Characterization of MEs:

Preparation of ME - based gels

Evaluation of gels

RESULTS AND DISCUSSION:

Characterization of MEs:

Preparation of ME - based gels

Evaluation of gels

RESULTS AND DISCUSSION:

Formulation

Average particle size (nm)

Polydispersity

F1

87± 2

0.291± 0.05

F2

61± 4

0.35± 0.02

The systems were observed for visual clarity and flow. The MEs were placed between two polarized plates (Cross polarized light microscopy) in series and then observed for light transmittance. After this, one of the plates was rotated relative to the other through 90⁰and then examined. MEs appear completely blank. ME systems were subjected to centrifugation at 4000 rpm for 30 minutes and then examined for any phase separation.

Globule size determination

Globule size determination

MATERIALS AND METHODS

MATERIALS AND METHODS

Because of this property MEs are being investigated for the delivery of protein and peptide drugs. Major limitations in realizing potential of microemulsion as drug delivery system are the narrow range of surfactants, cosurfactants, solvents that are accepted pharmaceutically^V^,
VI. Bacitracin zinc is an antibacterial drug, which is used mainly in the treatment of ophthalmic and dermatological infection^{VII, VIII, IX}. In most cases of dermal and subdermal primary and secondary skin bacterial infections, the disease treatment by topical drug application is not sufficient, and systemic antibiotic treatment is required. This is due to lack of permeability of drug molecules to deep skin layers and subdermal tissues from conventional experimental preparations ^{X, XI, XII}. However, systemic antibiotic administration may give rise to severe allergic reactions and general side effects ^XIII. Thus improved antibiotic delivery to the deep strata of the skin could be highly beneficial. Serious nephrotoxicity results from the parenteral use of this antibiotic. More over the drug gets inactivated on passage through the gastrointestinal tract due to degradation. Currently, intracellular infections are difficult to treat because penetration of antimicrobial agents through the cell membrane is insufficient. Another important issue in antimicrobial therapy is the treatment of infections caused by intracellular pathogens ^XIV. So improvement in the penetration of bacitracin zinc is a need of hour. Absorption of the drug via transdermal route is limited by the poor penetration of the drug through the stratum corneum due to its high molecular weight and size. Reducing the barrier properties of the stratum corneum by using the absorption enhancers increases the permeation of the drug. The addition of surfactants to the drug delivery system can also result in improved drug stability, clinical potency and drug absorption. In the present work an attempt has been made to develop a ME based topical drug delivery system for protein and peptide drug bacitracin zinc with a goal of developing a potential effective treatment for deep dermal and intracellular bacterial infections. The ME system has got the additional advantages of transparency, maximized solubilization of drug and thermodynamic stability. Currently the drug is marketed as ointments in U.S markets. Bacitracin zinc containing gels using MEs were designed and characterized for their delivery properties.

Figure 1 Phase diagram for various ratios of oleic acid/tween 80/propyleneglycol/saline.

Bacitracin zinc was purchased from Himedia chemicals, India, carbapol 934P, was obtained as a gift sample from Glenmark Industries, India, oleic acid, olive oil, ethylene glycol, propylene glycol, tween 80, triethanolamine, sorbitol, isooctanol, were purchased from Modern Scientifics, Nashik, India. All the materials used were as supplied by manufacturers without further purification after ascertaining that they were of required standards.

Construction of psuedoternary phase diagram:

Various ratios of surfactant/ cosurfactant were chosen and the corresponding mixtures were prepared. The ratios of 2:1, 3:1, and 4:1 were tested. The mixture of oil and surfactant/co-surfactant at predetermined weight ratios was diluted with water by sequential addition of 10 µl of water using a micropipette. The system was stirred using a magnetic stirrer to ensure thorough mixing. After each mixing the sample was allowed to settle and its physical condition (clarity and flowability) was reviewed. The sample was sonicated for 1 to 2 min to remove air bubbles and to enable a better visual examination. Mixtures that did not show a change in the meniscus after tilting to an angle of 90º were considered to be gels. Samples were examined under a microscope. The phase diagrams were shown below in Fig 1 and Fig 2.

Freeze thaw cycling

This test places stress on the ME, at temperature below freezing. Six heating/cooling cycles between 45⁰C and refrigeration temperature with storage at each temperature for not less than 48 hours, were carried out for the selected MEs.

MEs droplets smaller than 100 nm cannot be seen in the optical microscope. Coulter counter N4 was used in determining the globule size in the submicron range. The results were shown in table 1 below.

A weighed amount of polymer was soaked in the microemulsion system, stirred to disperse the polymer in the ME and left over night for gelling. To this the required quantity of triethanolamine was added for neutralizing the carboxylic acid groups in carbopol. Then the measured quantity of dimethlyformamide/benzyl alcohol was added. Sodium meta bisulphate was used in the preparation of gels so as to prevent the oxidation of tween 80 which may result in slight colour change. Percent w/w of various components in the formulations were shown in table 2 below.

All the gel formulations were evaluated for transparency, clarity, isotropic behavior, spreadability, pH, viscosity, drug content analysis, in-vitro permeation studies acceptability in humans and antibacterial activity.

Table1 Average particle size and polydispersity values of F1 and F2.

Formulation	Average particle size (nm)	Polydispersity
F1	87± 2	0.291± 0.05
F2	61± 4	0.35± 0.02

Table2. Percent w/w of various components in gel formulations.

Ingredients	F1 (%w/w)	F2 (%w/w)	F3 (%w/w)	F4 (%w/w)
Bacitracin zinc	1.3	1.3	1.3	1.3
Ethylene glycol	----	----	13.75	13.75
Propylene glycol	26.13	18.31	----	----
Tween 80	26.5	35.19	39.75	39.75
Oleic acid	12.37	16.5	16.5	16.5
Carbopol 934	0.4	0.35	---	---
Triethanol amine	0.015	0.015	---	---
Dimethyl formamide	1.55	---	---	1.55
Benzyl alcohol	---	2	2	---
Methyl paraben	0.200	0.200	0.200	0.200
Propyl paraben	0.02	0.02	0.02	0.02
Sodium metabisulphate	0.1	0.1	0.1	0.1
Lavender oil	0.100	0.100	0.100	0.100
Water	Up to 100	Up to 100	Up to 100	Up to 100

Table 3. Results of spreadability, pH, and drug content of different formulations

Parameters	F1 (mean ± s.d)	F2 (mean ± s.d)	F3 (mean ± s.d)	F4 (mean ± s.d)	MF (mean ± s.d)
Spreadability (seconds)	7±0.20	8±0.50	9±0.80	6±0.75	7±0.45
pH	7.1±0.3	6.8±0.2	7.2±0.1	7.5±0.1	7±0.3
Drug content	92.13±0.93	90.56±1.3	95.6±0.63	97.3±0.53	99.05±0.5
Viscosity (cps)	4085	2120	1575	1573	2635

Table 4. Cumulative release of the drug from the formulations

Time (Hours)	F1 (mean ± s.d)	F2 (mean ± s.d)	F3 (mean ± s.d)	F4 (mean ± s.d)	MF (mean ± s.d)
1	00.24±0.17	00.15±0.09	00.47±0.12	00.38±0.01	00.91±0.12
2	02.38±0.44	01.32±0.87	02.18±0.32	02.19±0.63	02.08±0.11
4	04.55±0.25	03.12±0.78	04.00±0.14	04.14±0.52	03.52±0.10
8	06.82±0.69	04.78±0.95	07.47±0.52	06.98±0.46	06.88±0.53
12	10.25±0.58	07.00±0.66	10.68±0.87	11.10±0.64	09.88±0.85
16	13.95±0.14	09.50±0.85	13.19±0.14	15.87±0.54	12.00±0.96
20	16.64±0.85	11.62±0.58	16.23±0.36	19.44±0.87	13.83±0.47
24	17.84±0.52	13.79±0.44	19.00±0.33	23.22±0.47	15.09±0.36

Table 5. Various parameters of the formulations

Formulations→	F1	F2	F3	F4	MF
Flux (J_SS) (mg/cm²*sec)	0.242 ± 0.0039^*	0.182 ± 0.0021^*	0.249 ± 0.0014^*	0.311 ± 0.0016^*	0.201 ± 0.0041
Permeability coefficient (cm/sec)	0.0091± 0.00023	0.00702± 0.00031	0.0098± 0.00011	0.0115± 0.00010	0.0077± 0.00013
Lag time (hours)	0.177 ± 0.0012	0.237 ± 0.0041	0.173 ± 0.0040	0.138 ± 0.0031	0.215 ± 0.0013
Diffusion coefficient(cm²/sec)	9.42E-5±2E-06	7.02E-05±1E-06	9.61E-05±4E-06	0.00012± 0.000006	7.73E-05±2E-06

Spreadability:

Carried out using Mutimer apparatus. The apparatus consists of two glass slides (20*4) cm. One of them being fixed onto the wooden board and other movable, tied to a string, which passes over a pulley carrying pan for weights. The height of the upper slide and pulley were kept at the same the same level. About 2 gm of gel was placed between the slide. A heavy weight was allowed to rest on the upper slide for few minutes to expel the entrapped air between the slides to provide a uniform film of gel. The weight was removed and the top slide was subjected to a pull of 20 gm. The time necessary for the top slide to travel the premarked 10 cm was noted. Generally a shorter time indicates better spreadability.

pH:

2.5 gms of the formulation was dissolved/dispersed in 25 ml of distilled water and the pH was determined by Elico pH meter, standardized by using pH 4 and pH 7 standard buffers prior to use.

Viscosity:

Viscosity measurements were carried out by using a Brookfield Digital Rheometer (Model DV-III, Brookfield Engineering Laboratories, USA) at different shear rates. 5 ml of microemulsion was filed in the sample adapter and SC 4-16 spindle was used.

Drug content analysis:

Three samples of about 1 gm of each gel were taken at random from each formulation in 100 ml volumetric flasks. The samples were mixed with a small amount of distilled water. For MBG, the solution was passed through sintered glass G-4 filter and the residue of aerosil 200/ HPMC and the filter was thoroughly washed with distilled water. The washings were collected in the volumetric flasks and the volume was made up to 100 ml with the same solvent. 1 ml aliquots were taken in 10 ml volumetric flasks and the same procedure was followed as described earlier using U. V. Spectroscopic method. The drug content was determined from the previously plotted calibration curve.

In-vitro permeation studies:

All the in-vitro permeation studies were carried out in Keshary-chien diffusion cells. It closely simulates the in-vivo situation since the membrane was exposed to ambient conditions while the receiver temperature is 37 ± 1⁰C. Human cadaver skin was used as a membrane for the experiments. The receptor compartment was filled with 15 ml of pH 7 phosphate buffer system. The solution in the receptor compartment was constantly stirred by means of Teflon coated magnetic bead on a magnetic stirrer, so that the hydrodynamic conditions of the system were maintained. Bacitracin zinc equivalent to 26 mg was applied uniformly on the membrane from both marketed and laboratory formulations. The active permeation area was 3.14 cm². The opening of the donor compartment was covered by foil, in order to prevent loss due to evaporation. An aliquot of 2 ml was removed from the receptor medium at intervals of 1, 2, 4, 8, 12, 16, 20 and 24 hours and replaced immediately with the same volume of the medium¹⁵. In vitro permeation profiles were also analyzed for the higuchi’s model. The results were shown in table 4 and table 5. One-way ANOVA followed by Dunnet’s test was used to compare different formulations with the marketed formulation (p <0.05) was considered to be significant.

Figure 2. Phase diagram for various ratios of oleic acid/tween 80/ethylene glycol/saline

Acceptability in humans:

Six volunteers were screened and test was performed on them. (The procedure was follows in accordance with the ethical standards of the responsible committee on human experimentation). An occlusive patch bearing the formulation was applied to the upper forearm for 23 hr. One hour after removal, skin sites were assessed for signs of skin irritation, which were rated with numerical scoring system. After assessment, an identical fresh patch was applied to the same area for a further 23 hr. The values assigned were as follows: vesicles- 5; odema-4; erythema-3; dryness-2; wrinkling-1; glazing-1. Each condition was scored according to the strength of observed reaction: 0- no visible reaction; 1- just present reaction; 2-slight reaction; 3-moderate reaction; 4- severe reaction. To obtain the value of the total reaction, the score strength of the reaction was multiplied by the corresponding rate and the resulting values were summed to provide a global score for the acceptability degree. One-way ANOVA was used to compare different formulations with the marketed formulation (p<0.05) was considered to be significant. The results are shown in table 6.

Table 6. Product of type of reaction observed and strength of reactions in human volunteers.

p>0.05, no significant difference was observed.

Table 7. Zones of various formulations (antimicrobial activity)

B. No	Sample	Zone of inhibition
1	F1	18±0.75^*
2	F2	20±0.63
3	F3	21±0.33^*
4	F4	21±0.23^*
5	MF	19.5±0.40

*p<0.05, was considered to be significant

Figure 3 Amount of drug permeated per unit area through the skin vs. time

Antimicrobial Activity:

Initially all the glass apparatus and nutrient agar medium were sterilized. Suspension of microorganism was inoculated in nutrient agar. Then medium was poured in to sterile petri dish. The wells were prepared in plate using borer of size 8-mm. Sample solutions were poured into wells of all plates. Then plates were kept at 4°C for 1 hour. After 1 hour, plates were incubated at 37°C for 24 hours¹⁶. Then zones of inhibition were measured. One way ANOVA was used to compare different formulations with the marketed formulation (p<0.05) was considered to be significant. The results are shown in table 7.

All the MEs formed were transparent and appeared like homogeneous single liquid, when observed for visual clarity against strong light. This indicates that the particle size of droplets was less than 100 nm. The droplets were not seen under optical microscope, which indicates that droplets were less than 200 nm in size. The systems appeared completely blank when observed the crossed polarizers and showed no birefringence. This proved that the systems were isotropic. And systems showed no phase separation during centrifugation and freeze-thawing cycles. Average particle size and polydispersity values of F1and F2 were shown in table 1 below. Polydispersity is measure of particle homogeneity and varies from 0- 1.0. The closer to zero the more homogeneous the particles are in the system¹⁷. The isotropic regions reveal that as the relative concentration of surfactant/cosurfactant increases the ME region also increases in size. The addition of ethylene glycol reduced the interfacial free energy and tension by incorporation into the interfacial layer. Cosurfactant can adjust the geometric packing of surfactant in the interface and thus reduce the tendency of surfactant to form a highly rigid film, thus allowing the interfacial film to take up different curvatures to form balanced ME. The percent w/w of various components used in gel formulations were shown in table 2. All the gels were clear and transparent. The systems appeared completely blank when observed under between the crossed polarizers and showed no birifringence. It proved that the systems were isotropic. The time taken indicates that all the formulations have good spreadability. All the formulations satisfied the pH conditions and drug content. The viscosities of chosen compositions were investigated at seven different shear rates at 298K.The shear viscosity decreases with shear rate for all the compositions. This indicates that the sample undergoes shear thinning. It is needless to say that thixotropy is a desirable characteristic of pharmaceutical dosage form. In this flow the molecules at rest entangle together with the association of immobilized solvent under the influence of the molecules tend to become disentangled and align themselves in the direction of flow. The molecules thus offer less resistance to flow and this occurs together with release of some entrapped water, which accounts for lower viscosity¹⁸. The results were shown in table 3 below. Interestingly, the in vitro release data as well as the percutaneous absorption studies were superior in the ME gel compared to the marketed and other ME based gel formulations. Maximum drug permeation and 1.53 times improvement in the drug release of formulation F4 compared to the marketed formulation. This may be due to the nano-size of the oil globules, which were embedded with the drug in the ME gel. The release profile of formulation F2 was comparatively less than that of the marketed formulation. The formulations F3 and F4 were similar in all aspects excepting that benzyl alcohol was added to formulation F3 and dimethyl formamide was added to formulation F4. But the formulation F4 was superior in all aspects to F3, which may due to the better penetration capacity of dimethyl formamide and also better solubility of drug in dimethyl formamide compared to benzyl alcohol. The amount of bacitracin zinc released from all the formulations studied above showed a linear relationship with zero-order release (regression coefficient= 0.99) as shown in table 4. Therefore the release of drug was independent of the concentration of drug in the receptor chamber. Amount of drug permeated from the formulations per unit area through the skin was shown in Fig 3. To explain the probable mechanism by which ME gel enhances the release and percutaneous absorption of drugs efficiently, the histological and histo-chemical structure of the stratum corneum must be taken in to consideration. Drugs can permeate through stratum corneum through two micropathways, one is intercellular and other is the transcellular way. Of these routes the intercellular route plays a major role in the percutaneous absorption of drugs. A dermally applied ME is expected to penetrate the stratum corneum and to exist intact in the whole horny layer, altering both the lipid and the polar pathways¹⁹. The lipophilic domain of the ME can interact in many ways with the stratum corneum. The drug dissolved in the lipid domain of the ME can directly partition in to the lipids of stratum corneum there by destabilizing its bilayer structure. The data was subjected to one-way ANOVA followed by Dunnet’s multiple comparision test. No significant difference was observed between marketed formulation and prepared formulations in skin irritation studies. Significant difference was observed between the marketed formulation and formulations F1, F3, and F4 in antibacterial activity.

CONCLUSION:

In summary this study sheds more light on the development of novel microemulsion based drug delivery system of bacitracin zinc, which is a polypeptide drug. The results presented here also suggest that significant amounts of polypeptide antibiotic can be administered in to the deep skin layers and more over the efficiency is also increased. Infact, no clear-cut mechanism could be considered in explaining the superiority of ME over the other systems excepting that it has both lipophilic and hydrophilic domains as well as the nanosize particles, which are responsible for better penetration and increased activity. The hydrophilic domain on the other hand hydrates the skin stratum corneum to a greater extent and there by plays an important role in the percutaneous absorption of drugs. Since same lipid chains are covalently attached to corneocytes, hydration of these proteins will also lead to the disorder of lipid bilayers which increases the penetration of oil domains in the microemulsion system. From the above study we can also conclude that the gels prepared showed pseudoplastic behaviour. ME gel is more advantageous for transdermal application of bacitracin zinc in comparison with the ME-based gel. The results suggested that the studied ME systems may be appropriate vehicle for the topical delivery.

ACKNOWLEDGEMENTS:

This work was partially supported by N.D.M.V.P. Samaj’s College of Pharmacy, Nashik, India by providing the necessary lab facilities. The authors are also thankful to Glenmark, India for providing gift samples.

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Received on 11.09.2009

Accepted on 01.11.2009

Research Journal of Pharmaceutical Dosage Forms and Technology. 1(3): Nov. – Dec. 2009, 217-221

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