made in the center of agar plates and formulations (0.5 g) were Þ lled in the cup.The prepared culture loops were streaked across the agar at right angle from ditch to the edge of the plate.The organogel formulations containing different penetration enhancers were compared with control.The bacterial growth was observed and percentage inhibition was calculated as follows: [7] Percentage Inhibition = L 2 /L 1 X 100, where L 1 = Total length of streaked culture, L 2 = length of inhibition.

Statistical analysis
The ß ux of the in vitro release studies was calculated using PCP Disso software.Flux at the end of 480 minute time period was used to compare the different formulations:

RESULTS AND DISCUSSION
Viscosity is an important parameter for characterizing the gels as it affects the extrudability and release of drug.Formulation P 6 showed the lowest viscosity and P 1 showed the highest viscosity.The results for viscosity, drug content, extrudability, homogeneity, and skin irritation of all the prepared organogel formulations are shown in Table 2.
The drug content of the gel preparations were found to be the highest in P 6 formulation i.e., 99.62 ± 0.015.The drug content determination also showed that the drug was uniformly distributed throughout the gel.The viscosity of the gels was found in the range of 15,440 to 1,023 cps for different formulations.All the formulations were homogenous in texture and showed no skin irritation upon application for eight hours.Among all the formulations, P 6 formulation showed excellent extrudability .
The in vitro release proÞ le of oxytetracycline HCl from its various organogel formulations are shown in Figure 2.

Flux of test formulation Fluctuation of control formulation
Enhancement ratio (ER) = It can be observed from the Þ gure that the drug release from formulations containing penetration enhancers were higher than its release from control.The drug release from organogels containing penetration enhancers can be ranked in the increasing order as per their respective penetration enhancers as propylene glycol < polyethylene glycol < ethanol < isopropanol < n-octanol < eucalyptus oil.The nonionic surfactant (propylene glycol) showed signiÞ cant enhancement compared to control formulation.Glycol is a sorption promoter and has high thermodynamic activity, which produces a driving force and thus carries the drug along with it by carrier mechanism; glycols when applied to skin also tend to partition into the skin and thereby promote the movement of drug.Alcohol shows greater release compared to glycol.Ethanol penetration enhancing effect can be attributed to two factors: (a) increase in thermodynamic activity due to evaporation of ethanol known as "push effect"; (b) "pull effect" in which permeation of drug molecules is increased due to reduction in barrier property.Propanol and octanol showed higher release rates than ethanol and this is probably because they contain longer carbon chain, which in turn increases their lipophilicity and hence partition coefÞ cient, which in turn enhances their push and pull effect.The highest percentage of drug release and ß ux is shown by formulation containing eucalyptus oil as penetration enhancer.Eucalyptus oil contains 1,8-cineole as its main constituent and a series of 17-monoterpene and terpenoids that have proven penetration enhancement effect for hydrophilic drugs. [8]e order of percentage drug release was: eucalyptus oil > n-octanol > isopropanol > ethanol > polyethylene glycol > propylene glycol.The result indicates that eucalyptus oil has the strongest penetration enhancement activity among the six penetration enhancers studied.When formulated in to organogel using isopropyl myristate with eucalyptus oil, the ß ux was found to be 1.18 and enhancement ratio was 5.57, which clearly indicates the synergistic effect [Figures 3 and 4].There is also potential synergistic effect observed between octanol and isopropyl myristate, the ß ux was approximately 0.90, the enhancement ratio was 4.11.Thus, it can well be concluded that the amount of drug released and ß ux show potential synergism when formulated with isopropyl myristate, but in case of eucalyptus oil have penetration enhancement ability of the oil predominant.
The antimicrobial activity was ranked in the same order as that of in vitro drug release.