materials and methods - shodhganga : a reservoir of indian...

Chapter 4 Materials and methods

Dept. of Pharmaceutics, KLE University’s College of Pharmacy, Belgaum 61

CHAPTER 4: MATERIALS AND METHODS

4.1 MATERIALS

4.1.1 Procurement of Drug and Excipients

The drugs, monomers, polymers, excipients, chemicals, reagents and equipments

used for various experiments used in this work are enlisted in following tables.

Drugs Gifted from / Purchased from

Salicylic acid S.D. Fine Chemicals Ltd., Mumbai

Ketoconazole FDC Limited, Raigad

Oxiconazole Nitrate KMWELL Biopharma Pvt Ltd., Bangalore

Materials Gifted / purchased from

Carbopol 934 NF Lubrizol Advanced Materials India Pvt. Ltd., 5th floor Omega, Hiranandani Business park, Powai, Mumbai – 400 076

Cellophane membrane Hi-media

Divinyl benzene Thermax Ltd., Pune

Ethylvinylbenzene Thermax Ltd., Pune

Eudragit RS 100 Degussa-Rohm GmbH & Co. (Germany)

Polyvinyl alcohol Sigma Aldrich, Germany

Styrene Thermax Ltd., Pune

Triethylcitrate Sigma Aldrich, Germany

All other chemicals and reagents used were of “analytical reagents” (AR) grade.



The following table explains details of equipments and glassware used in the

research work:

Instruments /

glassware

Supplier / Make Place of Work

3 Necked Reaction Vessel Blown USIC, Shivaji University,

Kolhapur

AUTOSORB-1C BET

analyzer Quantachrome, USA NCL, Pune

Mercury Intrusion

Porosimetry

Quantachrome

Equipments NCL, Pune

Differential Scanning

Calorimetry (SDT-2960) TA4000, Mettler, USA

College of Pharmacy,

Saswad, Pune

Franz diffusion cell Blown USIC, Shivaji University,

Kolhapur

FTIR Perkin Elmer, Spectrum

100 FTIR

Shivaji University,

Kolhapur

Malvern Particle Size

Analyzer (Mastersizer

2000, Version 2.0,)

Malvern Instruments

Ltd, UK

Poona College of

Pharmacy, Pune

Viscotech Rheometer

with Stress Rheologica

Basic software, version

5.0

Rheologica instruments

AB, Lund, Sweden

Poona College of

Pharmacy, Pune

Scanning Electron

Microscopy JEOL-JSM, 6360, Japan

Shivaji University,

Kolhapur

X- ray Diffractometry D8 Advanced, Bruker

AXS. Pune University, Pune



4.1.2 Drugs Profiles

1. Salicylic acid (Indian Pharmacopoeia, 1996)

Drug Category:

Keratolytic, Antiacne agent, Antiseborrheic, Antipsoriatic

Description:

Salicylic acid is a fine, white powder. It is used externally on the skin. It helps in

the treatment of athlete’s foot, ringworm of the scalp, and the removal of warts,

corns, and calluses. Salicylic acid is also incorporated into preparations for the

treatment of acne, dandruff, seborrhea, and insect bites.

Chemical IUPAC Name:

2-Hydroxybenzoic acid

Chemical Formula:

C7H6O3

Chemical Structure:

CAS Registry Number:

69-72-7

Average Molecular Weight:

138.121 g/mol

Physical Properties

Colour: Colourless or white

State/form: Solid-crystals



Description:

Colourless acicular crystals or a white crystalline powder. The synthetic form is

white but if prepared from natural methyl salicylate, it may have a slightly yellow

or pink tint. Salicylic acid is a white crystalline powder with a sweetish acrid

taste. If prepared from natural methyl salicylate, it may have a faint mint like

odour. It is available in forms of ointments, cream, gel, transdermal patches,

liquids and plaster.

Melting Point:

158-161°C

Solubility:

Freely soluble in ethanol (95%) and in ether; sparingly soluble in chloroform;

slightly soluble in water.

Existing topical therapy and its drawbacks:

Salicylic acid gel in the concentration of 0.5-5% once a day is used in the

treatment of acne and as a lotion in the concentration of 1.8-2% is applied on the

scalp once or twice a day for the treatment of dandruff. Because of its keratolytic

effect, salicylic acid is also used in the concentration of 2-10% cream for the

treatment of corns and calluses.

Salicylic acid is readily absorbed through the skin, slowly excreted in urine; and

systemic toxicity resulting from application to large area of the skin has been

reported. Topical application of 55% salicylic acid in Vaseline has shown good

skin penetration. However, it has a fairly strong affinity for polyethylene glycol

vehicle as it is adsorbed to polyethylene glycol, such that there is little release of

salicylic acid into sebaceous materials, so reducing the potential toxicity of the

percutaneous absorption. To minimize the absorption following topical

application, it should not be used for prolonged periods, in high concentration,

on large areas of the body, or on inflamed broken skin. Traditional formulations

of Salicylic Acid in ointment bases have disadvantages of being greasy and

irritant due to free crystals of the drug. (Alia A. Badawi, 2009)



Moderate or severe skin irritation; dryness and peeling of the skin; flushing and

redness of the skin are some of the commonly experienced side effects with

conventional topical dosage forms. Salicylism is sometimes also observed with

topical application.

2. Ketoconazole (Indian Pharmacopoeia, 1996)

Drug Category:

Antifungal

Description:

White to off-white, crystalline powder


Cis-1-acetyl-4-[2-(2, 4-dichlorophenyl)-2- (1-imidazolylmethyl)-1, 3-dioxolan-4-

ylmethoxy] phenyl-piperazine

Chemical Formula:

C26H28Cl2N4O4

Chemical Structure:


65277-42-1


531.44 g/mol

http://en.wikipedia.org/wiki/Carbon

http://en.wikipedia.org/wiki/Hydrogen

http://en.wikipedia.org/wiki/Chlorine

http://en.wikipedia.org/wiki/Nitrogen

http://en.wikipedia.org/wiki/Oxygen



Physical Properties

Colour: Colourless or white

State/form: Solid-crystals

Melting Point:

148-152°C

Solubility:

Freely soluble in dichloromethane; soluble in chloroform and in methanol;

sparingly soluble in ethanol (95%); practically insoluble in water and in ether.

Existing oral and topical therapy and its drawbacks:

It is widely recommended orally for the treatment of various fungal infections.

One of the major disadvantages of oral therapy in the treatment of skin infections

like Seborrhoeic dermatitis and Psoriasis is rapid relapse on the cessation of

therapy and the risk of hepatotoxicity.

Topical application of 2% cream or shampoo has shown beneficial effect in

Seborrhoeic dermatitis. However, topical application of ketoconazole causes

irritation, dermatitis or a burning sensation.

3. Oxiconazole Nitrate (FDA, 2012)

Drug Category:

Antifungal

Description:

Oxiconazole nitrate is a nearly white crystalline powder


2', 4’-dichloro-2-imidazol-1-ylacetophenone (Z)-[0-(2, 4-dichlorobenzyl) oxime],

mononitrate



Chemical Formula:

C18H13ON3CI4·HNO3

Chemical Structure:


64211-46-7


492.15g/mol

Melting Point:

137-138°C

Solubility:

Soluble in methanol; sparingly soluble in ethanol, chloroform and acetone, and

very slightly soluble in water.

Drawbacks of existing topical therapy:

Oxiconazole nitrate in the concentration of 1% as a cream and lotion is used in

the treatment of skin infections such as athlete’s foot, Jock itch and ringworm.

However, the topical treatment in some patients is associated with side effects

such as pruritis, burning sensation, irritation and allergic contact dermatitis,

folliculitis, erythema, fissure, maceration, rash, stinging and nodules.



4.1.3 Monomers and Polymers

1. Styrene

Other Names:

Phenylethene; vinyl benzene; cinnamene; styrol; ethenylbenzene; phenethylene;

diarex HF 77; styrolene; styropol


100-42-5

Description:

Styrene, is an organic compound with the chemical formula C6H5CH=CH2. Under

normal conditions, this aromatic hydrocarbon is a colourless oily liquid. It

evaporates easily and has a sweet smell, although high concentrations confer a

less pleasant odour.

Structure:

Molecular Formula:

C8H8

Molar Mass:

104.15 g/mol

Density:

0.909 g/cm³

Melting Point:

30°C (243.15 K)



Boiling Point:

145°C (418.15 K)

Solubility in Water:

< 1%

Stability:

Stable, but may polymerize upon exposure to light, normally shipped with a

dissolved inhibitor. Substances to be avoided include strong acids, aluminum

chloride, strong oxidizing agents, copper, copper alloys, metallic salts,

polymerization catalysts and accelerators.

Main Hazards:

Flammable: Vapour may travel considerable distance to ignition source.

Toxicology:

Toxic, carcinogen, mutagen, corrosive; causes burns to skin and eyes.

Lachrymator: harmful by inhalation, ingestion and through skin absorption.

Application:

Styrene is the precursor to polystyrene, an important synthetic material.

2. Divinylbenzene

Other Names:

Diethenylbenzene


1321-74-0

Description:

Divinylbenzene (DVB) is a clear yellow liquid with an aromatic odor. DVB is used

as a crosslinking agent, meaning it is used to join together individual chains

within a polymer. DVB is a mixture of isomers of divinylbenzene and



ethylvinylbenzene (EVB). Dow manufactures three DVB products: DVB-55, DVB-

63, and DVB-HP. They contain 56%, 63.5%, and 80% of the active cross-linker,

respectively.

Structure:

Molecular Formula:

C10H10

Melting Point:

-66.9 to -52°C

Boiling Point:

195°C

Solubility:

Insoluble in water; soluble in ethanol and ether.

Hazards:

DVB is a highly reactive chemical whose liquid and vapor are combustible. It is

stable under recommended storage conditions, which include maintaining

inhibitor concentration and effectiveness. Proper handling and storage

precautions must be observed when working with DVB. Exposure to elevated

temperatures can cause the material to polymerize or decompose. Avoid contact

with oxidizing materials, acids, metal halides, peroxides, brass, and copper.



Toxicology:

Eye contact with DVB may cause slight irritation with pain disproportionate to

the level of irritation to the eye tissues. Corneal injury is unlikely. Prolonged skin

contact may cause irritation with local redness, but is unlikely to result in

absorption of harmful amounts.

Repeated contact may cause skin burns. At room temperature, vapors are

minimal due to low volatility. Vapor from heated material may be hazardous on

single exposure.

Application:

DVB is an extremely versatile chemical cross-linking agent used to improve

polymer properties. DVB is used in the manufacture of adhesives, plastics,

elastomers, ceramics, biological materials, coatings, catalysts, membranes,

pharmaceuticals, specialty polymers, and ion exchange resins.

3. Eudragit RS 100

Nonpoprietary Names:

Ph.Eur.: Ammonio Methacrylate Copolymer, Type B

USP/NF: Ammonio Methacrylate Copolymer, Type B - NF

JPE: Aminoalkyl Methacrylate Copolymer RS

Synonyms: Acryl-EZE MP; Kollicoat MAE 30 D; polymeric methacrylates

Chemical Name:

Poly (ethyl acrylate-co-methyl methacrylate-co-trimethylammonioethyl

methacrylate chloride) 1:2:0.1

CAS Number: 33434 – 24 – 1



Chemical Structure

Description:

Polymethacrylates are synthetic cationic and anionic polymers of

Dimethylaminoethyl methacrylates, methacrylic acid and Methacrylate acid ester

in varying ratios. Several different types are commercially available and may be

obtained as dry powder, as an aqueous dispersion, or as an organic solution.

EUDRAGIT RS100 is a copolymer of ethyl acrylate, methyl methacrylate and a

low content of methacrylic acid ester with quaternary ammonium groups. The

ammonium groups are present as salts and make the polymer permeable. The

molar ratio of ethyl acrylate, methyl methacrylate and trimethylammonioethyl

methacrylate is approx. 1:2:0.1 in EUDRAGIT RS. It is a solid substance in the

form of colourless, clear to cloudy granules with a faint amine like odour.

Characteristics:

Customized release profile by combination of RL and RS grades in

different ratios

Suitable for matrix structures.

Dissolution:

Insoluble

Low permeability

pH independent swelling



Targeted drug release area: Time controlled release, pH independent

Alkali value: 152 mg KOH/ g polymer

Weight average molar mass: approx. 32,000 g/mol

Glass transition Temperature (Tg): ~65°C

Solubility

Eudragit RS 100 is soluble in methanol, ethanol and isopropyl alcohol

(containing approx. 3 % water), as well as in acetone, ethyl acetate and

methylene chloride to give clear to cloudy solutions. The substances are

practically insoluble in petroleum ether, 1 N sodium hydroxide and water.

Applications

Polymethacrylates are primarily used in oral capsule and tablet formulations as

film coating agent. Depending upon the polymer used, films of different

characteristics can be produced. Polymethacrylates are also used as binders in

both aqueous and organic wet-granulation process. Larger quantities (5-20%) of

dry polymer are used to control the release of an active substance from a tablet

matrix. Solid polymers may be used in direct-compression processes in

quantities of 10-50%. Polymethacrylate polymers may additionally be used to

form the matrix layers of transdermal delivery systems and have also been used

to prepare novel gel formulations for rectal administration.

4. Polyvinyl Alcohol

Nonproprietary Names:

PhEur: Poly (vinylis acetas),

USP: Polyvinyl alcohol

Synonyms:

Alcotex; Gelvatol; Lemol; Mowiol; Polyvinol; PVA; Vinyl alcohol polymer, PVOH,

INS No. 1203



Chemical Names: Ethenol homopolymer

C.A.S. Number: 9002-89-5

Definition:

Polyvinyl alcohol is a synthetic resin prepared by the polymerization of vinyl

acetate, followed by partial hydrolysis of the ester in the presence of an alkaline

catalyst. The physical characteristics of the product depend on the degree of

polymerization and the degree of hydrolysis.

Chemical Formula: (C2H3OR)n where R=H or COCH3 (randomly distributed)

Chemical Structure:

Empirical Formula: Weight range of approximately 20000-200000. (C2H4O)n.

The value of n for commercially available materials lies between 500 and 5000,

equivalent to a molecular weight.

Molecular Weight: 20000-200000.

Melting Point:

228°C for fully hydrolyzed grades; 180-190°C for partially hydrolysed grades

Solubility:

Soluble in water; slightly soluble in ethanol (95%); insoluble in organic solvents.

Dissolution requires dispersion (wetting) of solid in water at room temperature

followed by heating the mixture to about 900°C for approximately 5 min mixing

should be continued while the heated solution is cooled to room temperature.



Description

Polyvinyl alcohol is a water-soluble synthetic polymer. Polyvinyl alcohol for food

use is an odourless and tasteless, translucent, white or cream colored granular

powder. Typically a 5% solution of polyvinyl alcohol exhibits a pH in the range of

5.0 to 6.5. It has degree of hydrolysis of 86.5 to 89%. Polyvinyl alcohol has

excellent film forming, emulsifying, and adhesive properties. PVA is an atactic

material but exhibits crystallinity as the hydroxyl groups are small enough to fit

into the lattice without disrupting it.

Functional Category:

Coating agent; binder; sealing agent; lubricant; stabilizing agent; viscosity-

increasing agent and surface-finishing agent.

Applications:

Polyvinyl alcohol is used as an emulsion polymerization aid, as protective colloid,

to make polyvinyl acetate dispersions. It is used primarily in topical

pharmaceutical and ophthalmic formulations. It is used as stabilizing agent for

emulsions (0.25-3.0% w/v). Polyvinyl alcohol is also used as a viscosity-

increasing agent for viscous formulations such as ophthalmic products. It is used

in artificial tears and contact lens solutions for lubrication purposes, in sustained

release formulations for oral administration and in transdermal patches.

Polyvinyl alcohol may be made into microspheres when mixed with a

glutaraldehyde solution. It is also used as emulsifying agent.

Polyvinyl alcohol is the raw material to make other polymers like Polyvinyl

nitrate, Polyvinyl acetals, Polyvinyl butyral, and Polyvinyl formal.



4.2 METHODS

4.2.1 Preformulation Studies

Preformulation testing is the first step in the rational development of dosage

forms of a drug. It can be defined as an investigation of physical and chemical

properties of drug substance, alone and when combined with excipients. The

overall objective of preformulation testing is to generate information useful to

the formulator in developing stable and bioavailable dosage forms, which can be

mass-produced.

A thorough understanding of physicochemical properties may ultimately provide

a rationale for formulation design or support the need for molecular

modification or merely confirm that there are no significant barriers to the

compounds development. The goals of the program therefore are:

To establish the necessary physicochemical characteristics of a new drug

substance.

To determine its kinetic release rate profile.

To establish its compatibility with different excipients.

Hence, a preformulation study on the procured sample of drug includes

conducting physical tests and compatibility studies.

Characterization of Salicylic Acid, Ketoconazole and Oxiconazole Nitrate

Pure Drug

Organoleptic Properties

The drugs were tested for organoleptic properties such as appearance, colour,

taste, etc.

Melting Point Determination

The melting point of the drugs was determined by melting point apparatus.



Solubility Analysis

Preformulation solubility analysis was done to select a suitable solvent system to

dissolve the drug and also to test its solubility in the dissolution medium, to be

used.

Spectroscopic Studies

UV spectroscopy: (Determination of λ max)

The stock solutions (100μg/mL) of the drugs were prepared in ethanol (salicylic

acid) and methanol (Ketoconazole and Oxiconazole nitrate). The stock solutions

were appropriately diluted with the respective solvents to obtain a

concentration of 20μg /mL. The UV spectrum was recorded in the range of

200-400 nm on Schimadzu 1700 UV spectrophotometer to find the λ max.

IR Spectroscopy

The spectrum was recorded in the wavelength region of 4000 to 400 cm-1. A dry

sample of the drug and potassium bromide were mixed uniformly and filled into

the die cavity of sample holder and an IR spectrum was recorded using diffuse

reflectance FTIR spectrophotometer.

Construction of Calibration Curve for Drugs

The stock solution (100 μg/mL) was prepared by dissolving 10 mg of the drug in

ethanol (Salicylic acid) and methanol (Ketoconazole and Oxiconazole nitrate) in a

100 mL volumetric flask. From the stock solution, solutions containing 2, 4, 6, 8,

10, 12, 14, 16, 18 and 20 μg/mL of the drugs were prepared by appropriate

dilutions. Absorbance of these solutions were measured at 296 nm for Salicylic

acid, 238 nm for Ketoconazole and 211 nm for Oxiconazole nitrate against

respective blank solvents.



DRUG-EXCIPIENT COMPATIBILITY STUDIES

Drug-excipients compatibility studies were carried out for one month. The drug

with excipients Eudragit RS 100 & PVA were subjected to storage at room

temperature and elevated temperature at 45°C/ 75% RH in stability chamber for

one month. After 7, 14, 21 and 30 days the samples were taken to check the

following parameter.

Physical change

The samples were checked for physical changes such as discoloration, odor etc.

FTIR study

The dry sample of drug and potassium bromide were mixed uniformly and filled

into the die cavity of sample holder and an IR spectrum was recorded using

diffuse reflectance FTIR spectrophotometer.

4.2.2 FORMULATION DEVELOPMENT OF MICROSPONGES

4.2.2.1 Free Radical Polymerization Reactions: Fundamentals

It is possible to form addition polymers from monomers containing

C=C double bonds; many of these compounds polymerize spontaneously unless

polymerization is actively inhibited.

The simplest way to catalyze the polymerization reaction that leads to an

addition polymer is to add a source of a free radical to the monomer. The term

free radical is used to describe a family of very reactive, short-lived components

of a reaction that contain one or more unpaired electrons.

In the presence of a free radical, addition polymers form by a chain reaction

mechanism that contains chain initiation, chain propagation, and chain

termination steps.



a. Chain initiation

A source of free radicals is needed to initiate the chain reaction. The free radicals

are usually produced by decomposing peroxide such as di-tert-butyl peroxide or

benzoyl peroxide, shown below. In the presence of either heat or light, the

peroxides decompose to form a pair of free radicals that contain an unpaired

electron.

b. Chain propagation

The free radical produced in the chain initiation step adds to an alkene to form a

new free radical.

The product of this reaction can then add additional monomers in a chain

reaction.

c. Chain termination

Whenever pairs of radicals combine to form a covalent bond, the chain reactions

carried by these radicals are terminated.



The formation of branched polymers

We might expect the product of the free radical polymerization of ethylene to be

a straight chain polymer. As the chain grows, however, it begins to fold back on

itself. This allows an intra-molecular reaction to occur in which the site at which

polymerization occurs is transferred from the end of the chain to a carbon atom

along the backbone.

When this happens, branches are introduced onto the polymer chain. Free

radical polymerization of ethylene produces a polymer that contains branches in

between 1% and 5% of the carbon atoms. Of these branches, 10% contain two

carbon atoms, 50% contain four carbon atoms and 40% are longer side chains.

4.2.2.2 Preparation of Salicylic acid Microsponges by Liquid-liquid

Suspension Polymerization method:

Styrene and divinylbenzene in quantities as mentioned in the Table 5, for 9

formulations were taken in round bottom flask. 0.24 g of Dicalcium phosphate,

0.06 g of polyvinyl alcohol, 6.0 g of sodium sulphate and 0.6 g of benzoyl

peroxide were added. To this 360 mL of water was added, speed of the stirrer

was adjusted to 450-500 rpm to get the dispersion of monomer mixture. The

flask was flushed with nitrogen during stirring. To this, Salicylic acid equivalent

to 20% of monomer concentration was added through the neck of the flask to get

drug-loaded microsponges.



After adding the drug, the temperature was maintained at 84±2°C and the

reaction was then allowed to continue for 24 hrs maintaining the temperature

and speed of rotation constant. After 24 hrs, the particles were filtered and

washed with several portions of water and allowed to dry at 70°C. Unloaded

microsponges were prepared by following the same procedure except for the

addition of drug.

Figure 6: Reaction vessel for microsponge preparation by liquid-liquid

suspension polymerization

Based on 32 factorial design 9 unloaded microsponge formulations containing

styrene and divinylbenzene were prepared. Weight of styrene taken was 50, 60,

70 g with proportionate concentration of DVB (10, 15, 20% of styrene), as

described in the following table No. 5.



Table 5: Formulation batches of Salicylic acid microsponges

Formulation code Styrene Divinylbenzene

Level Amount (g) Level Amount (g)

F1 +1 70 +1 7

F2 0 60 0 6

F3 -1 50 -1 5

F4 +1 70 +1 10.5

F5 0 60 0 9

F6 -1 50 -1 7.5

F7 +1 70 +1 14

F8 0 60 0 12

F9 -1 50 -1 10

The microsponges were prepared by liquid-liquid suspension polymerization

method, the temperature and speed of stirrer was optimized.

Ethanol entrapment

Blank microsponges prepared by the above procedure, with different

crosslinking density and particle size using mineral oil as porogen instead of

drug, were entrapped by the following procedure:

1.5 g of drug was dissolved in 3 g ethyl alcohol. The first half of the drug solution

was added to the 1.5 g blank microsponges in an amber bottle. The bottle was

arranged on a roller mill and mixed for 1 hr. The mixture was dried in an oven at

65°C for 2.5 hrs. This process was repeated for a second entrapment step for the

remaining drug solution and the drying process was held at 50°C for 24 hrs.

(Mine Orlu., 2006).



4.2.2.3 Preparation of Ketoconazole and Oxiconazole nitrate Microsponges

by Liquid-liquid Suspension Polymerization method

Ketoconazole and Oxiconazole were found to be sensitive to reaction conditions

of suspension polymerization techniques. Hence quasi-emulsion solvent

diffusion method was chosen to prepare Eudragit based microsponges.

Quasi-emulsion Solvent Diffusion method: (Eudragit microsponges)

The processing flow chart is presented in Figure No. 7. To prepare the inner

phase, Eudragit RS 100 was dissolved in 3 mL of methanol and triethylcitrate

(TEC) was added at an amount of 20% of the polymer in order to facilitate the

plasticity. The drug was then added to the solution and dissolved under

ultrasonication at 35°C. The inner phase was poured into the PVA (72000)

solution in 200 mL of water (outer phase). The resultant mixture was stirred for

60 min, and filtered to separate the microsponges. The microsponges were

washed and dried at 40°C for 24h. (D’souza J. I., 2008)

Figure 7: Preparation of microsponges by quasi- emulsion solvent diffusion method



Seven different ratios of drug to Eudragit RS 100 (1:1, 3:1, 5:1, 7:1, 9:1, 11:1 and

13:1) were employed to determine the effects of drug : polymer ratio on physical

characteristics and dissolution properties of microsponges. Agitation speed

employed was 500 rpm using three blade propeller stirrers.

Table 6: Microsponge formulations using Eudragit RS100

Constituents Ketoconazole Microsponges

F10 F11 F12 F13 F14 F15 F16

Oxiconazole nitrate Microsponges

F17 F18 F19 F20 F21 F22 F23

Inner phase

Ketoconazole/

Oxiconazole nitrate 2.5 2.5 2.5 2.5 2.5 2.5 2.5

Eudragit RS 100 (g) 2.5 0.83 0.50 0.36 0.28 0.23 0.19

Methanol (mL) 3 3 3 3 3 3 3

Outer phase

Distilled water (mL) 200 200 200 200 200 200 200

PVA 72000 (mg) 50 50 50 50 50 50 50



4.2.3 EVALUATION OF MICROSPONGES

4.2.3.1 Determination of Production Yield and Loading Efficiency

The production yield of the microparticles was determined by calculating

accurately the initial weight of the raw materials and the last weight of the

microsponge obtained (Kilicarslan M., 2003).

The loading efficiency (%) of the microsponges can be calculated according to

the following equation:

4.2.3.2 Particle Size Analysis

Particle size analysis of prepared microsponges was carried by using Malvern

Particle Size Analyzer Hydro 2000 MU (A). Microsponges were dispersed in

double distilled water before running sample in the instrument, to ensure that

the light scattering signal, as indicated by particles count per second, was within

instrument’s sensitivity range.

Figure 8: Malvern Mastersizer 2000: Malvern particle size analyzer Hydro 2000 MU (A)

It is a flexible, modular and fully integrated, particle sizing system with assured

measurement performance from submicron to millimeter. It can measure



particle size of wet or dry particles from milligram quantities of precious

pharmaceuticals.

Figure 9: Fundamentals of particle size analysis

During the measurement, particles are passed through a focused laser beam.

These particles scatter light at an angle that is inversely proportional to their

size. The angular intensity of the scattered light is then measured by a series of

photosensitive detectors. The map of scattering intensity versus angle is the

primary source of information used to calculate the particle size. The scattering

of particles is accurately predicted by the Mie scattering model. The Mastersizer

2000 software, allows accurate sizing across the widest possible dynamic range.

4.2.3.3 Scanning Electron Microscopy

For morphology and surface topography, prepared microsponges were coated

with platinum at room temperature so that the surface morphology of the

microsponges could be studied by SEM.

The SEM, a member of the same family of imaging is the most widely used of all

electron beam tools (Goldstein J. I., 2003). The SEM employs a focused beam of

electrons, with energies typically in the range from a few hundred eV to about 30

keV, which is rastered across the surface of a sample in a rectangular scan

pattern. Signals emitted under this electron irradiation are collected, amplified,

and then used to modulate the brightness of a suitable display device which is

being scanned in synchronism with probe beam.



Figure 10: Scanning electron microscopy; JEOL-JSM, 6360, Japan

This arrangement has several important benefits:

1. The magnification, which is the ratio of the areas scanned on the display

device and on the sample, is obtained geometrically and so does not rely

on lenses.

2. Any emission from the sample that can be collected can be used to form

an image.

3. Multiple images using different signals can be collected simultaneously.

4. Because the signal is acquired sequentially it can be processed and

enhanced before it is displayed.

The resolution of the SEM cannot be better than the dimension of the pixels used

to display the image, and this limitation controls the imaging performance for all

magnifications lower than about 20,000 Χ. At higher magnifications, however,

the resolution may be determined by other considerations including the type of

signal that is being employed, the signal to noise ratio, the physical size of the

electron probe, and the width of the electron-solid interaction. Effects such as

sample charging and beam induced damage also, increasingly, influence

performance and the choice of operating conditions.



4.2.3.4 Infrared Spectroscopy

FTIR spectroscopy was conducted using Perkin Elmer, Spectrum 100 FT-IR

spectrometer. Spectrum was recorded in the wavelength region of 4000 to 400

cm-1. The procedure consisted of dispersing a sample in excess of potassium

bromide nearly at the ratio 1:100, mixed well, after which the mixture was kept

into the sample holder for analysis.

Figure 11: Perkin Elmer, Spectrum 100 FT-IR Spectrometer

Fourier-transform infrared (FTIR) spectroscopy (Griffiths pP. R., 1986) is based

on the idea of the interference of radiation between two beams to yield an

interferogram. The latter is a signal produced as a function of the change of path

length between the two beams. The two domains of distance and frequency are

interconvertible by the mathematical method of Fourier-transformation. The

radiation emerging from the source is passed through an interferometer to the

sample before reaching a detector. Upon amplification of the signal, in which

high-frequency contributions have been eliminated by a filter, the data are

converted to digital form by an analog-to-digital converter and transferred to the

computer for Fourier-transformation.



Figure 12: Schematic of a Michelson interferometer

This method is widely used in the pharmaceutical industry for the qualitative

and quantitative analysis of active and non-active ingredients. Infrared

spectroscopy can provide valuable additional structural information, such as the

presence of certain functional groups.



4.2.3.5 Differential Scanning Calorimetry (DSC)

Figure 13: Differential scanning calorimetry (SDT-2960), TA Inc., USA

Thermal analysis is an important evaluation technique to find any possible

interaction between the drug and used polymers. Any of such interaction may

reduce the drug entrapment efficiency of the polymer and may also alter the

efficacy of the drug. Such interaction can be identified by any change in

thermogram.

Thermograms of pure Salicylic acid, blank styrene microsponge, Salicylic acid

entrapped microsponge, pure Eudragit RS 100, pure ketaconazole, oxiconazole

nitrate and drug entrapped microsponges were obtained using DSC instrument,

Differential Scanning Calorimetry (SDT-2960); TA4000, Mettler, Japan. Indium

standard was used to calibrate the DSC temperature and enthalpy scale. The

powder sample of microsponges was hermetically kept in the aluminum pan and

heated at constant rate 5°C/min, over temperature range of 100 C to 250°C. An

inert atmosphere was maintained by purging nitrogen at the flow rate of 100

mL/min.



4.2.3.6 Powder X-ray Diffraction Studies

Figure 14: Powder X-Ray Diffractometer; D8 Advance, Bruker AXS

Diffraction techniques are perhaps the most definitive method of detecting and

quantifying molecular order in any system of pharmaceutical relevance (Saleki-

Gerhardt A., 1994). Conventional X-ray powder diffraction, also known as PXRD

can be used to quantify noncrystalline material down to the levels of 5%.

Furthermore, with temperature and environmental control, it can also be used to

follow the kinetics of phase transformation. However, it is important to consider

that the diffraction techniques only ‘see’ molecular order, and thus the disorder

is only implied by the absence of order (Hancock B. C., 1997).

PXRD is one of the most widely attempted quantification techniques because of

its simplicity and it measures differences in periodicities of atoms/molecules in a

powder sample (Stephenson G. A., 2001). PXRD patterns of crystalline forms

show strong diffraction peaks, whereas amorphous ones exhibit diffuse and halo

diffraction patterns.



Figure 15: Schematic of a PXRD

According to Bragg’s law, diffraction will occur when the conditions of the

following equation are satisfied:

nλ=2d.sinθ

Where d is the distance between the planes in a crystal, expressed in angstrom

units, n is the order of reflection (an integer), and λ is the wavelength of X-rays.

To verify the physical state of drug in pure state and the changes in the

crystallinity of the components of formulation, the PXRD study was carried out

by using X ray diffractometer. The voltage of 40 kV and a current of 40 mA for

generator were applied with Cu as the tube anode material. The samples of pure

drug and microsponge formulation were analyzed between 5° to 50° (2θ).



4.2.3.7 Characterization of Pore Structure

Physical and chemical gas adsorption and mercury intrusion porosimetry (MIP)

are the most widely used techniques to characterize powders and solid

materials. With nitrogen gas adsorption, depending on the equipment used pore

diameter range of 0.3–300 nm, i.e. mesopores and macropores, are determined.

Low-pressure mercury porosimetry determines macropores (pore diameter 14–

200 µm), and high-pressure porosimetry mesopores and macropores (pore

diameter 3 nm–14 µm), depending on the equipment.

Both of these techniques can provide reliable information about the pore

size/volume distribution, the particle size distribution, the bulk density and the

specific surface area for porous solids regardless of their nature and shape.

However, the applicable pore size ranges of each technique are different.

Figure 16: Pore size limits of gas adsorption (BET) and mercury intrusion

porosimetry

Gas sorption and mercury porosimetry can be complementary techniques.

Physical adsorption techniques can extend the lower size measurement down to

about 0.00035 mm diameter, thus probing the intra-particle microstructure.

Mercury porosimetry is paired with the gas sorption technique to obtain

porosity information in the large size range (greater than about 0.3 mm diameter

up to about 360 mm), which is not attainable by gas sorption. When using two

different techniques, one should not expect necessarily to obtain the same

results in the overlapping or common range of both instruments. However,

comparable results have been reported for some materials (Westermarck S.,

2000).



Mercury intrusion porosimetry (MIP)

Porosity parameters of microsponges such as intrusion–extrusion isotherms,

pore size distribution, total pore surface area, average pore diameters, shape and

morphology of the pores, bulk and apparent density can be determined by using

mercury intrusion porosimetry. Incremental intrusion volumes can be plotted

against pore diameters that represented pore size distributions. The pore

diameter of microsponges can be calculated by using Washburn equation

(Washburn E. W., 1921).

Pore morphology was characterized from the intrusion–extrusion profiles of

mercury in the microsponges as described by Orr (Orr J. C., 1969).

A typical MIP test involves placing a sample into a container, evacuating the

container to remove contaminant gases and vapors (usually water) and, while

still evacuated, allowing mercury to fill the container. This creates an

environment consisting of a solid, a non-wetting liquid (mercury), and mercury

vapor. Next, pressure is increased toward ambient while the volume of mercury

entering larger openings in the sample bulk is monitored. When pressure has

returned to ambient, pores of diameters down to about 12 mm have been filled.

The sample container is then placed in a pressure vessel for the remainder of the

test. A maximum pressure of about 60,000 psia (414 MPa) is typical for

commercial instruments and this pressure will force mercury into pores down to

about 0.003 mm in diameter. The volume of mercury that intrudes into the

sample due to an increase in pressure from Pi to Pi+1 is equal to the volume of the

pores in the associated size range ri to ri+1, sizes being determined by

substituting pressure values into Washburn’s equation (Dees P. J., 1981).



Figure17: Cross section of a penetrometer in which pressure has forced some

mercury into the pores of the sample and about 50% of the stem capacity has

been used

The measurement of the volume of mercury moving into the sample may be

accomplished in various ways. However, electronic means of detecting the rise

and fall of mercury within the capillary are much more sensitive, providing even

greater volume sensitivity down to less than a microliter. The measurement of a

series of applied pressures and the cumulative volumes of mercury intruded at

each pressure comprises the raw data set. A plot of this data is called the

intrusion curve. When pressure is reduced, mercury leaves the pores, or

extrudes. A plot of this data is called the extrusion curve. According to the shape

of the pores and other physical phenomena, the extrusion curve usually does not

follow the same plotted path as the intrusion curve. The intrusion and extrusion

curve gives information about the pore network.

The sample cup has a capillary stem attached and this capillary serves both as

the mercury reservoir during analysis and as an element of the mercury volume

transducer. Prior to the beginning of each analysis, the sample cup and capillary



are filled with mercury. After filling, the main source of mercury is removed

leaving only the mercury in the sample cup and capillary stem, the combination

being referred to as the penetrometer. Pressure is applied to the mercury in the

capillary either by a gas (air) or a liquid (oil). The pressure is transmitted from

the far end of the capillary to the mercury surrounding the sample in the sample

cup.

Figure 18: Typical intrusion-extrusion curve in mercury porosimetry

The capillary stem is constructed of glass (an electrical insulator), is filled with

mercury (an electrical conductor), and the outer surface of the capillary stem is

plated with metal (an electrical conductor). The combination of two concentric

electrical conductors separated by an insulator produces a co-axial capacitor.

The value of the capacitance is a function of the areas of the conductors, the

dielectric constant of the insulator, and other physical parameters. In the case of

this particular capacitor, the only variable is the area of the interior conductor as

mercury leaves the capillary and enters the sample voids and pores, or as it

moves back into the capillary when pressure is reduced. This is mechanically

analogous to a mercury thermometer in which case mercury moves in and out of

a calibrated capillary from a large bulb at one end. A small volume of mercury

entering or leaving a small capillary causes the length (and area) of the mercury



column to change significantly, thus providing volume-measuring sensitivity and

resolution. In the case of the thermometer, the change in volume is proportional

to the change in temperature by the coefficient of volumetric expansion of

mercury.

The capacitance value of the stem is monitored by a capacitance detector that,

similar to the pressure transducer electronics, produces an electrical signal that

is proportional to capacitance. Capacitance measurements are transformed into

volume measurements by knowledge of the diameter of the precision capillary

and the equation governing coaxial capacitors.

BET (Nitrogen adsorption) method

The AUTOSORB-1C (Quantachrome, USA) analyzer is microprocessor controlled

with a Windows 95, 98, 2000 based PC utilizing Quantachrome’s state-of-the-art,

data acquisition and data reduction software.

Surface area and pore size of the particles were determined using following

methods:

• Adsorption and desorption isotherms.

• Multi and single point BET surface area (including C constant and

correlation coefficient).

• Mesopore volume and area distribution (BJH and DH methods).



Figure 19: AUTOSORB-1C (Quantachrome, USA) analyzer

An empty sample cell was weighed, placed 0.043 g of the sample and then

inserted quartz-wool in it. The sample was activated at 115°C for 12 hours using

Nitrogen as adsorbate. After 12 hours the sample cell was removed from

activation station and was cooled down to room temperature and the weight was

taken again. The weight of sample cell was substracted from the final weight to

get the exact weight of the sample, which was 0.04 g. The sample was then

placed at analysis station and analysis was started at liquid nitrogen temperature

(-196°C). During the entire period of analysis P/Po tolerance and equilibrium

time were maintained to three and two respectively. During the analysis the

system was purged with helium. The data obtained was analyzed using

appropriate software Autosorb for Windows version 1.24.



4.2.3.8 In-vitro Release Study of Microsponges

Accurately weighed loaded microsponges (5 mg) were placed in 50 ml of

ethanol/methanol in 100 ml glass bottles. The later were horizontally shaken at

37°C at predetermined time intervals. Aliquot samples were withdrawn

(replaced with fresh medium) and analysed UV spectrophotometrically at 296

nm for Salicylic acid, 238 nm for Ketoconazole and 211 nm for Oxiconazole

nitrate. The contents of drugs were calculated at different time intervals up to

6hrs.

4.2.3.9 Stability Profile of Microsponge Formulation

The purpose of stability testing is to provide evidence on how the quality of an

active substance or pharmaceutical product varies with time under the influence

of a variety of environmental factors such as temperature, humidity, and light.

(Vadas E. B., 2000)

In any, rationale design and evaluation of dosage forms for drugs, the stability of

the active component is the major criteria in determining their acceptance or

rejection. During the stability studies the product is exposed to normal

conditions of temperature and humidity. However, the studies take a longer time

and hence it would be convenient to carry out the accelerated stability studies

where the product is stored under extreme conditions of temperature. To assess

the drug and formulation stability, stability studies were done according to ICH

and WHO guidelines. Optimized formulation sealed in aluminum packaging

coated inside with polyethylene, and various replicates were kept in the

humidity chamber maintained at 40±2°C and 75±5% RH for 6 months. The

samples were analyzed for the physical changes and in-vitro release profile at an

interval of 1 month for 6 months.



4.2.4 FORMULATION OF GEL LOADED WITH MICROSPONGES AND PLAIN

DRUG

Table 7: Composition of gels

Ingredients Quantity (% w/w)

Drug

(free or entrapped, equivalent to)

Salicylic Acid : 6

Ketoconazole : 2

Oxiconazole nitrate : 1

Propylene glycol 40

Methanol 8

Menthol 0.04

Methyl paraben 0.18

Sodium metabisulphite 0.10

Disodium edentate 0.10

Carbopol 934 1.00

Triethanolamine q. s.

Purified water q. s. to make 100

A clear dispersion of carbopol was prepared in water using moderate agitation.

Intermittent sprinkling of carbopol prevents lump formation resulting in clear

homogenous dispersion. Drug or drug containing microsponge formulation was

dispersed in propylene glycol and methanol. Various ingredients viz. paraben,

sodium metabisulphite and disodium edetate were dissolved in water and added

to the drug solvent system. Triethanolamine was used to neutralize and adjusted

to final weight with water. Gels prepared were degassed by ultrasonication

(Amin P. D., 1994).



4.2.5 EVALUATION OF GEL LOADED WITH MICROSPONGES AND PLAIN

DRUG

4.2.5.1 Determination of Viscosity

Viscosity of the formulated gels was determined by Brookfield Viscometer using

Spindle type 93/T-C.

4.2.5.2 Drug Diffusion from Microspongic Gels

The in-vitro measurement of drug permeation through cellophane membrane

was performed in Franz Diffusion cell (Mehdi A., 2006). 1 g of gels containing

free or entrapped drug were placed in the donor compartment, while the

receptor compartment contained 12 mL of the receptor phase. Aliquots of 0.5 mL

samples were withdrawn at suitable intervals from the receptor compartment

and the drug was assayed spectrophotometrically.

4.2.5.3 Safety Considerations (Draize Skin Irritation Testing)

The irritation potential of the gels containing free drug and drugs entrapped in

microsponges were evaluated in comparison to marketed gel by carrying out the

Draize patch test on rabbits (Draize J. H., 1944; Verneer B. J., 1991; Joshi M. D.,

2006). Animal care and handling throughout the experimental procedure was

performed in accordance to the CPCSEA guidelines. The experimental protocol

was approved by the Institutional Animal Ethical Committee. White New Zealand

rabbits weighing 2.5-3 kg were obtained and acclimatized before the beginning

of the study.

Primary Dermal Irritation Test

A. Rabbit screening procedure

1. A group of at least 6 New Zealand White rabbits were screened for the

study.

2. All rabbits selected for the study were in good health (rabbit exhibiting

snuffles, hair loss, loose stools, or apparent weight loss was rejected and

replaced).



3. 18 hrs. prior to application of the test substance, each rabbit was

prepared by clipping the hair from the back and sides using a small

animal clipper.

4. Six animals with skin sites that were free from hyperemia or abrasion,

due to shaving were selected for each group.

B. Study procedure

1. Four areas of skin, two on each side of the rabbit's back, were utilized for

sites of application.

2. Each animal serves as its own control.

3. Besides the test substance (marketed/in-house gels containing free drug

and gels containing drug entrapped in microsponges), a positive control

substance (a known skin irritant, formalin) and a negative control

(untreated patch) were applied to the skin.

4. The four intact (flee of abrasion) sites of administration were assigned a

code number:

5. Test substances applied were:

Group No. of

animals

Test substance applied at

Site 1 Site 2 Site 3 Site 4

Group 1 6 Positive control

Salicylic acid microspongic gel

Marketed product

Negative control


Ketoconazole microspongic gel

Marketed product

Negative control


Oxiconazole microspongic gel

Marketed product

Negative control



1. The pattern of administration makes certain that the test substance and

controls were applied to each position at least once.

2. Each test or control substance was held in place with a 1 Χ 1 sq. in. 12-ply

surgical gauze patch. The gauze patch was applied to the appropriate skin

site and secured with 1 in. wide strips of surgical tape at the four edges,

leaving the center of the gauze patch non-occluded.

3. 0.5 g of gel was weighed and placed on the gauze patch. The test

substance patch was placed on the appropriate skin site and secured. The

patch was subsequently moistened with 0.5 mL of physiological saline.

4. The negative control site was covered with an untreated 12-ply surgical

gauze patch (1 Χ1 sq. in).

5. The positive control substance and vehicle control substance were

applied to a gauze patch in the same manner.

6. The entire trunk of the animal was covered with an impervious material

for a 24 hrs. period of exposure, secured by wrapping several long strips

of athletic adhesive tape around the trunk of the animal. The impervious

material aids in maintaining the position of the patches and retards

evaporation of volatile test substances.

7. An Elizabethan collar was fitted and fastened around the neck of each test

animal. The collar remains in place for 24 hrs. exposure period. The

collars were utilized to prevent removal of wrappings and patches by the

animals, while allowing the animal’s food and water ad libitum.

8. The wrapping was removed at the end of the 24 hrs. exposure period. The

test substance skin site was wiped to remove any test substance still

remaining.

9. Immediately after removal of the patches, each 1 Χ 1 sq. in. test or control

site was outlined with an indelible marker by dotting each of the four

corners. This procedure delineates the site for identification.



C. Observations

1. Observations were made of the test and control skin sites 1 hr. after

removal of the patches (25 hrs. post-initiation of application). Erythema

and edema were evaluated and scored on the basis of the designated

values presented in Table 8.

Table 8: Evaluation of skin reactions

SKIN REACTION VALUE

Erythema and eschar formation

No erythema 0

Very slight erythema (barely perceptible) 1

Well-defined erythema 2

Moderate to severe erythema 3

Severe erythema (beet redness) to slight eschar formation (injuries in depth)

4

Necrosis (death of tissue) +N

Eschar (sloughing or scab formation) +E

Edema formation

No edema 0

Very slight edema (barely perceptible) 1

Slight edema (edges of area well defined by definite raising) 2

Moderate edema (raised approximately 1 mm) 3

Severe edema (raised more than 1 mm and extending beyond the area of exposure)

4

Total possible score of primary irritation 8



2. Observations were made again at 48 and 72 hrs. after application and

scores were recorded.

3. If necrosis was present or the dermal reaction needs description, the

reaction should be described.

4. Necrosis should receive the maximum score for erythema and eschar

formation (4) with a (+N) to designate necrosis.

5. When a test substance produces dermal irritation that persists for 72 hrs.

post-application, daily observations of test and control sites were

continued on all animals until all irritation caused by the test substance

resolves or until Day 14 post-application.

D. Evaluation of Results

1. A subtotal irritation value for erythema or eschar formation was

determined for each rabbit by adding the values observed at 25, 48, and

72 hrs. of post application.

2. A subtotal irritation value for edema formation was determined for each

rabbit by adding the values observed at 25, 48, and 72 hrs. of post

application.

3. A total irritation score was calculated for each rabbit by adding the

subtotal irritation value for erythema or eschar formation to the subtotal

irritation value for edema formation.

4. The primary dermal irritation index (PDII) was calculated for the test

substance or control substance by dividing the sum of total irritation

scores by the number of observations, 18 (3 days Χ 6 animals =18

observations).



Table 9: The categorization of dermal irritation; modification of the

original classification described by Draize (1944)

Score (PDII) Interpretation

0.0 Nonirritant

>0.0 and ≤ 0.5 Negligible irritant

>0.5 and ≤2.0 Mild irritant

>2.0 and ≤5.0 Moderate irritant

>5.0 and ≤ 8.0 Severe irritant

4.2.5.4 Antifungal Activity of Ketoconazole and Oxiconazole nitrate Gels

The antifungal activity of Ketoconazole and Oxiconazole nitrate from the

optimum formula (microspongic-gels) as well as the free Ketoconazole and

Oxiconazole nitrate and marketed formulations of the same were determined

using Candida albicans as a representative fungus, adopting the cup plate

method. The mean inhibition zone was calculated for each plate, and this value

was taken as an indicator for the antifungal activity.

Standard calibration curve of Ketoconazole and Oxiconazole nitrate using

cup plate method

A single well-isolated colony of Candida albicans of at least 1 mm in diameter

was picked from the culture plate (Sabouraud dextrose agar) using a disposable

plastic loop (10 μL) and suspended into a tube containing 10 mL of Sabouraud

dextrose broth. The resulting suspension was mechanically shaken for 30

seconds, and then incubated at 35°C for 24 hrs. One mL of the inoculum was

mixed with the melted Mueller-Hinton agar, then poured into a sterile petri dish,



and allowed to solidify. Wells were made by using sterile cork-borers. Six

concentrations of Ketoconazole and Oxiconazole nitrate were made by dissolving

the desired amount of Ketoconazole and Oxiconazole nitrate in a sterile Dimethyl

sulphoxide (DMSO). Each concentration was placed in each well, and the plates

were incubated aerobically at 37°C for 24 hrs. After incubation, the inhibition

zone diameter around each well was measured using a ruler, and a graph of

inhibition zone versus drug concentration was plotted.

Microbiological assay of Ketoconazole and Oxiconazole nitrate

One gram each of free Ketoconazole and Oxiconazole nitrate gel and gel

containing microspongic Ketoconazole and Oxiconazole nitrate and

Ketoconazole and Oxiconazole nitrate marketed formulations were placed in

each well with a control (blank gel). Mean inhibition zone of Ketoconazole and

Oxiconazole nitrate released from 5 plates for each formula was calculated.

Statistical analysis using ANOVA test followed by Dunnett’s Multiple Comparison

Test at level of significance of 0.05 was carried out to determine the degree of

significance between the test and the reference standard. (Ellaithy H. M., 2002).

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