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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.
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Grade A and Grade B formulation will remain as nano- emulsion 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
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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
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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
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in the solubilising and colloidal stabilizing environment of the gut. Pouton‟s study
reveal that such formula- tions 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
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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
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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%.
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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.
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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.
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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
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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).
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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
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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.
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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.)
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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
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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
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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.
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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.
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4F.1.6 Drug content
Drug content was analysed by taking amount of drug equivalent to 8 mg and
diluting suitaibly with acetonitrile and anaysing the drug content by UV
spectroscopic analysis.
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
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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
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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
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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
Particle size was measured by using a photon correlation spectroscopy using a
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.2.5.4 Drug content
Drug content was analysed by taking amount of drug equivalent to 10 mg and
diluting suitaibly with acetonitrile and anaysing the drug content by UV
spectroscopic analysis.
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
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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
spectroscopic analysis.
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.
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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
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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
Particle size was measured by using a photon correlation spectroscopy using a
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
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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.3.5.4 Drug content
Drug content was analysed by taking amount of drug equivalent to 10 mg and
diluting suitaibly with acetonitrile and anaysing the drug content by UV
spectroscopic analysis.
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
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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.
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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.
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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
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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.
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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.