formulation and evaluation of topical emulgel of

www.wjpr.net Vol 6, Issue 10, 2017.

567

Bachhav et al. World Journal of Pharmaceutical Research

FORMULATION AND EVALUATION OF TOPICAL EMULGEL OF

KETOCONAZOLE BY CUBOSOMAL TECHNIQUE

Jyoti K. Bachhav1*, Bhushan A. Bhairav

2 and Ravindra B. Saudagar

3

1,2

Department of Quality Assurance Techniques, R. G. Sapkal College of Pharmacy,

Anjaneri, Nashik-422213, Maharashtra, India.

3Department of Pharmaceutical Chemistry, R. G. Sapkal College of Pharmacy, Anjaneri,

Nashik- 422213, Maharashtra, India.

ABSTRACT

This novel particle are prepared by either hydrophilic, lipophilic or

amphiphilic molecule prepared by high speed homogenization

technique. It is time consuming technique. The aim of this study is to

enhance ketoconazole solubility and drug delivery of ketoconazole in

biological membrane by preparing ketoconazole emulgel formulation.

Emulgel were prepared by using Glycerin monooleate and poloxomer-

407. The prepaid formulations were characterized by Nano size

analyzer, poly dispersity, entrapment efficiency, zeta potential, in-vitro

drug release. In design formulation optimize batch is selected on the

basis of particle size and entrapment efficiency. The experimental

result demonstrates that successful development of cubosomal

nanoparticle containing ketoconazole for fungal infection targeting

formulation.

INTRODUCTION

Ketoconazole is an antifungal drug that inhibits the fungal infection. It is used to superficial

candiditis Cubosomes are discrete, sub-micron, nano-structured particles of the bicontinuous

cubic liquid crystalline phase. Such novel particles are utilized to encapsulate molecules

which are poor water soluble drugs. Ketoconazole is poorly water soluble drug so solubility

is main constraint for bioavailability. The aim of this study is to investigate the potential of

the cubosomal technique which is Nano-carrier to improve the solubility of the ketoconazole.

Cubosomes are discrete submicron nanostructure formulation of bicontinuous cubic liquid

crystalline phase whose size range is 10-500 nm in diameter they appear like dot square

World Journal of Pharmaceutical Research SJIF Impact Factor 7.523

Volume 6, Issue 10, 567-588. Research Article ISSN 2277– 7105

Article Received on

14 July 2017,

Revised on 03 August 2017,

Accepted on 24 August 2017

DOI: 10.20959/wjpr201710-9221

*Corresponding Author

Jyoti K. Bachhav

Department of Quality

Assurance Techniques, R.

G. Sapkal College of

Pharmacy, Anjaneri,

Nashik-422213,

Maharashtra, India.


568


shaped each dotes corresponds to presence of pore size 5-10nm. Cubosomal gel have great

potential in formulating nano size particulate system for topical delivery they show best

advantages such as high drug pay load due to high internal surface area and cubic liquid

structure. The purpose of present study to develop the ketoconazole topical gel to increase the

drug solubility by avoiding the first pass metabolism of the drug released.

Liquid Cubosomes Precursors

There is difficulty and expense of high shear dispersion of viscous bulk cubic phase to form

cubosomes because it aggressive process of manufacturing. Due to high energy process and

expensive raw materials it is difficult to scale up and also harmful to the fragile temperature

sensitive active ingredient like protein. In cubosomes a strong driving force are exist for

development of liquid phase to cubosomes to avoid the high energy processing and produced

them in situ hence the hydro trophy dilution process are found to be consistently which

produced smaller more stable cubosomes the particles and growth are employed by

nucleation crystallization and precipitation method.9 This is achieved by dissolving the

monoolein in a hydro trope, such as ethanol, that prevents liquid crystalline formation.

Subsequent dilution of this mixture spontaneously “crystallizes” precipitates of the

cubosomes.

Powdered Cubosomes Precursors

Powdered cubosome precursors are composed of dehydrated surfactant coated with polymer.

Such powders offer advantages to liquid phase hydrotropic cubosome precursors. Hydration

of the precursor powders forms cubosomes with a mean particle size of 600 nm as confirmed

by light scattering and cryo-TEM8 the lipids used to make cubosomes are waxy, sticky

solids. Water-soluble non-cohesive starch coating on the waxy lipid prevents agglomeration

and allows control of particle size. Spray drying is excellent processes for this purpose the

encapsulation of particles are done. The process provide easy route to preload active drug in

to cubosomes prior to driving9. Spray-drying experiments are required a Pulvis Basic Unit.

That is a cylindrical chamber having a cyclone collector and air existed. The nozzle having

liquid orifice size of 0.1 cm is used to incorporate liquid to spray-dried into the top of the

spray dryer body. The air pressure having the 300 Kpa is pump by which orifice size is 0.25-

cm. Drying of liquid feed done by the heated, drying air at 2000C that flows down and passes

to the nozzle. That prevents any oxidation of the monoolein at the elevated temperatures and


569


liquid crystalline materials are forms that provide the high shear to disperse the high-

viscosity.

Cubosomes Application

High drugs payload due to high internal surface area and cubic crystalline structures.

Relatively simple method of preparation.

Control released of solublized substance is the most popular application of cubosomes.

It provides the drug in Nano size 10-500nm.

It mostly used in melanoma therapy.

Enhances the solubility of the poor water soluble drugs.

Capability of encapsulating hydrophilic, hydrophobic and amphiphilic substances.

Target release auctioned is shown.

It produces high level of dilution.

Manufacture of Cubosomes

Cubosomes can be manufactured by two distinct methods:

Top down technique

Bottom up technique[1,2]

Top down technique

Bulk cubic phase is first produced by the application of high energy such as high pressure

homogenization; it is processed into cubosomes nanoparticles. Bulk cubic phase resembles a

clear rigid gel formed by water-swollen cross linked polymer chains. The cubic phases are

differ in that they are a single thermodynamic phase and have periodic Liquid crystalline

structure Cubic phase’s ruptures in a direction parallel to the shear direction; the energy

required is proportional to the number of tubular Network.

The cubic phase’s exhibits yield stress that increases with increasing amount of bilayer

forming surfactant and oils. Warr & Chen gave the cubic phases may behave as lamellar

phases during dispersion with increasing shear, dispersed liquid crystalline particles are

forming at intermediate shear rates, whereas defect free bulk phase reforms at higher shear

rate.


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Bottom up Technique

In this cubosomes are allowed to form or crystallize from precursors. The bottom-up

approach first forms the Nanostructure building blocks and then assembles them into the final

material. It is more recently developed technique of cubosome formation, allowing

cubosomes to form and crystallize from precursors on the molecular length scale. The key

factor of this technique is hydro trope that can dissolve water insoluble lipids into liquid

precursors. This is a dilution based approach that produces cubosomes with less energy input

when compared top down approach. This method is more robust in large scale production of

cubosomes. The cubosomes at room temperature is by diluting monoolein-ethanol solution

with aqueous poloxamer 407 solution. The cubosomes are spontaneously formed by

emulsification. Another process is also developed to produce the cubosomes from powdered

precursors by spray drying technique. Spray dried powders comprising monoolein coated

with starch or dextran form cubosomes on simple hydration. Colloidal stabilization of

cubosomes is immediately provided by the polymers.

Preparation method of cubosomes

The cubosomes dispersion carried out by the fabrication method and emulsification method.

Fabrication method

GMO/P407 cubic gel GMO 5% and P407 1.0% were firstly melted at the 600 C in hot water

bath the X amount of drug is kept in to the melted mass and stirred continuously to dissolve.

Deionized water is added drop by drop and vortex mixers are set to the homogenization. It

kept in to 48 hrs at the room temperature the optically isotropic cubic gel are form and it

disturbed by mechanical stirring the crude dispersion was subsequently fragmented by

sonicater probe having the energy 200W under the cool temperature at then 200C in water

bath for the 20 min.

Emulsification method

In this method the GMO and P407 are put in to the water and it followed the ultra-sonication

the 5% GMO and1% P407and 5% ethanol in 89% water are taken GMO and P407 are melted

at the 600C and mixed the ethanoic solution was added to the melting. The resultant mixture

is added drop wise to deionized water preheated at the 700C. Ultra sonicated at maximum

power130 kW for 50min at the same temperature the disperse solution are kept in ambient

temperature and protected from light. Most compounds face the solubility problems. Hence,

with the advancement of chemical science, the need of development of pharmaceutical.


571


Technologies are also increasing and it depends upon drug to drug. The ketoconazole is

poorly water soluble drug. Solubility is main consent of the class II drugs during the oral

administration it shows the systemic adverse effect, hepatotoxicity and skin rash. The

marketed formulation of ketoconazole 2% topical gel was available but in MSDS data shows

the safety and efficacy area. To avoiding this evidence the ketoconazole by preparing the

1%ketoconazole emulgel that show the emollient and show onset of action fast.

MATERIAL AND METHOD

Kaetoconazole was gifted from Aarti Pharmaceuticals Ltd. Mumbai, Poloxomer 407 LR

Research-Lab Fine Chem. Industry – Mumbai. Glyceryl monooleate was gifted from LR

Estelle chemical Pvt. Ltd. Industry-Ahmadnagar and Sodium phosphate (monobasic &

dibasic) was gifted from LR Research-Lab Fine Chem. Industry – Mumbai, Methanol LR

Merck Specialties Pvt. Ltd. Mumbai.

Preparation of cubosomes

Polymer mixed and melted in a water bath at 60˚c to this mixture

Add drug.

Stir until completely dissolved, and then to this solution add drop by drop preheated

(up to 70 ˚ c) distilled water of suitable quantity (95%) by continuous stirring,

After complete Addition of water kept aside for one day to attained equilibration, there

is formation two phase systems Disturbed by stirring.

This whole system is taken into subjected for homogenization at 8000-10000 rpm for 2 hr.

under at room temperature Thus formed liquid dispersion of cubosomes was kept at a room

temperature, avoids direct sunlight and which will used for further study.


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Composition of cubosomes formulation

Table 1: Composition of Cubosomes Formulation.

Formulation Glyceryl Monooleate Poloxomer 407 Ketoconazole (gm) Water %

F1 4.8% 0.2% 1 100

F2 4.6% 0.4% 1 100

F3 4.4% 0.6% 1 100

F4 4.2% 0.8% 1 100

F5 4.0% 1.0% 1 100

F6 3.8% 1.2% 1 100

F7 3.6% 1.4% 1 100

F8 3.4% 1.6% 1 100

F9 3.2% 1.8% 1 100

Cubosomes biphasic solution

Figure 1: Cubosomes biphasic solution.

Preparation of cubosomes emulgel formulation[7,8]

Cubosomes emulgel obtain by the addition of the weighted amount of the carbopol 2% in

distilled water and kept it for half day for gelling and swelling of carbopol and then addition

of 1% sodium hydroxide which maintain the pH and obtain gelling consistency. The obtain

gel is diluted with the cubosomes solution containing only entrap consistency of cubosomes it

stir continuously and make its homogeneous emulgel formulation.

Evaluation of ketoconazole cubosomes

1. pH measurement.

2. Particle size analysis.

3. Zeta potential.

4. Entrapment efficiency.


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1. pH measurement

pH of formulation is determined by using digital pH meter by immersing the electrode in gel

formulation and pH is measure.

2. Particle size, polydispersity index

Particle size analysis determine by the (Nano ZS, Malvern, Worcestershire, UK) instrument

at 25ºC, Which is based on the Brownian motion. Sample were diluted in particle free

purified water to scattering intensity approximately 150-300keps. The mean z-average

diameter and polydispersity indices were obtain by cumulative analysis using MALVERN

software.

3. Zeta potential

Zeta potential is key indicator of the stability of formulation. The magnitude of zeta potential

indicates the degree of electronic repulsion between adjusts, similarly charge particle in

dispersion.

4. Entrapment efficiency

Entrapment efficiency is defined as the percentage amount of drug which is entrapped by the

cubosomes. For the determination of entrapment efficiency, the un-entrapped drug was first

separated by centrifugation at 15000 rpm for 30 minutes. The resulting solution was then

separated and supernatant liquid was collected. The collected supernatant was then diluted

appropriately and estimated using UV visible spectrophotometer at 242 nm.

Total amount of drug- unentraped drug

% Entrapment efficiency = ----------------------------------------------------- X 100

Total amount of drug

Evaluation of the ketoconazole cubosomale mulgel formulation

1. pH measurement.

2. Viscosity measurement.

3. Drug content.

4. Clarity.

5. Diffusion study.

6. Drug kinetic release.

7. Antimicrobial activity.

8. Accelerated stability study.

9. Statistical analysis.


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1. pH measurement

The obtain cubosomal emulgel were analyzed by digital pH meter by immersing the electrode

in gel formulation and pH is measure which is previously calibrated by the pH 7.

2. Viscosity measurement

Viscosity of the different formulation was determined at room temperature using a Brook

field viscometer. A Cole parmer viscometer was used to measure the viscosity of the

prepared gel bases. The spindle was rotated at 10 rpm to 100 rpm and viscosity were observe.

3. Drug content

Drug loaded cubosomes were mixed with methanol and sonicated for 10 min to obtain a clear

solution. Concentrations of drug were determined spectro-photometrically at λmax 242 nm.

Actual yield

Drug Content = ----------------------------------- X 100

Theoretical yield

4. Clarity test

The formulations were visually checked for clarity.

5. Diffusion study

In vitro skin permeation studies were performed by using a franze diffusion cell with a

receptor compartment capacity of 50ml. The synthetic cellophane membrane was mounted

between the donor and receptor compartment of the diffusion cell. The formulated

cubosomes gel of 1gm was placed over the drug release membrane (In the donor

compartment) and the receptor compartment of the diffusion cell was filled with phosphate

buffer pH 7.4. The whole assembly was fixed on a magnetic stirrer, and the solution in the

receptor. And the solution in the receptor compartment was constantly and continuously

stirred using magnetic beads at 50 rpm; the temperature was maintained at 37 ± 0.50C by

surrounding water in jacket. The samples of 1ml were withdrawn at time interval of 1, 2, 3, 4,

6, 8 and 12 hours and analyzed for drug content UV. Spectrophotometric ally at 287nm

against blank. The receptor phase was replaced with an equal volume of phosphate buffer at

each time of sample withdrawal. The cumulative amounts of drug from cubosomes

permeated through synthetic membrane plotted.


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6. Drug kinetic analysis

Drug kinetic analysis done for the ketoconazole emulgel were considered for various kinetic

models for the optimized batch F3.

7. Antimicrobial study

An agar diffusion method used for determination the antifungal activity of formulation.

Standard petri dish 9 cm containing medium to depth of 0.5cm were used. The sterility of the

lots was controlled before used. Inocula were prepared by suspending 1-2 colonies of candida

albicans (NCIM NO.3102) FROM 24 hr. Cultures in sabouraud’s medium in to tube contain

10 ml of sterile saline. The tubes were diluted with saline. The inoculum spread over the

surface of agar medium. The plate was dried at 35º C for 15 min prior to placing the

formulation. The boars of 0.5 cm diameter were prepaid and 20μl sample of formulation (1

%w/v) were added in the bores. After incubation at 35ºC for 24 hr. the zone of inhibition

around the boars are measure.

8. Stastical analysis

In order to compare the results ANOVA (Design expert dx 7) was used. Stability data were

compared using ANVA test. Data reported A statistically significant difference was

considered at is not less than 0.05.

RESULT AND DISCUSSION

Compatibility study

Fourier Transform Infrared Spectroscopy

Overlay FT-IR spectra of physical mixture of drug with Poloxomer 407, and Glyceryl

monooleate, showed matching peaks with the pure drug spectra. The characteristic peaks of

drug were also seen in the spectra of drug in combination with polymers which indicate

compatibility of drug with polymers.


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I.R. Spectrum of ketoconazole

Figure 2: IR spectrum of Ketoconazole.

The absorption bands of ketoconazole are characteristic of group present in its molecular

structure. The presence of absorption band corresponding to the functional group present in

structure of ketoconazole confirms the identification and purity of gifted ketoconazole

sample.

Compatibility spectrum of ketoconazole with excipients

Figure 3: Compatibility spectrum of ketoconazole with excipients.

Evaluation of Ketoconazole cubosomes

1. pH measurement: The pH of the cubosomes formulation from F1to F9 was found to be in

the range.


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Table 2: Observation table of pH.

Sr. No. Formulation code Observe pH (S.D)

1 F1 6.67±0.01

2 F2 7.02±0.02

3 F3 7.1±0.012

4 F4 6.98±0.01

5 F5 7.42±0.01

6 F6 6.44±0.02

7 F7 6.96±0.04

8 F8 7.51±0.01

9 F9 7.02±0.01

Particle size analysis: Particle sizes of cubosomes dispersion were found out by the Malvern

instrument it shows the result.

Effect of GMO: It is seen increase the GMO concentration particle size is reduced and

entrapment efficiency also increases.

Table 3: Observation table of pH.

Sr. No. Formulation code Observe pH (S.D)

1 F1 6.67±0.01

2 F2 7.02±0.02

3 F3 7.1±0.012

4 F4 6.98±0.01

5 F5 7.42±0.01

6 F6 6.44±0.02

7 F7 6.96±0.04

8 F8 7.51±0.01

9 F9 7.02±0.01

Graph of size distribution and PDI. Ratio of cubosomes particles.

Table 4: Ratio of cubosomal particle size.

Ratio of cubosomal particle size

Diam. (nm) % Intensity Width (nm)

Z-Average (d.nm): 111.7 Peak 1: 139.1 100.0 69.15

PdI: 0.193 Peak 2: 0.000 0.0 0.000

Intercept: 0.925 Peak 3: 0.000 0.0 0.000

Result quality: Good


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Figure 4: Cubosomal size distribution by Intensity.

Zeta potential

Zeta potential shows the stability of the cubosomes under the stress condition according ICH

guideline of stability study of various pharmaceutical formulation. Zeta potential is affected

by increased the particle size reduces the zeta potential vice versa in reduction in particle size

zeta potential is increases.

Graph of Zeta potential

Table 5: Zeta potential.

Mean (mV) Area (%) Width (mV)

Zeta Potential (mV): 34.2 Peak 1: 34.2 100.0

Zeta Deviation (mV): 5.09 Peak 2: 0.000 0.0 0.000

Conductivity (mS/cm): 0.413 Peak 3: 0.000 0.0 0.000

Result quality: Good

Figure 5: Graph for Zeta Potential.


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Entrapment efficiency

Entrapment efficiency are taken after centrifugation on high speed, The GMO concentration

which response the entrapment efficiency. The percent entrapment efficiency of 9

formulation are range were found between 59-92% that indicates that obtain result are depend

upon the selected independent variables.

Figure 6: Entrapment efficiency of cubosomes.

Evaluation of ketoconazole emulgel formulation

Viscosity measurement of the ketoconazole emulgel formulation

Viscosities are taken by Brook field viscometer it depend upon the polymeric concentration.

Table 6: Viscosity of Ketoconazole.

Rpm Viscosity(cps)

10 7209

20 6124

30 5186

50 4621

100 3702

Diffusion study

In vitro drug study shows that the how drug behaves in vitro, release study required for the

predicting the reproducibility of rate and drug released the cumulative drug released chart

shown in graph.


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Figure 7: Diffusion of emulgel compare with marketed drug.

Cumulative Drug Release of marketed vs. emulgel

Table 7: Viscosity of Ketoconazole.

Sr.no. Evaluation test Observation

1 pH measurement 6.8-7.4

2 Clarity Opaque

3 Homogencity Homogeneous

4 Drug content 97%

The obtain result shows that the cubosomal emulgel show efficient result then marketed one.

The in-vitro drug release shows the onset of fast diffusion along the biological membrane due

to high penetration of the glyceryl monooleate and drug class itself and decreased in particle

size of the drug shows good results.

Drug kinetic release: The kinetic analysis of the release data is shown in different kinetic

model. Emulgel obeys the zero order kinetics.

Figure 8: Drug kinetic release.


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Antimicrobial activity

The zone of inhibition observes for selected micro-organism is show in fig. result obtain from

antifungal activity F3 batch formulation were taken. The standard value of ketoconazole

against Candida albican for zone of inhibition is 18mm.The study indicates that Cubosomal

emulation and 100% efficacy.

Figure 9: Zone of inhibition of Ketoconazole emulgel.

Accelerated stability study of F3 batch

Stability study were taken after 3 month according to ICH guideline through accelerated

stability studies for optimized gel formulation F3 batch show compatible result pH of

formulation and zeta potential that two factor are consider.

Stability study.

Sr.No. Consider parameter Observation

1 Zeta potential 29.3 Mv

2 pH 7.2

Table 8: Zeta Potential.

Zeta potential

Mean (mV) Area

(%)

Width

(mV)

Zeta Potential (mV): 29.3 Peak 1: 29.3 100.0 4.27

Zeta Deviation (mV): 4.27 Peak 2: 0.00 0.0 0.00

Conductivity (mS/cm): 0.411 Peak 3: 0.00 0.0 0.00


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Figure 10: Graph of Zeta Potential.

Statistical Analysis

Statistical analysis of the result was determine using analysis of the variances (ANOVA) to

determine the significance of difference between groups in that following two variables are

consider Poloxomer and GMO. Statistical analysis revealed that increasing the GMO

concentration had significant statistical effect on entrapment efficiency. (F= 17.95 p value

<0.0029) whereas increasing the poloxomer concentration also had a significant effect on the

entrapment efficiency. (F= 87.38 p value < 0.0001).

Entrapment efficiency percentage for the prepared drug cubosomes ranged from 59.24% to

92.22%. The calculated E.E. values were analyzed using polynomial quadratic model with

adequate precision of 22.55 and reasonable difference between predicted R2 (0.9102) and the

adjusted R2 (0.9551).

The calculated equation for the EE analysis was

EE= 107.87 – 3.71 (GMO) – 15.3937 (Poloxomer)

Figure 11: 3D surface plot of Entrapment Efficiency.


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Particle size of the prepared cubosomal formulae ranged between 111.7 and 181.0 nm.

Particle values were subjected to polynomial analysis using quadratic model. Adequate

precision was calculated by the Design-Expert software to demonstrate the signal to noise

ratio, whereas the ratio greater than 4 is desirable indicating the validity of the utilized model

to navigate the design space. On the other hand, predicted R2 was calculated as a measure of

how good the model could predict a response value by comparing the calculated value with

the adjusted R2. Adequate precision was 8.813 with reasonable difference between the

predicted R2(0.5352) and the adjusted R2(0.7737).

The calculation equation for the PS analysis was:

PS= 2.14718- 0.013742(GMO) + 0.082778(Polyxomar)

Figure 12: 3D surface plot of Particle Size distribution.

CONCLUSION

Cubosomes can be formed by simple combination of biologically compatible lipids (GMO)

and water and are thus well suited for pharmaceutical and body tissue. The ability to form

cubosomes during manufacture offers enhanced flexibility for product development efforts.

The above research specifies cubosomal utility as immediate release drug carrier. Onset

released is achieved when they are formulated in gel form maintaining the cubosomes

structure. Although they possess advantageous characteristics, there is a still long way to go

before their clinical application.


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