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FORMULATION AND EVALUATION OF ETHOSOMES
CONTAINING ANTIFUNGAL AGENT
M.Pharm Dissertation Protocol Submitted To
Rajiv Gandhi University of Health Sciences
Karnataka
BY
Ms. DAVE REECHA AIMPRASAD B.pharm
Under the guidance of
Dr. ROOPA KARKI M.pharm, PhD
Professor and Head of the department
Department of Industrial Pharmacy
Acharya & B.M. Reddy College of Pharmacy
Bangalore – 560 090
2008-2010
1
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
KARNATAKA, BANGALORE.
ANNEXURE II
PROFORMA FOR REGISTRATION OF SUBJECT FOR
DISSERTATION
1
2
Name of the
candidate and
address
DAVE REECHA AIMPRASAD
Name of the
Institution
ACHARYA & B.M. REDDY
COLLEGE OF PHARMACY
Jivantika Adya Sthan,
2, Rajputpara,
Rajkot – 360001
Gujarat.
Soldevanahalli,
Hesarghatta Main Road,
Chikkabanavara Post ,
Bangalore-560 090.
3
Course of the
study and subject
M. Pharmacy
(Industrial Pharmacy)
4
Date of admission
25.05.2008
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Title of the project:FORMULATION AND EVALUATION OF ETHOSOMES
CONTAINING ANTIFUNGAL AGENT
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BRIEF RESUME OF THE INTENDED WORK:-
6.1
NEED FOR THE STUDY:
Topical antifungal drugs are medicines applied to the skin
to treat skin infections caused by a fungus. Antifungal drugs are
commonly used for the treatment of Athlete’s Foot(Tinea pedis),
Ringworm and Tinea Versicolor1.
Superficial fungal infections are among the most widespread
diseases known to man. They target parts of the body are the
skin, the nail, the buccal cavity, the eye and the vagina.
Dermatologic fungal infections are usually described by their
location on the body: tinea pedis (infection of the foot), tinea
unguium (infection of the nails), tinia capitis (infection of the
scalp). Three types of fungus are involved in most skin
infections: Trichophyton, Epidermophyton and Microsporum1,2.
Most topical antifungal drugs require four weeks of
treatment. Infections in some areas, particularly the spaces
between toes, may take up to six weeks for cure. Most topical
antifungal agents are well tolerated. Topical antifungal drugs
have no recognized drug-drug or food-drug interactions. In order
to
elicit
a
pharmacologic
response
following
topical
administration, antifungal agents must enter into and diffuse
across the target biologic tissues, which have distinct
architectures and compositions depending on their function. The
rate and extent of transport will depend on the interplay between
the drug's molecular properties and the characteristics of the
biologic tissue. The drug may also interact with specific proteins
or other membrane components2,3.
3
In dermal and transdermal delivery, skin is used as a portal
of entry for drugs, for localized and systematic treatment.
Because of barrier properties of the outer layer of the skin, in
many cases, permeation enhancing agents are required to achieve
therapeutic levels of drug. Transdermal route is, therefore, a
better route to achieve constant plasma levels for prolonged
periods of time, which additionally could be more advantageous
because of less frequent dosing regimens. Classic liposomal
systems were found to be effective at forming drug reservoir in
the upper layers of skin, for localized treatment4.
Recently, it was found that ethosomal carriers, phospholipid
vesicular system, containing relatively high concentration of
alcohol, were very effective at enhancing dermal and
transdermal delivery of both lipophilic and hydrophilic
molecules5,6. Ethosomes are soft, malleable, noninvasive
delivery carriers for enhanced delivery of active agents into
deep skin layers and/or the systemic circulation. The size of the
ethosomes can be modulated to range anywhere from 30 nm to a
few microns3. In comparison to many dermal and transdermal
delivery systems ethosomes have several advantages such as,
enhanced permeation, platform for the delivery of large and
diverse group of drugs, various application in pharmaceutical,
cosmetic and veterinary products, covered by International
patents, efficient and versatile, safe and approved components,
passive noninvasive delivery system, low risk profile, high
patient compliance and high cost to benefit ratio7.
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6.2
REVIEW OF LITERATURE:
 They
have
developed
therapy
with
ethosomal
erythromycin and applied to the skin of S. aureusinfected mice. It was as effective as systemically
administered erythromycin, suggesting a new possibility
to treat deep dermal infections by local application of
antibiotic in ethosomal carrier. On the contrary, no sub
dermal healing was observed in infected animals treated
with topical hydroethanolic erythromycin solution4.
 They have developed
bacitracin and fluorescently
labeled bacitracin (FITC-Bac) ethosomes
and were
characterized for shape, lamellarity, fluidity, size
distribution and entrapment capacity. Confocal laser
scanning microscopy (CLSM) experiments revealed that
ethosomes facilitated the co penetration of antibiotic and
phospholipids
into
cultured
Swiss
albino
mice
fibroblasts. Efficient delivery of antibiotics to deep skin
strata from ethosomal applications could be highly
beneficial, reducing possible side effects and other
drawbacks
associated
with
systemic
treatment.
Furthermore, ethosomal delivery systems could be
considered for the treatment of a number of dermal
infections5.
 They have prepared ethosomes of zidovudine and
characterized in vitro and in vivo. The effect of different
formulation variables on skin permeation of zidovudine
was studied using locally fabricated Keshry-Chien type
of diffusion cell. To understand the mechanism of better
skin permeation of ethosomes, vesicle skin interaction
study was carried out. To confirm the better skin
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permeability of ethosomes, fluorescence microscopy
using rhodamine 123 as fluorescence probe was
performed. Results were compared with those obtained
after administration of liposomes and hydroethanolic and
ethanolic solution of drug. The optimized ethosomal
formulation showed transdermal flux of 78.5± 2.5
µg/cm/h across the rat skin. It can be concluded from the
study that ethosomes can increase the transdermal flux,
prolong the release and present an attractive route for the
sustained delivery of zidovudine. Results indicate that the
ethosomal system may be a promising candidate for
transdermal delivery of number of problematic drug
molecules6.
 They have developed a novel transdermal drug delivery
system, that facilitates the skin permeation of finasteride,
encapsulated in novel lipid-based vesicular carriers
(ethosomes). Finasteride ethosomes were studied for their
morphological characteristics, particle size, zeta potential
and the entrapment capacity. In contrast to liposomes,
ethosomes were found to be more efficient delivery
carriers with high encapsulation capacities. In vitro
percutaneous permeation experiments demonstrated that
the permeation of finasteride through human cadaver skin
was significantly increased when ethosomes were used.
The finasteride transdermal fluxes from ethosomes
containing formulation were 7.4, 3.2 and 2.6 times higher
than
that
of
finasteride
from
aqueous
solution,
conventional liposomes and hydroethanolic solution
respectively.
Furthermore,
ethosomes
produced
a
significant finasteride accumulation in the skin. The study
demonstrated that ethosomes are promising vesicular
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carriers for enhancing percutaneous absorption of
finasteride8.
 They
have
formulated
ethosomes
containing
5-
aminolevulinic acid (ALA) and they were characterized
and
examined
for
enhance
skin
production
of
protoporphyrin IX (PpIX), and results were compared
with traditional liposomes. Results showed that the
average particle sizes of the ethosomes were less than
those of liposomes. The results indicated that the
penetration ability of ethosomes was greater than that of
liposomes9.
 They have investigated the transdermal potential of novel
ethanolic liposomes (ethosomes) bearing Melatonin
(MT). MT loaded ethosomes were prepared and
characterized
for
vesicular
shape
and
surface
morphology, vesicular size, entrapment efficiency,
stability, in vitro skin permeation and in vivo skin
tolerability. % Entrapment efficiency of MT in ethosomal
carrier was found to be 70.71 ± 1.4. Stability profile of
prepared system assessed for 120 days revealed very low
aggregation and growth in vesicular size (7.6 ± 1.2%).
MT loaded ethosomal carriers also provided an enhanced
transdermal flux of 59.2 ± 1.22 μg/cm2/h and decreased
lag time of 0.9 h across human cadaver skin. Fourier
Transform-Infrared (FT-IR) data generated to assess the
fluidity of skin lipids after application of formulation
revealed a greater mobility of skin lipids on application
of ethosomes as compared to that of ethanol or plain
liposomes. Further, a better skin tolerability of ethosomal
suspension on rabbit skin suggested that ethosomes may
offer a suitable approach for transdermal delivery of
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melatonin10.
 They have investigated the basic properties and the in
vitro release rate kinetics of azelaic acid, alternatively
vehiculated in different phospholipids-based vesicles
such as ethosomes and liposomes. Azelaic acid diffusion
from ethosomal or liposomal dispersions and from
ethosomes and liposomes incorporated in a viscous gel
was investigated by a Franz cell assembled with synthetic
membranes. The release rate was more rapid from
ethosomal systems than liposomal systems11.
 They have found that ethosomal carriers, phospholipid
vesicular
systems
containing
relatively
high
concentrations of alcohol, were very effective at
enhancing dermal and transdermal delivery of both
lipophilic and hydrophilic molecules. Fluorescent probes
delivered from ethosomal systems reached the deep strata
of
the
skin.
Delivery
of
minoxidil,
acyclovir,
testosterone, and trihexyphenidyl hydrochloride from
ethosomes were much greater compared to delivery from
classic liposomes. Lastly, the transdermal delivery of
insulin from an ethosomal carrier resulted in lower blood
glucose levels in normal and diabetic rats in vivo.
Regarding ethosomes, results indicated that ketotifen
should be incorporated in ethosomal vesicles for
optimum skin delivery12.
 They have evaluated the efficacy in a 2-armed, doubleblind, randomized clinical study, in the treatment of
recurrent herpes labialis of 5% acyclovir ethosome in
comparison with that of a commercial 5% acyclovir
cream(Zovirax) and that of a drug-free vehicle. In a
crossover arm in which the 2 active preparations were
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compared, the time to crusting of lesions was
significantly shorter with the ethosomal acyclovir (1.8
days) than with the cream (3.5 days). In a parallel arm in
which all 3 preparations were compared, the time to
crusting with the ethosomal acyclovir (1.6 days) was
significantly shorter than the time with the acyclovir
cream (4.3 days) and the time with the drug-free vehicle
(4.8 days). No adverse effects were reported. This pilot
study suggests the improved clinical efficacy of the new
ethosomal preparation in comparison with Zovirax cream
in the treatment of recurrent herpes labialis13.
6.3
OBJECTIVES OF THE STUDY:
Following are the objectives of the present study
 To formulate ethosomes containing anti psoriatic
drug fluconazole.
 To characterize the prepared formulations using
various in vitro methods which includes vesicular
shape and surface morphology, vesicular size and
size distribution, storage-physical stability of
ethosomes, entrapment efficiency.
 To conduct the in vivo studies like skin irritation
studies.
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7
MATERIALS & METHODS:-
7.1
SOURCES OF DATA:
1) Review of literature from:
a) Journals such as
i) Indian Journal of Pharmaceutical Sciences
ii) European Journal of Pharmaceutical
Sciences
iii) Journal of Control Release
iv) International Journal of Pharmaceutics
v) Drug Development and Industrial
Pharmacy
vi) Indian Drugs
vii) International Journal of Cosmetic Science
b) World Wide Web.
c) I.I.Sc Library, Bangalore
d) J-Gate@Helinet.
7.2
METHODS OF COLLECTION OF DATA:
1) Characterization of ethosomal
formulations:
a) Vesicular shape and surface
morphology
 Scanning electron microscopy
(SEM)
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b) Vesicular size and size distribution
 Dynamic light scattering (DLS)
2) Storage-physical stability of ethosomes will be
determined by the ability of vesicles to retain the drug (i.e.,
drug-retentive behavior) will be assessed using the
suspensions at different temperatures, i.e., 4±2ºC for
different periods of time (1, 20,40,60,80 and 120days). The
vesicular
suspensions
were
kept
in
sealed
vials
(10mlcapacity) after flushing with nitrogen. The stability of
ethosomes will be assessed quantitatively by monitoring size
and morphology of the vesicles over time using DLS and
TEM.
3) Entrapment efficiency will be determined as
prepared ethosomal vesicles were separated from the free
(unentrapped) drug by a Sephadex G-50 minicolumn
centrifugation technique. The method will be separated at
least three times with fresh syringe packed with gel each
time until the fraction collected will be free from
unentrapped drug. The vesicles will be lysed by TritonX-100
(0.5%w/w) and entrapped drug will be estimated using
HPLC.
4) Skin irritation study
It is done by using wistar rats. The back skin of area
of 5cm2 is used. If the formulation produced scores of 2 or less
they are considered to have no skin irritation.
Erythema scale: none-0, slight-1, well defined-2, moderate-3,
scar formation-4.
Edema scale: none-0, slight-1, well defined-2,
moderate-3, severe-4.
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7. 3
DOES THE STUDY REQUIRE ANY INVESTIGATION
OR INVESTIGATION TO BE CONDUCTED ON
PATIENT OR OTHER HUMANS OR ANIMALS?
“YES”
7.4
HAS ETHICAL CLEARANCE BEEN OBTAINED FROM
YOUR INSTITUTION IN CASE OF 7.3?
“YES. COPY ENCLOSED.”
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8
REFERENCES:-
1) Majdeline E, Robert M, Yogeshvar N. Ingenta connect new
development in topical antifungal therapy. Amer J Drug Del., 2006;
4(4): 231-47.
2) http://www.google.com/wikepedia
3) http://www.rxlist.com
4) Mustufa M, Ossama Y, Viviane F, Nawal K et al. Deformable
liposomes and ethosomes: mechanism of enhanced skin delivery. Int
J Pharm., 2006;322(1- 2): 60-6.
5) Godin B, Touitou E, Rubinstein E, Athamna A, Athamna M, A new
approach for the treatment of deep skin infection by ethosomal
antibiotic preparation: an in vivo study. J Antimicrob chemo.,
2005;55(6):989-94.
6) Godin B and Touitou E. Mechanism of bacitracin permeation
enhancement through the skin and cellular membranes from an
ethosomal carrier. J Control Rel., 2004;94(2-3):365-79.
7) http://ntt-inc.com
8) Rao Y et al., In vitro percutaneous permeation and skin accumulation
of finasteride using vesicular ethosomal carriers. AAPS PharmaSci
Tech., 2008;July 23. (Under press)
9) Yi-Ping Fang, Yi-Hung Tsai, Pao-Chu Wu, Yaw-Bin Huang,
Comparison of 5-aminolevulinic acid encapsulated liposomes versus
Ethosomes for skin delivery of photodynamic therapy. Int J Pharm.,
2008;356:144-52.
10) Vaibhav D, Dinesh M, Jain N, Melatonin loaded ethanolic liposomes:
characterization and enhanced transdermal delivery. Eur J Pharm and
Biopharm., 2007;67(2):398-405.
11) Umamaheshwari R, Bhadra D, Jain N et al. Ethosomes a novel
vesicular system for enhanced transdermal delivery of anti-HIV drug.
Ind J Pharma Sci., 2004;66(1):72-81.
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12) Touitou E, Dayan N, Bergelson L, Eliaz M et al. Ethosomes-novel
vesicular carriers for enhanced delivery: characterization and skin
penetration properties. J Control Rel., 2000;65(3):403-18.
13) Horwitz E, Pisanty S, Czerninski R, Helser M, Eliav E, Touitou E et
al. A clinical evaluation of a novel liposomal carrier (ethosome) for
acyclovir in the topical treatment of recurrent herpes labialis. Oral
Surg Oral Med Oral Pathol Oral Radiol Endod., 1999; 87(6):700-5.
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9
Signature of the candidate:-
10
Remarks of the Guide:-
11
Name and Designation of:-
11.1
Institutional Guide:
Dr.Roopa karki
Professor and HOD
Dept. of Industrial Pharmacy
11.2
Signature:
11.3
Co-Guide:
11.4
Signature:
11.5
Head of the Department:
NIL
Dr. Roopa Karki
Dept. of Industrial Pharmacy
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11.6
Signature:
12.1
Remarks of the Principal:
12.2
Signature:
Dr. DIVAKAR GOLI
PRINCIPAL
ACHARYA & B. M.
REDDYCOLLEGE OF
PHARMACY,
CHIKKABANVARA POST,
HESARGHATTA MAIN ROAD,
SOLDEVANAHALLI,
BANGALORE-90
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