Evaluation Most commonly SMEDDS are evaluated by...

EXPERIMENTAL

SPTM, SVKM’S, NMIMS, MUMBAI 78

Evaluation

Most commonly SMEDDS are evaluated by142

Thermodynamic Stability Studies:

The physical stability of a lipid based formulation is also important to its

performance, which can produce adverse effect in the form of precipitation of the

drug in the excipient matrix. In addition, the poor physical stability of the

formulation can lead to phase separation of the excipient, which affects not

only formulation performance, as well as visual appearance of formulation. In

addition, incompatibilities between the formulation and the gelatin capsules shell

can lead to brittleness or deformation, delayed disintegration, or incomplete release

of drug.

Thermodynamic stability studies can be conducted by exposing the systems to

1. Heating cooling cycle

2. Centrifugation test

3. Freeze thaw cycle

Dispersibility Test

The efficiency of self-emulsification of oral nano or micro emulsion is assessed by

using a standard USP XXII dissolution apparatus 2 for dispersibility test.

One milliliter of each formulation was added in 500 mL of water at 37 ± 1 0C. A

standard stainless steel dissolution paddle is used with rotating speed of 50 rpm

provided gentle agitation. The in vitro performance of the

formulations is visually assessed using the following grading system

Grade A: Rapidly forming (within 1 min) nanoemulsion, having a clear or bluish

appearance.

Grade B: Rapidly forming, slightly less clear emulsion, having a bluish

white appearance.

Grade C: Fine milky emulsion that formed within 2 min

Grade D: Dull, greyish white emulsion having slightly oily appearance that is slow

to emulsify (longer than 2 min).

Grade E : Formulation exhibiting either poor or minimal emulsification with large

oil globules present on the surface.

EXPERIMENTAL


Grade A and Grade B formulation will remain as nanoemulsion when dispersed in

GIT. While formulation falling in Grade C could be recommend for SEDDS

formulation.

Turbidimetric Evaluation

Nephelo /turbidimetric evaluation is done to monitor the growth of emulsification.

Fixed quantity of Self- emulsifying system is added to fixed quantity of

suitable medium (0.1N hydrochloric acid) under continuous stirring (50 rpm)

on magnetic hot plate at appropriate temperature, and the increase in turbidity is

measured, by using a turbidimeter. However, since the time required for complete

emulsification is too short, it is not possible to monitor the rate of change of

turbidity (rate of emulsification).

Viscosity Determination

The SEDDS system is generally administered in soft gelatin or hard gelatin

capsules. So, it can be easily pourable into capsules and such systems should not

be too thick. The rheological properties of the micro emulsion are evaluated by

Brookfield viscometer. This viscosities determination conform whether the system

is w/o or o/w. If the system has low viscosity then it is o/w type of the system and if

a high viscosity then it is w/o type of the system.

Droplet Size Analysis and Particle Size Measurements

The droplet size of the emulsions is determined by photon correlation

spectroscopy (which analyses the fluctuations in light scattering due to Brownian

motion of the particles) using a Zetasizer able to measure sizes between 10 and 5000

nm. Light scattering is monitored at 25°C at a 90° angle, after external

standardization with spherical polystyrene beads. The nanometric size range of the

particle is retained even after 100 times dilution with water which proves

the system‟s compatibility with excess water.

Refractive Index and Percent Transmittance

Refractive index and percent transmittance proved the transparency of formulation.

The refractive index of the system is measured by refractometer by putting a drop

of solution on slide and it comparing it with water (1.333). The percent

EXPERIMENTAL


transmittance of the system is measured at particular wavelength using UV-

spectrophotometer by using distilled water as blank. If refractive index of system is

similar to the refractive index of water (1.333) and formulation have percent

transmittance > 99 percent, then formulation have transparent nature.

Electro Conductivity Study

The SEDD system contains ionic or non-ionic surfactant, oil, and water. This

test is performed for measurement of the electro conductive nature of system.

The electro conductivity of resultant system is measured by electro conductometer.

In conventional SEDDS, the charge on an oil droplet is negative due to presence of

free fatty acids.

In vitro Diffusion Study

In vitro diffusion studies w e re carried out to study the drug release behavior of

formulation from liquid crystalline phase around the droplet using dialysis

technique.

Drug Content

Drug f r o m pre-weighed SEDDS is extracted by dissolving in suitable

solvent. Drug content in the solvent extract was analyzed by suitable

analytical method against the standard solvent solution of drug.

Zeta potential

The charge of the oil droplets of SMEDDS is another property that should be

assessed.The charge of the oil droplets in conventional SMEDDS is negative due

to the presence of free fatty acids; however, incorporation of a cationic lipid,

such as oleylamine at a concentration range of 1.0-3%, will yield cationic

SMEDDS. Thus, such systems have a positive n-potential value of about 35-45

mV. This positive n-potential value is preserved following the incorporation of the

drug compounds.

Polarity

Emulsion droplet polarity is also a very important factor in characterizing

EXPERIMENTAL


emulsification efficiency. The HLB, chain length and degree of unsaturation of the

fatty acid, molecular weight of the hydrophilic portion and concentration of the

emulsifier have an impact on the polarity of the oil droplets. Polarity represents the

affinity of the drug compound for oil and/or water and the type of forces

formed. Rapid release of the drug into the aqueous phase is promoted by polarity.

Drug precipitation /stability on dilution

There are chances of precipitation of drug from SMEDDS upon dilution with

aqueous fluid.143-144

The ability of SMEDDS to maintain the drug in solubilised

form is greatly influenced by the solubility of the drug in oil phase. If the

surfactant or co-surfactant is contributing to the greater extent in drug

solubilisation then there could be a risk of precipitation, as dilution of SMEDDS

will lead to lowering of solvent capacity of the surfactant or co-surfactant, hence it

is very important to determine stability of the system after dilution. This is usually

done by diluting a single dose of SMEDDS in 250 ml of 0.1N HCl solution. This

solution is observed for drug precipitation if any. Ideally SMEDDS should keep the

drug solubilized for four to six hours assuming the gastric retention time of two

hours.

FACTORS AFFECTING SMEDDS

Nature and dose of the drug

Drugs which are administered at very high dose are not suitable for SMEDDS

unless they exhibit extremely good solubility in at least one of the components of

SMEDDS, preferably lipophillic phase. The drugs which exhibit limited solubility in

water and lipids (typically with log P values of approximately 2) are most difficult

to deliver by SMEDDS. The ability of SMEDDS to maintain the drug in

solubilised form is greatly influenced by the solubility of the drug in oil phase.

As mentioned above if surfactant or co-surfactant is contributing to the greater

extent in drug solubilisation then there could be a risk of precipitation, as dilution

of SMEDDS will lead to lowering of solvent capacity of the surfactant or co-

surfactant. Equilibrium solubility measurements can be carried out to anticipate

potential cases of precipitation in the gut. However, crystallisation could be slow

EXPERIMENTAL


in the solubilising and colloidal stabilizing environment of the gut. Pouton‟s study

reveal that such formulations can take up to five days to reach equilibrium and

that the drug can remain in a super-saturated state for up to 24 hours after the initial

emulsification event. It could thus be argued that such products are not likely to

cause precipitation of the drug in the gut before the drug is absorbed, and indeed

that super-saturation could actually enhance absorption by increasing the

thermodynamic activity of the drug. There is a clear need for practical methods to

predict the fate of drugs after the dispersion of lipid systems in the gastro-intestinal

tract.

Polarity of the lipophillic phase

The polarity of the lipid phase is one of the factors that govern the drug release

from the microemulsions. The polarity of the droplet is governed by the HLB, the

chain length and degree of unsaturation of the fatty acid, the molecular weight of

the hydrophilic portion and the concentration of the emulsifier. In fact, the

polarity reflects the affinity of the drug for oil and/or water, and the type of forces

formed. The high polarity will promote a rapid rate of release of the drug into the

aqueous phase. This is confirmed by the observations of Sang-Cheol Chi, who

observed that the rate of release of idebenone from SMEDDS is dependant upon

the polarity of the oil phase used. The highest release was obtained with the

formulation that had oil phase with highest polarity.

Application

Improvement in Solubility and Bioavailability If drug is formulated in SEDDS, then it increases the solubility

145 because it

circumvents the dissolution step in case of Class-П drug (Low solubility/high

permeability). 146-148

Protection against Biodegradation

The ability of self-emulsifying drug delivery system to reduce degradation as well

as improve absorption may be especially useful for drugs, for which both low

solubility and degradation in the GI tract contribute to a low oral bioavailability.

Many drugs are degraded in physiological system, may be because of acidic PH in

EXPERIMENTAL


stomach, hydrolytic degradation, or enzymatic degradation etc. Such drugs when

presented in the form of SEDDS can be well protected against these degradation

processes as liquid crystalline phase in SEDDS might be an act as barrier

between degradating environment and the drug.

4E.2 Literatures review

1. Bok Ki Kang et al in 2004 reported enhancement in bioavailability of

simvastatin by using the technique of self microemulsifying drug delivery

systems. 149

2. Weu Yu et al reported enhanced bioavailability of silymarin by making use of

self microemulsifying drug delivery systems in 2006. 150

3. Ping zhang et al reported improvement in biovailability of oridonin by

preparing slelf microemulsifying drug delivery systems for the same in

2008.151

4. Jong Soo Woo reported reduction in food effect and improvement in

biovailability of itraconazole in 2008.152

5. M.Cirri et al reported development of liquid formulations of xibernol, an

lipopilic drug, by enhancing solubility through formulation of self-micro

emulsifying drug delivery systems in 2007.153

6. Rene holm et al in 2003 and Shui-Mei Khoo et al in 1998, reported

enhancement of in vivo absorption of halofantrine, a poorly soluble

compound by formulating it into self microemulsifying drug delivery system

in 2003. 154-155

7. Patel A.R et al reported improvement in in-vivo absorption of finofibrate by

self microemulsifying drug delivery systems in 2007.156

EXPERIMENTAL


4E.3 Materials

Materials used for preparation of SMEDDS were

Table 4E.2 Excipients used for formulation of SMEDDS

Labrafil M 2125 Labrasol

Maisin 35-1

Labrafac PG

Labrafil M 1944

Lauroglycol FCC

Cremophore RH 40

Lauroglycol 90

Cremophore EL

Peceol

Transcutol

Propylene glycol

Polyethylene glycol 400

Isopropyl myristate

Tween 80

4E.4 Solubility studies in modified oils, surfactants and co surfactants

Excess amounts of drug was added to 1gm of modified oils, surfactants and co

surfactants in glass vials .Solution was vortexed for 2 minutes using cyclomixer and

then shaken in rotary shaker for 2 days at 37◦C .Resultant solutions were then

centrifuged for 15 minutes at 2000 rpm. Supernatant was taken diluted suitably with

methanol, filtered through whatmann filter paper 0.45 μm pore size and absorbance

was taken. Each experiment was carried out in triplicate.

4E.5 Preparation of SMEDDS

Based on the results of solubility following combinations of oils,surfactants and co-

surfactants were tried.

Different combinations of surfactant and co-surfactant(S/Cos) were tried in ratios of

surfactant: co-surfactant such as 1:1, 1:2, 1:3, 2:1, 3:1 and 4:1 respectively.

Concentration of oil in s/cos system was varied from 10%-50%.

EXPERIMENTAL


Table 4E.3 Combinations for formulation of SMEDDS

Sr.No. Oil Surfactant Co-surfactant

1 Labrafil M 2125 Cremophore RH40 Transcutol

2 Maisin 35-1 Cremophore RH40 Transcutol

3 Labrafil M 1944 Cremophore RH40 Transcutol

4 Labrafil M 2125 Cremophore RH40 PEG 400

5 Maisin 35-1 Cremophore RH40 PEG 400

6 Labrafil M 1944 Cremophore RH40 PEG 400

7 Labrafil M 2125 Cremophore RH40 Ethanol

8 Maisin 35-1 Cremophore RH40 Ethanol

9 Labrafil M 1944 Cremophore RH40 Ethanol

10 Labrafil M 2125 Cremophore EL Transcutol

11 Maisin 35-1 Cremophore EL Transcutol

12 Labrafil M 1944 Cremophore EL Transcutol

13 Labrafil M 2125 Cremophore EL PEG 400

14 Maisin 35-1 Cremophore EL PEG 400

15 Labrafil M 1944 Cremophore EL PEG 400

16 Labrafil M 2125 Cremophore EL Ethanol

17 Maisin 35-1 Cremophore EL Ethanol

18 Labrafil M 1944 Cremophore EL Ethanol

4E.6 Characterization of systems

4E.6 .1 Thermodynamic stability of the systems

A. Heating cooling cycle

Test systems were exposed to six cycles between 4 ˚C and 40˚C with storage at

each temperature was not less than 48 hours.

Systems showing turbidity at the end of test period were discarded. Selected stable

systems were subjected to centrifugation test.

B. Centrifugation test

Test formulations were subjected to centrifugation test by rotating them at 3500

rpm for 30 minutes. Systems which were not showing any phase separation were

tested for freeze –thaw stress test.

EXPERIMENTAL


C. Freeze thaw testing

Test formulations were exposed to three cycles of not less than 48 hours at -21º C

and +25ºC. Formulations stable in the testing were considered for further

evaluation.

4E.6 .2 Visual observation of self microemulsifying properties

Method reported by Nazzal et al for visual assessment of self microemulsifying

properties was used with modification.157

Initial screening of self

microemulsifying properties of the systems were judged by dispersing them in

volumetric flask of 50 ml water and adding 0.5 gm of prepared system, time

required for complete dispersion in terms of volumetric flask inversions and

appearance of the dispersion was observed followed by dispersing same amount

of SMEDDS in 900 ml of water in USP type II apparatus at 50 RPM and

maintaining temperature at 37±0.5C.Depending on time required for dispersion

and appearance, systems were classified into following categories-

Grade A-Rapidly forming (within 1 min) nanoemulsion , having a clear or bluish

appearance

Grade B- Rapidly forming (within 1-2 Minutes) nanoemulsion , having a clear or

bluish appearance

Grade C- Nanoemulsion , having a clear or bluish appearance formed in more

than 2 minutes

Grade D-Rapidly forming, slightly less clear emulsion, having a bluish white

appearance

Grade E- Fine milky emulsion that formed within 2 minutes

Grade F – Dull, grayish white emulsion having slightly oily appearance that is

slow to emulsify (longer than 2 minutes)

Grade G- Formulation, exhibiting either poor or minimal emulsification with

large oil globules present on surface.

The formulations that passed stability and also dispersibility test in grade A were

selected for further studies.

EXPERIMENTAL


4E.6 .3 Pseudoternary phase diagram

Pseudo ternary phase diagrams of oil ,surfactant /cosurfactant ratio (s/cos), and

water were developed using the water titration method.The mixtures of oil and

s/cos at certain weight ratios were diluted with water in a dropwise manner.158

For each phase diagram , selected mixtures were titrated with water and visually

observed for phase clarity .Boundaries of self microemulsifying region of the

selected systems was found out by noting the amount of water required for

transparent to turbid and turbid to transparent transitions of the given systems.

Phase diagrams were constructed using chemix ternary phase diagram software.

4E.6 .4 Particle size measurement/Globule size analysis

The average droplet size and zeta potential of selected systems were determined

by photon correlation spectroscopy using Malvern Zetasizer159

. The selected

formulations were dispersed in water (diluted 100 times) and placed in

electrophoretic cell for measurement.

4E.6 .5 Drug loading capacity

Drug laoding capacity in selected systems was determined by adding increasing

amount of drug in systems. Effect of increase in drug loading on self micro

emulsifying region and globule size were also determined.

4E.6 .6 In vitro multimedia dissolution studies

In vitro multimedia dissolution studies in different solvents as water, 0.1N HCl

phosphate buffer pH 6.8 and OGD medium by following the procedure described

in dissolution method.

4E.6 .7 Saturation solubility testing

Saturation solubility testing was carried out in water, 0.1 N HCl and phosphate

buffer 6.8 by taking 10 ml of each each solvent and adding excess amount of drug

present in the form of SMEDDS. All mixture were vortexed for 2 minutes using

cyclomixer and then kept in rotary shaker for 48 hours at 37º C. Resultant

solutions were then centrifuged for 15 minutes at 2000 rpm, supernatant was

EXPERIMENTAL


taken, diluted suitably and absorbance was taken. Concentration in each was

calculated

4E.6 .8 Drug content

Amount equivalent to 8 mg dissolved in methanol by standard diluting procedure

to get a concentration of 8 ppm and injected into HPLC. Concentration of sample

was calculated from peak area.

4F Solubility enhancement by preparation of nanoparticles

Various methods are reported for the preparation of nanoparticles as described before.

Based on the literature survey following methods for the preparation of nanoparticles

were selected for the drug

Ionic gelation using chitosan and tripolyphosphate

Antisolvent precipitation using supercritical carbon dioxide

Nanoencapsulation with polymers

4F.1 Preparation of nanoparticles with supercritical Antisolvent precipitation

4F.1.1 Introduction

Micronization is an important procedure used in the pharmaceutical industry to

reduce the particle size of active pharmaceutical ingredients, resulting in increase of

their dissolution rate, and hence bioavailability. However, conventional techniques

like jet milling and spray drying neither neither produce very neither narrow and

controlled size and distribution of particle nor prevent drug from thermal

degradation. Therefore, several supercritical fluids based techniques have been

proposed for the production of micronic and nanometric particles of pharmaceutical

compounds.

Recently, particle formation processes based on the use of supercritical fluids as

solvents or antisolvents have been introduced as a viable means of controlling

particle formation.

A SCF exists as a single phase above its critical temperature (Tc) and pressure (Pc).

EXPERIMENTAL


SCFs have properties useful to product processing because they are

intermediate between those of pure liquid and gas (i.e., liquid-like density, gas-like

compressibility and viscosity and higher diffusivity than liquids). Moreover, the

density, transport properties (such as viscosity and diffusivity), and other physical

properties (such as dielectric constant and polarity) vary considerably with small

changes in operating temperature, pressure, or both around the critical points.

Hence, it is possible to fine- tune a unique combination of properties necessary for a

desired application. Commonly used supercritical solvents include carbon

dioxide,nitrous oxide, ethylene, propylene, propane,n-pentane, ethanol, ammonia,

and water.Carbon dioxide is one of the most commonly used SCFs because of its

low critical temperature (Tc = 31.10C) and pressure (Pc = 73.8 bar). Apart

from being nontoxic, nonflammable, and inexpensive, the low critical temperature

of CO2 makes it attractive for processing heat- labile molecules.160

Figure 4F.1 Diagram of supercritical region

Table 4F.1 Critical conditions for some solvents

Substance Tc, K Pc,

atm

Density

(g/ml) Ammonia 405.6 112.5 0.24 Benzene 562.1 48.3 0.30

Carbon dioxide 304.2 72.9 0.47

Ethane 305.5 48.2 0.20

Ethanol 516.6 63.0 0.28

Methane 190.6 45.8 0.16

Propane 370.3 41.9 0.22

Chloroform 299.3 47.9 0.62

Water 647.3 218.3 0.32

EXPERIMENTAL


Basic techniques in SCF technology 161-162

1) Rapid Expansion of Supercritical Solutions

A supercritical solvent saturated with a solute of interest is allowed to expand at a

very rapid rate, causing the precipitation of the solute.

Figure 4F.2 RESS technique

2) Gas Antisolvent Recrystallisation

The solubility of pharmaceutical compounds in supercritical solvents can be

decreased by using SFs in gaseous form as antisolvents. It is possible to induce

rapid crystallization by introducing the antisolvent gas into a solution containing

dissolved solute. One of the requirements for this approach is that the carrier solvent

and the SF antisolvent must be at least partially miscible. This process works in a

semi batch mode, with the supercritical solvent introduced into an already existing

stationary bulk liquid phase.

EXPERIMENTAL


Figure 4F.3 Precipitation with compressed fluid antisolvent

3) Precipitation with Compressed Fluid Antisolvent

The s o l u t e can be crystallized from a solution using antisolvents in two

ways

• Gas antisolvent recrystallisation (GAS) method; or

• By spraying liquid into the SF antisolvent. In the latter, the antisolvent

rapidly diffuses into the liquid solvent and the carrier liquid solvent a schematic

view of the rapid expansion of supercritical solutions (RESS) process.

Figure 4F.4 Schematic representation of Gas antisolvent or SAS laboratory

scale apparatus ( C) CO2 cylinder; L) liquid solution; N) N2 cylinder; H)

heat exchanger; M) saturator; P) precipitator; S) condenser.)

EXPERIMENTAL


4) Impregnation or infusion of polymers with bioactive materials

Some gases cause swelling of polymers or drug carriers at high pressures. This

swelling behavior can be exploited for various such as control delivery of drugs.

Substances such as fragrances, pest control agents, and pharmacologically

active materials can be impregnated with a solid polymer, which is exposed to a

supercritical fluid during the impregnated process.

5) Solution enhanced dispersion by Supercritical Fluid

This technique was developed at the University of Bradford to overcome some of

the limitations of the RESS and GAS methods. The drug solution and the

SF are introduced simultaneous into the arrangement causing rapid dispersion,mixing

and Extraction of the drug solution solvent by SF leading to very high super

saturation ratios.

The temperature and pressure together with accurate metering of flow rates of

drug solution and SF through a nozzle provide uniform condition for particle

formation. This helps to control the particle size of the product and by choosing

an appropriate liquid solvent it is possible to manipulate the particle morphology

Applications of SCFs to increase the solubility of poorly soluble drugs

1) Micro particles and Nanoparticles

SCF technology is useful in obtaining micro and nanoparticles of drugs.Reverchon et

al in 1999, reported formation of micro and nano particles of antibiotics using SAS

process.163

2) Inclusion complexes

For many nonpolar drugs, previously established inclusion complex preparation

methods involved the use of organic solvents that were associated with high

residual solvent concentration in the inclusion complexes.

Earlier, cyclodextrins were used for the entrapment of volatile aromatic

compounds after supercritical extraction. Based on this principle, several attempts

were reported to form inclusion complexes of drugs by supercritical fluids.164-165

EXPERIMENTAL


3) Solid Dispersions

SCF techniques can be applied to the preparation of solvent-free solid dispersion

dosage forms to enhance the solubility of poorly soluble compounds. Traditional

methods suffer from the use of mechanical forces and excess organic solvents.A

solid dispersion of carbamazepine in polyethylene glycol 4000 (PEG-4000) increased

the rate and extent of dissolution of carbamazepine.

4) Solubilization of pharmaceuticals

RESS technology has been used for most of pharmaceutical compounds below 600C

and 300 b a r s showed a considerable higher solubility.

5) Micronization of Pharmaceuticals

The RESS process has been shown to be capable of forming micron-sized particals.

Krukonics et al., 1984, first extensively studied RESS in micronization of a

wide variety of materials, including pharmaceuticals, biological and polymers.

He produced uniform submicron powder of estradiol.

4F.1.2 Literature review

1. Improvement in dissolution rate of a poorly soluble drug cilostazol by

supercritical antisolvent process was reported by Min-Soo Kim et al in

2007.166

2. D.Wong et al in 2005 reported improvement in physicochemical

characterization,solubility and dissolution rate of felodipine solid dispersions

prepared by supercritical antisolvent precipitation process in 2005.167

3. Kalogiannis C. et al ,reported formation of amorphous amoxicillin by the use

of antisolvent precipitation technique.168

4. Kikic et al reported enhancement in solubility of atenolol by processing in

2006.169

5. Reverchon et al reported formation of amorphous rifampicin nanoparticles in

2002.170

6. Reverchon et al reported formation of amorphous tetracycline nanoparticles in

1999.171

EXPERIMENTAL


7. E.Reverchon et al in 2006 reported formation of spherical amorphous particles

of cephalosporin with improved kinetic property.172

8. Y.Chang et al in 2008, reported formation of submicronic particles of

sulphamethoxazole. 173

9. Min–Soo Kim et al, reported enhanced bioavailability of atorvastatin calcium

due to change in nature to amorphous in 2008. 174-175

4F.1.3 Materials

Drug, methanol, acetone

4F.1.4 Preparation of nanoparticles

Solution of drug in methanol (2%) was prepared

The SAS process was performed using the experimental equipment as previously

described. Briefly the SC-CO2 was pumped to the top of the particle precipitation

vessel through the outer capillary of the two flow ultrasonic spray nozzle by syringe

pump.The drug solution was introduced into the particle precipitation vessel by an

HPLC liquid pump through the two flow ultrasonic spray nozzle. Meanwhile the SC-

CO2 continued to flow through the vessel to maintain the steady state. The conditions

of particle precipitation vessel were investigated at temperature ranging from 60-72º

C and pressure 100 bars. The residual solvent was drained out of the particle

precipitation vessel by the backpressure regulator. After spraying of drug solution into

the particle precipitation vessel completed, an additional SC-CO2 continued to flow

into the vessel at same rate for further 120 minutes to remove residual solvent from

precipitated particles and then slowly depressurized to atmospheric pressure. The

precipitated particles were collected on the wall and bottom of the particle

precipitation vessel and then stored in a desiccator at room temperature.

Evaluation

Prepared nanoparticles were evaluated for particle size analysis, drug content,

saturation solubility studies, in-vitro release study, morphological characteristic and

physicochemical characterization.

EXPERIMENTAL


4F.1.5 Evaluation and characterization176

4F.1.5.1 Saturation solubility testing

Nanoparticles containing amount of drug equivalent to 8 mg was added to vials

containing 5 ml each of 0.1 N HCl, phosphate buffer pH 6.8 and water; and

rotated in rotary shaker for 48 hours at 37◦ C. Solutions were then centrifuged at

2000 rpm for 15 minutes and supernatant was analysed for drug content using

UV spectroscopic analysis.

4F.1.5.2 Dissolution testing

Release was checked in all previously mentioned medias for 1 hour and drug

content was analysed using UV spectroscopic analysis.

4F.1.5.3 Drug content

Drug content was analysed by taking amount of drug equivalent to 8 mg and

diluting suitably with acetonitrile and analyzing the drug content by UV

spectroscopic analysis.

4F.1.5.4 Physicochemical characterization

4F.1.5.4.1 XRD Analysis

X-ray diffraction analysis was carried out as per the procedure discussed in the

section 4A.

4F.1.5.4.2 IR Analysis

An IR spectrum of the drug was collected by the same procedure mentioned in

sections 4A.

4F.1.5.5 Particle size, zeta potential and polydispersity index

Particle size was measured by using a photon correlation spectroscopy using a

zetasizer the instrument used for this study was Beckman coulter counter.

EXPERIMENTAL


4F.1.6 Drug content


diluting suitaibly with acetonitrile and anaysing the drug content by UV


4F.1.7 Percentage yield

Percentage yield was calculated using following formula

% yield= Amount in gms of nanoparticles obtained 100 4F.1

Total amount of drug added

4F.2 Preparation of nanoparticles using ion gelation technique

4F.2.1 Introduction

The potential use of polymeric nanoparticles as drug carriers has led to the

development of many different colloidal delivery vehicles. The advantage of this kind

of systems lie to their capacity to cross biological barriers, to protect macromolecules

from degradation into in biological media and to deliver drugs or macromolecules to a

target site with following controlled release. In the last years several synthetic as well

as natural polymers have been examined for pharmaceutical applications.

Chitosan is a cationic polysaccharide obtained by partial deacetylation of chitin , the

major component of crustacean shells. In contrast to other polymers, chitosan is a

hydrophilic polymer with positive charge that comes from weak basic groups, which

give it special characteristics from the technological point of view.177

Chitosan microspheres can be prepared by reacting chitosan with controlled amounts

of multivalent anion resulting in cross linking between chitosan molecules. The cross

linking may be achieved in acidic ,neutral or basic environment depending on the

method applied.Eventhough several techniques such as cross linking with

anions,precipitation,complex coacervation,modified emulsification and ion gelation,

precipitation-chemical cross linking,glutaraldehyde cross linking and thermal cross

linking178

, are reported for the formation of chitosan microparticles,principally two

techniques are usually employed to obtain chitosan microparticles, in one method,

chitosan chains can be chemically cross linked leading to quite stable matrixes, where

the strength of covalent bonds stands out. Glutaraldehyde is broadly used as s cross

EXPERIMENTAL


linking molecule in covalent formulations. In other method chitosan hydrogels can

also be obtained by ionic gelation,where micro or nanoparticles are formed by means

of electrostatic interactions between the positively charged chitosan chains and

polyanions employed as cross linkers.179

The most extensively used polyanion is the

tripolyphosphate (TPP). Due to the proved toxicity of glutaraldehyde and other

organic molecules used in the synthesis of gels covalently stabilized, only the second

synthesis technique can be used for pharmaceutical applications. One of the most

important properties of any nanogel is the extent of swelling. This means that its

structure can undergo volume phase transition from swollen to collapsed state.The

vextent of swelling depend on several external conditions such as temperature, pH or

ionic strength of the medium.

Ionic gelation method is reported to be used for the preparation of chitosan

nanoparticles180

for the delivery of proteins and peptides including insulin and also for

cyclosporin.

As ion gelation method was used mainly for the delivery of proteins and peptides and

no reports were available for the preparation of nanoparticles of CC by ion gelation

method, an attempt was tried to check the suitability of the method for the delivery of

CC.

4F.2.2 Literatures review

1. Muhammed Rafeeq et al reported formation of nanoparticles of isoniazide for

enhanced bioavailability in 2010.181

2. Sanju Dhawan et al in 2004 reported formation of mucoadhesive chitosan

microspheres.171

3. Amit Dustgani et al in 2008 reported formation of chitosan nanospheres loaded

with dexamethasone sodium phosphate.182

4. Ionic gelation method was reported to be used for the formation of nanoparticles of

ampicillin trihydrate by Saha et al.

183

5. Literature reports formation of vaccinnes by the method of ionic gelation of

chitosan using tripolyphosphate.184

EXPERIMENTAL


4F.2.3 Materials

Table 4F.2 Excipients used for formulation of nanoparticles by ion gelation

method

Chitosan Acetic acid

Sodium

Tripolyphosphate

Sodium hydroxide

4F.2.4 Preparation of nanoparticles by ion gelation

Nanoparticles were prepared using a factorial design of 23.

Nanoparticles were prepared by the method given by Lopez leon et al.185

Solution of chitosan in acetic acid (0.1%)was prepared in three concentrations as

mentionedin table No. and pH of the solution was adjusted to 4 by using sodium

hydroxide (10%).Solutions of STTP were also prepared in three concentrations as

shown in table no. in distilled water. Amount of drug equivalent to 10 mg was added

to 2 ml solution of STTP and drug was solubilised by adding a solution of sodium

hydroxide (10%). Drug solution was added to chitosan solution dropwise with stirring

on a magenetic stirrer at room temperature using a hypodermic needle.Nanoparticles

were concentrated by rotating them at12000 rpm for 30 minutes and then air dried

overnight.186

Total 9 systems were prepared.

Table 4F.3 Formulation design for preparation of nanoparticles by ion gelation

method

Concentration

of STTP

Concentration

of chitosan

0.05%W/V 0.075%W/V 0.1%W/V

0.05%W/V S1 S2 S3

0.1%W/V S4 S5 S6

0.2%W/V S7 S8 S9

Amongst all prepared systems, only systems (In bold) which were having

comparative clear appearance were chosen for further studies.

EXPERIMENTAL


4F.2.5 Evaluation187

Prepared nanoparticles were evaluated for particle size analysis, drug content, drug

loading efficiency, in-vitro release study and morphological analysis.

4F.2.5.1 Measurement of particle size and zetapotential


zetasizer.

4F.2.5.2 Drug loading efficiency

Drug loading efficiency was calculated by analyzing amount of drug present

in supernatant by UV spectroscopic analysis by using following formula-

4F.2

4F.2.5.3 Percentage yield

Percentage yield was calculated using following formula-

% yield= amount in gms of nanoparticles obtained 100 4F.3

Total amount of drug+polymers added





4F.2.5.5 Solubility studies

Nanoparticles containing amount of drug equivalent to 10 mg was added to

vials containing 5 ml each of 0.1 N HCl, phosphate buffer pH 6.8 and water

and rotated in rotary shaker for 48 hours at 370 C. Solutions were then

centrifuged at 12000 rpm for 30 minutes and supernatant was analysed for

drug content using UV spectroscopic analysis.

EXPERIMENTAL


4F.2.5.6 In vitro release study

Release was checked in all previously mentioned medias for 1 hour and

subsequently for 2, 4 and 8 hours and drug content was analysed using UV


4F.2.5.7 Morphological analysis

Surface morphology was studied by using TEM analysis.

4F.3 Preparation of nanoparticles using nanoencapsulation technique

4F.3.1 Introduction

As reported before the aim of preparing nanoparticles by nanoencapsulation was to

retard the release in stomach. Various polymers are available which are used as

enteric coated polymers.Eudragit polymers are also a group of polymers which are

repeatedly reported to be used in the formulation of nanoparticles as well as

controlled or sustained release per oral systems. Many nanoparticles are used for

targeted as well as in ocular therapy.Various grades of eudragits are available but

most commonly used areE100, RL100, RS 100,EPO, RSPO and RLPO.

Various grades of eudragits are used for various purposes such as Eudragit 100and

eudragit E 12.5 are yellow in color, are soluble in gastric fluid up to pH 5 and are used

for film coating. Eudragit NE 30 is a swellable grade yellow in color and used for

sustained release. Eudragit L100, L 12.5 and L 12.5 P are a white free flowing

powder, soluble in intestinal fluid from pH 6 and used for enteric coatings.

Eudragit L 30 D-55, L100-55, Eastacryl 30D, Kollicoat MAE 30 D, Kollicoat MAE

30 DP are white or milky white in color ,soluble in intestinal pH from pH 5.5 which

are used for enteric coatings. Eudragit S100, S12.5, S12.5 P are white free flowing

powders ,soluble in intestinal fluid from pH 7 are used for the purpose of enteric

coatings.

Eudragit RL 100188

,RLPO,RL 30 D,RL 12.5 are high permeability nonbiodegradable

polymers used for sustained release whereas Eudragit RS 100189

,RSPO,RS30D,RS

12.5 are low permeability polymers used for sustained release. 190

As release of this formulation was expected in intestine, finally it was decided to use

Eudragit L 100 because it was reported to enhance the solubility and biovailability of

many poorly soluble drugs.

EXPERIMENTAL


4F.3.2 Literature review

1. Chander Dora et al reported in 2010, formation of nanoparticles of

glibenclamide with eudragit L100 which enhanced the bioavailability of drug.

191

2. S. Mudgal et al reported formation and evaluation of nanoparticles of 5-

flurouracil with eudragit L100 in 2010.192

3. P. Devarajan et al reported formation of gliclazide nanoparticles for sustained

release of drug with Eudragit L100.193

4. M.Cetin et al reported formulation of nanoparticles of diclofenac sodium

using eudragit L100.194

5. Nanoparticles containing corticosteroids were reported to prepared by novel

method of flow reactor by H.Erikainen et al.195

6. Gonzalez et al reported formation of eudragit L100 with combination of

eudragit L30 D55 nanoparticles of acetyl salicylic acid to prevent the contact

of gastric fluid with active.196

4F.3.3 Materials

Table 4F.4 Excipients used for formulation of nanoparticles by

nanoencapsulation method

Eudragit L 100 Methylene chloride

Ethanol Polyvinyl alcohol

4F.3.4 Preparation of nanoparticles by nanoencapsulation

Nanoparticles were prepared by using o/w emulsification solvent evaporation

technique.

Polymer was dissolved in a 5 ml mixture of methylene chloride: ethanol (3:1).After

dissolving polymer, drug (10mg) was added in the mixture and dissolved by using

ultrasonicator. This solution was poured in 1% w/w PVA solution by keeping

different ratio‟s of phase to volume. A o/w emulsion was formed with extensive

stirring with a magnetic stirrer.system was kept on stirring until methylene chloride

was evaporated.System was centrifuged at 12000 rpm for 1 hour and resultant

nanoparticles were collected and lyophilized.Total 9 systems were prepared , out of

that best system was evaluated particle size, polydispersity index, zeta potential, drug

EXPERIMENTAL


loading efficiency, drug content, percentage yield, drug content, Saturation solubility

testing, in vitro multimedia study and surface morphology.

Table 4F.5 Formulation design for preparation of nanoparticles by

nanoencapsulation method

Sr. No. Drug:Polymer Organic phase: Aqueous phase

1 1:10 1:2

2 1:10 1:3

3 1:10 1:4

4 1:20 1:2

5 1:20 1:3

6 1:20 1:4

7 1:30 1:2

8 1:30 1:3

9 1:30 1:4

Amongst all prepared systems, only systems (In bold) which were having

comparative clear appearance in the respective ratio and were chosen for further

studies.

4F.3.5 Evaluation197

Prepared nanoparticles were evaluated for particle size analysis, drug content, drug

loading efficiency, in-vitro release study and morphological analysis.

4F.3.5.1 Measurement of particle size and zeta potential


zetasizer.

4F.3.5.2 Drug loading efficiency

Drug loading efficiency was calculated by analyzing amount of drug present

in supernatant by UV spectroscopic analysis by using following formula-

4F.4

EXPERIMENTAL


4F.3.5.3 Percentage yield

Percentage yield was calculated using following formula

% yield= amount in gms of nanoparticles obtained 100 4F.5

Total amount of drug+ polymers added





4F.3.5.5 Solubility studies

Nanoparticles containing amount of drug equivalent to 10 mg was added to

vials containing 5 ml each of 0.1 N HCl, phosphate buffer pH 6.8 and water

and rotated in rotary shaker for 48 hours at 37ºC. Solutions were then

centrifuged at 12000 rpm for 30 minutes and supernatant was analysed for

drug content using UV spectroscopic analysis.

4F.3.5.6 In vitro release study

Release was checked in all previously mentioned medias for 1 hour and

subsequently for 2 ,4 and 8 hours in phosphate buffer pH 6.8 and drug release

was analysed using UV spectroscopic analysis.

4F.3.5.7 Morphological analysis

Surface morphology was studied by using SEM analysis.

4G Optimization of formulation

From all the methods tried two formulations were selected as showing improved

saturation solubility and better release profiles in multimedia dissolution studies.

4G.1 Complexation with cyclodextrin using lyophilization technique

4G.1.1 Optimization of formula

In this method drug complexation with hydroxyl- propylated beta cyclodextrins in the

ration 1:2 was chosen as the formulation of choice.

EXPERIMENTAL


Formulation was filled manually in a hard gelatin capsule (No. 4). To avoid plug

formation of complex in capsule shell and for the better release some amount of

diluent and disintegrating was added into the capsule.

4G.1.2 Evaluation

Prepared optimized formulations were studied for drug content, multimedia

dissolution and weight variation.

4G.1.2.1 Drug content

Drug content was analysed by emptying contents of capsule in methanol,

sonicating them for 10 minutes, filtering through whatmann filter paper (0.45

micron pore size) and diluting the sample with methanol to get concentration

of 8 ppm. Drug content was analyzed by using HPLC method described in

analytical method development.

4G.1.2.2 Multimedia dissolution

Multimedia dissolution studies were carried out in afore mentioned media and

drug release was checked by HPLC.

4G.1.2.3 Weight variation

Weight variation test was carried out according to the IP 2007.

4G.2 SMEDDS

4G.2.1 Optimization of formula

No optimization of formula was done. SMEDDS system 1 equivalent to 8 mg of

drug was filled into hard gelatin capsule.

4G.2.2 Evaluation

Prepared optimized formulations were studied for drug content, multimedia

dissolution and weight variation.

EXPERIMENTAL


4G.2.2.1 Drug content

Drug content was analysed by emptying contents of capsule in methanol,

sonicating them for 10 minutes, filtering through whatmann filter paper (0.45

micron pore size) and diluting the sample with methanol to get concentration

of 8 ppm. Drug content was analysed by using HPLC method described in

analytical method development.

4G.2.2.2 Multimedia dissolution

Multimedia dissolution studies were carried out in afore mentioned media and

drug release was checked by HPLC.

4G.2.2.3 Weight variation

Weight variation test was carried out according to IP 2007.

4H. In –vivo studies

4H.1 Development and validation of analytical method for analysis of

candesartan cilexetil in plasma

4H.1.1 Literature review

There are various methods used for analysis of candesartan cilexetil in plasma.by

HPLC as well as few reported methods by LCMS.198-203

As the bioavailability of drug is reported to be very low (15%) of the given dose

,expected plasma levels were also low. Finally to get the accurate results a LC-

MS/MS method was decided to be used and the same was validated for linearity,

accuracy and precision, percentage extraction yield, stock solution stability, stability

of analyte in plasma and ruggedness.

EXPERIMENTAL


4H.1.2 Development and Validation of method

4H.1.2.1 Specificity

The specificity of the intended method was established by screening the standard

blank (without spiking with CANDESARTAN of different batches/lots of

commercially available rabbit blank plasma). Seven different batches of plasma (K2

EDTA) including one haemolysed plasma were screened.

4H.1.2.2 Plasma Linearity (Calibrant samples)

The linearity of the method was determined by using a 1/x2

weighted least square

regression analysis of standard plots associated with and seven-point standard curve.

4H.1.2.3 Precision and Accuracy

The precision of the CANDESARTAN assay was measured by the percent coefficient

of variation and % Nominal over the concentration range of LQC, MQC and HQC

samples during the course of validation.

4H.1.2.3.1 Between Batch Precision

The between batch accuracy and precision of CANDESARTAN was found

out over the range of the low, middle and high quality control samples.

4H.1.2.3.2 Within Batch Precision

The within batch accuracy and precision of CANDESARTAN was found out

over the range of the low, middle and high quality control samples.

4H.1.2.4 Percentage Extraction Yield

Recovery of CANDESARTAN

The percentage mean recoveries were determined by measuring the response

of the extracted plasma quality control samples at LQC and HQC against

aqueous extracted quality control samples at LQC and HQC.

EXPERIMENTAL


4H.1.2.5 Stock Solution Stability

4H.1.2.5.1 Long Term Stock Solution Stability

Long term stock solution stability for CANDESARTAN at concentration 100.00

ng/mL and was determined by using aqueous standard after the storage for 15 days

and 30 days at 2 - 8oC. Stability was assessed by comparing against the initially

injected CANDESARTAN standard stock solution of concentration 100.00.

4H.1.2.6 Stability of Analytes in Plasma

Stability studies in plasma were conducted in the various conditions using three

replicates of LQC and HQC samples as described below-

4H.1.2.6.1 Freeze Thaw Stability

Freeze thaw stability of the spiked quality control samples was determined

during three freeze thaw cycles stored at below -20 ± 5°C. Stability was

assessed by comparing against the freshly spiked quality control samples.

4H.1.2.6.2 Long Term Stability in Matrix

Long term stability of the spiked quality control samples in matrix was

determined for 15 days and 30 days for CANDESARTAN which was stored at

-20 ± 50C temperature. Stability was assessed by comparing against the freshly

thawed quality control samples.

4H.1.2.7 Ruggedness

Ruggedness was performed by using three Quality control batches. One batch was

analyzed by using different column, second batch was analyzed by different analyst,

third batch was analyzed by using different analysis.

EXPERIMENTAL


4H.2 In-vivo bioavailability studies

In vivo bioavailability testing of prepared systems was carried out in mixed

population New Zealand white rabbits, each weighing around 1.8-2.2 kg. Each

formulation was tested in a group comprising 6 rabbits. A dose equivalent to 8mg of

drug was administered orally to each rabbit. Eleven blood samples were withdrawn

from each rat over a period of 48 hours, collected at time intervals of 0 h,1 h, 2 h, 3 h,

4 h, 6 h, 8 h,10 h,12 h, 24 h and 48 h. Plasma was separated using K2EDTA. All

plasma samples were stored in deep freezer till analysis. Plasma samples were

analysed for finding out various pharmacokinetic parameters such as Cmax, Tmax,

AUC (0-t), AUC (0-∞) etc.

4I Stability studies

Stability study was conducted as per ICH guidelines for two final formulations.

Capsules were packed in 30cc thick walled HDPE bottles with CRC caps. Each bottle

containing 30 capsules and 2 g of silica bag were kept for real time (25°C/ 60% RH)

and accelerated (40°C/ 75% RH) stability study. Samples were analyzed for

dissolution and total drug content by validated HPLC method.

Evaluation Most commonly SMEDDS are evaluated by...

Documents

Transcript of Evaluation Most commonly SMEDDS are evaluated by...