Chapter 7

Summary
CHAPTER-VII

SUMMARY

Acne is a common skin disorder afflicting more than 85% of adolescents

and can persist or develop over time to affect up to 50% of adults older than 20
years of age. The largest population-based survey of acne involved 105
dermatology residents and just over 20,000 non institutionalized people in the
USA. This study generated an overall US population acne prevalence estimate of
13%. Substantial healthcare resources and consumer expenditures are committed
to the treatment of acne. In 2002, the total cost burden of acne and its treatment
was estimated to exceed US$1 billion annually.

Isotretinoin was approved in the United States in 1982 as a treatment for

severe recalcitrant nodular (cystic) acne that is unresponsive to conventional
therapy including systemic antibiotics. Isotretinoin is 13-cis retinoic acid or 13
cis-Vitamin A, its isomers and some of its analogs are widely known to leave a
therapeutic activity in the treatment of several skin disorders like acne,
hypertropic lupus erythmatosus, keratinization disorders. Some evidences also
have been brought about the activity of Isotretinoin in basal cell carcinoma and
squamous cell carcinoma.

Isotretinoin decomposes in the presence of light and atmospheric oxygen.

Isotretinoin is very poorly soluble in water what makes its bioavailability quite
low after an oral intake of about 25% in fasted condition and 40% in fed
conditions. The maximum concentration (Cmax) is reached after 2-4hrs, while the
Cmax of the active metabolite, 4-oxo-isotretinoin is reached after 6hrs. The
elimination half life of Isotretinoin is about 7 to 37 hrs while the half life t 1/2 of

Design and Development of a Self Nano Emulsifying Drug

Delivery System (SNEDDS) for Isotretinoin with enhanced Oral
Bioavailability and Improved Stability 221
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Summary
active metabolite is of 11 to 50 hrs. The steady state concentration is reached after
1 week of treatment.

Very few publications and/or patents about the pharmaceutical formulation

of Isotretinoin are available. The drug is available in most markets in the form of a
soft gelatin capsule containing a fatty liquid formulation of Isotretinoin. The
reported literatures show a maximum in-vitro drug release of about 60-70% even
after 5 h.

It is increasingly being recognized by the pharmaceutical industry that for

these molecules, drug delivery systems play an important role for improving
bioavailability.

Although the process of passive diffusion is responsible for absorption of

non ionized lipophilic molecules via the transcellular pathway, specialized
absorption mechanisms, first pass metabolisms and efflux systems at the GI wall
appear to play a major role for lack of absorption and poor bioavailability for
some molecules.

Nowadays, an increasing number of new chemical entities and many

existing drugs exhibit low solubility in water, which may lead to poor oral
absorption, high intra- and inter-subject variability and lack of dose
proportionality. Thus, for such compounds of BCS II type, the absorption rate and
degree from the gastrointestinal tract (GIT) are usually controlled and limited by
dissolution process. To overcome the problem, various formulation strategies have
been adopted including the use of cyclodextrins, nanoparticles, solid dispersions
and permeation enhancers.

In recent years, much attention has been paid to self-emulsifying drug

delivery systems (SEDDS), which have shown lots of reasonable successes in
improving oral bioavailability of poorly soluble drugs. SEDDS are usually
Design and Development of a Self Nano Emulsifying Drug
Delivery System (SNEDDS) for Isotretinoin with enhanced Oral
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Chapter 7
Summary
composed of a mixture of oil and surfactant or cosurfactant and are capable of
forming fine oil-in-water emulsions upon gentle agitation provided by the GIT
motion. After oral administration, SEDDS can maintain the poorly soluble drugs
dissolved in the fine oil droplets when transiting through the GIT.

Nanoemulsions have been reported to make the plasma concentration

profiles and bioavailability of poorly soluble drugs more reproducible.
Nanoemulsions have also been reported as one of the most promising techniques
for enhancement of transdermal permeation and bioavailability of poorly soluble
drugs. Nanoemulsions are thermodynamically stable transparent (translucent)
dispersions of oil and water stabilized by an interfacial film of surfactant and
cosurfactant molecules having a droplet size of less than 100 nm. Potential
advantages of these systems include enhanced oral bioavailability (enabling dose
reduction), more consistent temporal profiles of drug absorption, selective drug
targeting toward a specific absorption window in the GI tract, and drug protection
from the hostile environment in the gut.

The concept of developing this novel formulation is derived from the

burgeoning problems faced during the development of the bioequivalent generic
formulation. The commercially available marketed product itself constituted batch
to batch variability which further contributed to the variability caused by the
nature of the molecule itself. This inadvertently created further hurdles in
developing the generic version of Roacutane 20mg soft gelatin capsule product.

This variation occurs mainly due to the particle size and solid state
characteristics of Isotretinoin. The particle size of the active is mainly controlled
by the API manufacturer during the recrystallization or purification step. The size
reduction of the bigger particles causes serious blow in the purity and its physical
characteristics due to the energy intensive milling process. Hence such process is
mostly discouraged in the formulation industry but the quality of the product is
Design and Development of a Self Nano Emulsifying Drug
Delivery System (SNEDDS) for Isotretinoin with enhanced Oral
Bioavailability and Improved Stability 223
Chapter 7
Summary
built through the input and in-process controls by controlling the API particle size
in a narrow range at the vendor level itself. This makes the condition more
complex and increases the cost of the active material and ultimately adds on the
cost of the final formulation and makes the treatment expensive.

The research work entitled "Design and Development of a Self Nano

Emulsifying Drug Delivery System (SNEDDS) for Isotretinoin with enhanced
Oral Bioavailability and Improved Stability " was undertaken with the following
objective.

The objective of this study was to develop and formulate a novel

composition which shall be manufactured through easily scalable process at the
formulation manufacturer site without involving sophisticated and energy
intensive milling process irrespective of the particle size of the active material
procured from the API manufacturer, to enhance the oral bioavailability of the
Isotretinoin which will reduce the dose needed and decrease the risk of adverse
side effects, to improve Tmax and reduce inter and intra subject variability and to
address the stability issues associated with the Isotretinoin molecule.

The study has been presented in 8 chapters with introduction in chapter 1,

followed by literature review in chapter 2. The Drug and polymer profile was
discussed in chapter 3. The objectives of the study were briefed in chapter 4 and
elaborate methodology in chapter 5. Results and discussion was made in chapter
6. Summary was presented in chapter 7. Conclusion was finally presented in
chapter 8 with up to date bibliography till 2010 related to the study at the end.

The important criterion for selection of components is their pharmaceutical

acceptability. It has been demonstrated that only very specific pharmaceutical
excipient combinations lead to efficient nanoemulsion formulations. One
important consideration when formulating a self emulsifying formulation is

Design and Development of a Self Nano Emulsifying Drug

Delivery System (SNEDDS) for Isotretinoin with enhanced Oral
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Summary
avoiding precipitation of the drug on dilution in the gut lumen in-vivo. Therefore,
the components used in the system should have high solubilization capacity for
the drug, ensuring the solubilization of the drug in the resultant dispersion. Results
from solubility studies reveal that Peppermint oil and Triacetin showed the highest
solubilization capacity for Isotretinoin, followed by Polysorbate-80, Cremophore
RH40, and Gelucire 44/14 among the surfactants and Transcutol HP, PEG 600 and
Tetraglycol among the co-surfactants used.

Self-nanoemulsifying systems form fine oil-water emulsions with only

gentle agitation, upon their introduction into aqueous media. Surfactant and
cosurfactant get preferentially adsorbed at the interface, reducing the interfacial
energy as well as providing a mechanical barrier to coalescence. The decrease in
the free energy required for the nanoemulsion formation consequently improves
the thermodynamic stability of the nanoemulsion formulation. Therefore, the
selection of oil and surfactant, and the mixing ratio of oil to S/CoS, play an
important role in the formation of the nanoemulsion.

For the development of a self-emulsified formulation, a right blend of low

and high HLB (hydrophile lipophile balance) surfactant is necessary for the
formation of a stable nanoemulsion. Therefore, high HLB surfactants (Polysorbate
80, Cremophore RH40, and Gelucire 44/14 with HLB value above 10.0) and a low
HLB co-surfactant (Transcutol HP with a HLB of 4.2) were selected. It is
important that the excipients used are listed as GRAS (generally regarded as safe)
in the Handbook of Pharmaceutical Excipients (published by the American
Pharmaceutical Association) or they have a history of use in marketed
formulations.

In the present study both, Peppermint oil alone and mixture of Peppermint
oil and Triacetin (1:1) were tested for phase behaviour studies with Polysorbate-
80, Cremophore RH40 and Gelucire 44/14 as surfactants and Transcutol HP as the
Design and Development of a Self Nano Emulsifying Drug
Delivery System (SNEDDS) for Isotretinoin with enhanced Oral
Bioavailability and Improved Stability 225
Chapter 7
Summary
cosurfactant to form S/CoS mixture. Based on solubility data, about 24 mg of
Isotretinoin is soluble in 1 g of Peppermint oil. In order to maximize the drug
loading capacity in minimum quantity of Peppermint oil, it was combined with
Triacetin (1:1) to produce a similar solubility effect with less usage of Peppermint
oil. The nanoemulsion existence area increased as the S/CoS ratio increased.
Thus, an S/CoS ratio between 3:1 and 4:1 was selected for the formulation study.
The selected S/CoS ratio between 3:1 and 4:1 were evaluated for titration after
drug loading. Titration studies revealed that 3:1 S/CoS ratio produced microfine
crystals upon standing. Surfactant is anticipated to enter the water phase and
redistribute mainly between the water phase and the emulsion-water interface,
resulting in a loss of solvent capacity of the vehicle. Thus, the problem of
precipitation was solved by increasing the surfactant proportion (S/CoS [4:1]) in
the system.

As seen from the ternary plot, Peppermint oil alone or in combination with
Triacetin in the ratio of 1:1 with Polysorbate 80 as the surfactant and Transcutol
HP as the cosurfactant gave a wider nanoemulsion region at a S/CoS ratio of 4:1.
Thus, Peppermint oil alone or in combination with Triacetin in the ratio of 1:1
with Polysorbate 80 as the surfactant and Transcutol HP as the cosurfactant was
selected as the preferred vehicle for the optimized formulation. The inclusion of
drug narrowed the nanoemulsion existence area, because inclusion of the drug in
the lipid phase led to expansion of the lipid phase and consequently a need for a
higher S/CoS ratio for stabilization.

A series of SNEDDS formulations were prepared using Polysorbate-80,

Cremophore RH40, and Gelucire 44/14 as surfactants and Transcutol HP, PEG
600 and Tetraglycol as the cosurfactants in the S/CoS ratio of 4:1. Peppermint oil
and Triacetin were used as oils; Peppermint oil was used either alone or in
combination with Triacetin in the ratio of 1:3 and 1:1 (Peppermint oil/Triacetin).

Design and Development of a Self Nano Emulsifying Drug

Delivery System (SNEDDS) for Isotretinoin with enhanced Oral
Bioavailability and Improved Stability 226
Chapter 7
Summary
The components were mixed by gentle stirring and vortex mixing. The contents
were heated upto 40ºC on a magnetic stirrer and then sonicated for 10 minutes in
Ultrasonic bath until Isotretinoin was perfectly dissolved. The preparation
obtained (equivalent to 20 mg Isotretinoin) were filled into hard gelatin size ‘0’
capsules. The filled capsules were stored at room temperature until their use in
subsequent studies.

It was observed that a decrease in the proportion of Peppermint oil in the

composition (Peppermint oil/Triacetin (1:3)) resulted in generation of nonclear
dispersion. This could be due to loss of solvent capacity of Triacetin. Thus the
problem of precipitation was solved by increasing the proportion of Peppermint
oil in the system (Peppermint oil/Triacetin (1:1)). It was also observed that only
Transcutol as cosurfactant produced a clear dispersion when compared to the other
cosurfactants employed. This may be attributed to its fluid like property which
may easily penetrate and occupy the sphere like gap between the interfacial
surfactant molecules.

It was observed that Peppermint oil alone or in combination with Triacetin

in the ratio of 1:1 as oil, Polysorbate-80 as surfactant and Transcutol HP as co
surfactant gave the highest drug loading capacity which was taken for further
evaluation. It can be seen that there is a correlation between the drug content and
clarity of the SNEDDS formulations. Those formulations which had the highest
self emulsification gave maximum drug loading.

Nanoemulsions are characterized by the droplet size in nanometer size

range. Therefore particle size analysis was performed to see whether the resultant
emulsions are indeed nanoemulsions. The selected formulations were in the size
range of 100 – 150 nm. The amount of Isotretinoin dissolved in 45 min (t45
values) increases when the particle size decreased.

Design and Development of a Self Nano Emulsifying Drug

Delivery System (SNEDDS) for Isotretinoin with enhanced Oral
Bioavailability and Improved Stability 227
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Summary
It is clear that increasing the concentration of both surfactant and co-
surfactant lead to decreasing particle size which means that the particle size
distribution is inversely proportional to the concentration of surfactant and co-
surfactant. An increase in the ratio of the oil phase (Peppermint oil) resulted in a
proportional increase in particle size, because of the simultaneous decrease in the
S/CoS proportion. It is well known that the addition of surfactants to the
nanoemulsion systems causes the interfacial film to stabilize and condense, while
the addition of cosurfactant causes the film to expand; thus, the relative proportion
of surfactant to cosurfactant has varied effects on the droplet size. Therefore,
using a proper ratio of surfactant and co-surfactant results in production of
formulation in nano range particle size.

Turbidity values have been reported to be of use in SNEDDS

characterization. It can be seen that there is a good correlation between the visual
observation and turbidity of all formulations. It can be observed that there is a
correlation between the viscosities and the rate of dissolution of the drug from the
formulation. The formulation which had the lowest viscosity showed a faster onset
on drug release.

FT-IR spectrums were mainly used to determine if there was any

interaction between the drug and any of the excipient used. The presence of
interaction was detected by the disappearance of peaks corresponding to important
functional groups of the drug. Isotretinoin compatibility with the ingredients of
self-nanoemulsified formulations was tested using FT-IR. The absorbance
spectrums of Isotretinoin showed several characteristic peaks. The spectrum of
Isotretinoin SNEDDS formulation had the feature of each of the components and
did not change the infrared spectrum of Isotretinoin. This indicated that there is no
chemical interaction in the ternary system and that the molecular structure of

Design and Development of a Self Nano Emulsifying Drug

Delivery System (SNEDDS) for Isotretinoin with enhanced Oral
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Summary
Isotretinoin remained completely intact. Therefore Isotretinoin did not interact
with any one of the self-nanoemulsified components.

The release of Isotretinoin from the investigated SNEDD formulations was

markedly different from one formulation to another. Drug release from the
SNEDD formulations was found to be significantly higher as compared with that
of conventional marketed formulation (soft gel capsules). It could be suggested
that the SNEDD formulation resulted in spontaneous formation of a nanoemulsion
with a small droplet size, which permitted a faster rate of drug release into the
aqueous phase, much faster than that of marketed formulation. Thus, this greater
availability of dissolved Isotretinoin from the SNEDD formulation could lead to
higher absorption and higher oral bioavailability.

Freeze thawing process showed that there was very slight recrystallization
at freezing temperature. So in order to solve this stability issue, solidifying agents
such as PEG 6000 and Poloxamer 407 were added to the SNEDDS formulation to
make a semisolid composition.

It was observed that there was no significant change in the drug release by
adding solidifying agents. However it was observed that PEG 6000 had better
solidifying capacity and faster drug release. Based on the above findings, the final
composition with solidifying agent PEG 6000 was prepared and subjected for
stability studies.

Generally, SNEDD formulations are put into hard gelatin capsules as the
final dosage form. However, liquid-filled hard gelatin capsules are susceptible to
leakage, and the entire system has a very limited shelf life owing to its liquid
characteristics and the possibility of precipitation of the drug from the system.
Thus, the developed formulation with solidifying agent PEG 6000 was subjected
to stability studies to evaluate its stability and integrity of the dosage form. The

Design and Development of a Self Nano Emulsifying Drug

Delivery System (SNEDDS) for Isotretinoin with enhanced Oral
Bioavailability and Improved Stability 229
Chapter 7
Summary
formulation was found to be stable for 6 months at accelerated conditions and 12
months at long-term conditions. There was no significant change in the drug
content, drug release (t90%), or particle size of the resultant nanoemulsion. It was
observed that the related impurities were very minimal at the end of accelerated
stability which proves that peppermint oil as carrier enhanced the stability of the
composition without the need for any antioxidants in the formulation. It was also
seen that the formulation was compatible with the hard gelatin capsule shells, as
there was no sign of capsule shell deformation. There were also no significant
changes in the appearance or nanoemulsifying property. Furthermore, the
formulation was found to show no phase separation, drug precipitation, or capsule
leaks. Thus, these studies confirmed the stability of the developed formulation and
its compatibility with hard gelatin capsules.

The results of bioavailability studies showed that the SNEDDS

composition containing 10 mg of the active showed a 6-fold increase in the
relative bioavailability. The idea of fast release was proved in this study by the
fact that the maximum concentration for the test product was at 1.0 hour compared
to 2.67- 4.0 hours for the reference product with less inter subject variability.

The novel composition of Isotretinoin achieved its goal in providing a

enhanced bioavailability and significantly shorter Tmax, which suggests a reduction
in the regular dose of this drug to potentially eliminate its side effects.

Design and Development of a Self Nano Emulsifying Drug

Delivery System (SNEDDS) for Isotretinoin with enhanced Oral
Bioavailability and Improved Stability 230