Documente Academic
Documente Profesional
Documente Cultură
Dr. R. P. Patel
M. Pharm., Ph.D.
H.O.D.
Department of Pharmaceutics and
Pharmaceutical Technology,
Shree S. K. Patel College of Pharmaceutical Education and Research,
Ganpat University 382711.
Forwarded through:
Dr. N. J. Patel
M. Pharm., Ph.D.
Principal,
Head of Department of Pharmacology,
Shree S. K. Patel College of Pharmaceutical Education and Research,
Ganpat University 382711.
Date:
Place: Ganpat University
DECLARATION
I hereby declare that the topic entitled Formulation, Development, Optimization and
Evaluation of Novel Topical Anti-acne Drug Delivery System which is submitted to
the Ganpat University, Kherva, in partial fulfillment for the award for Degree of Master
of Pharmacy in Department of Industrial Pharmacy, is the result of the work done by me
in Formulation and Development Department of Cadila Pharmaceuticals Limited,
Ahmedabad under the guidance of Dr. Hamsaraj Karanth and in Department of
Industrial Pharmacy under the guidance of Dr. R. P. Patel, Head of the Department
of Pharmaceutics & Pharmaceutical Technology, Shree S. K. College of pharmaceutical
Education and Research, Ganpat University, Kherva.
I further declare that the results of this work have not been previously submitted for any
degree of fellowship.
It is great pleasure for me to acknowledge all those who have contributed towards
the conception, origin and nurturing of this project.
This work was carried out at the Formulation and Development Department of
Cadila Pharmaceuticals Limited, Ahmedabad and Department of Pharmaceutics &
Pharmaceutical Technology, S. K. Patel College of Pharmaceutical Education
and Research, Kherva, during the years 2009-2010 which is affiliated with the
Ganpat University.
You want to do the right thing and you want to do it for the right reasons but if you
dont have the right guidance you can never hit the right target.
I am grateful to our Principal Dr. N. J. Patel for providing the facilities in the
institute for completion of this work.
My deepest gratitude to Dr. Dinesh Shenoy, Dr. Rahul Kasar, Mr. Gopalakrishan
Sarwanan, Dr. Imraan Khan, Dr. Sandeep Paranjape, Mr. Saurin Amin, Mr.
Deepak Gajare for sharing their views, constant guidance, scientific insight and
invaluable support as well as help in conducting my study. I am extremely thankful
to Mr. Ashish Gogia, Mr. Amit Mukharya, Mr. Bankimchandra Bhatt, Mr.
Deepak Bhatt, Mr. Rajneesh Shrivastava, and Dr. J. Suri Babu for providing
critical suggestions on my topic of dissertation and continuous guidance throughout
the investigation.
I sincerely acknowledge Col. Vijay K. Sharma, Mr. J. N. Pathak and other staff of
Cadila Pharmaceuticals Limited, Dholka, Ahmedabad, for their constant
encouragement, co-operation and valuable suggestions during my work.
It would have never been possible for me to take this project to completion without
the ideas and support of all staff members of S. K. College of pharmaceutical
Education and Research, Ganpat University, Kherva as well as all research
scientists of Formulation and Development Department of Cadila Pharmaceuticals
Limited.
Your sorrows get divided and your happiness gets multiplied with your friends A
friend is a person who understands your feelings, emotion and helps you to be what
you to be. I thank a lot my friends Kaushal Patel, Chirayu Pathak who helped me
in hardship through the sweet fragrance of friendship without which I could not have
won all the battles.
Last, but not the least, I express my gratitude and apologize to everybody whose
contributions, I could not mention in this page.
May the candle be lightened forever, the joy is not of light alone, but of presence of
those, who played the role behind the curtain.
GRAND FATHER
&
GRAND MOTHER
1.0 ABTRACT
Tretinoin is widely used in topical acne treatment at 0.025% and 0.05%
concentrations. Tretinoin is very effective in 0.05% strength but it causes skin
erythema on the applied area. The Niosomes seems to be promising drug delivery in
modern drug delivery systems. The main benefit over liposome is that the lipids are
replaced by non-ionic vesicles and hence the preparation is totally non-antigenic. The
non-ionic surfactants like SPANs and TWEENs are obtained from synthetic sources
and hence the quality is maintained same all the time. The tretinoin was incorporated
into niosomes using SPAN 60 and cholesterol was used as a stabilizer. Various ratios
of SPAN 60, cholesterol and tretinoin were tried and optimized. Various process
parameters were also optimized for the rotary flask evaporation method. The niosomal
dispersion was incorporated in to carbopol 971NF gel. The gel was kept for 3 months
accelerated stability studies. The niosomal dispersion was evaluated for various
parameters like vesicle size, shape and morphology by Transmission electron
microscopy (TEM). In-vitro and in-vivo studies were carried out. The drug release
pattern from gel was evaluated on the basis of in-vitro studies and skin irritation
studies on rat skin. The in-vitro study shows sustained release gel effects whereas the
in-vivo study shows no signs of irritation on the applied skin area.
2.0 INTRODUCTION
The ideal drug delivery system delivers drug at rate dictated by the need of the body
over the period of treatment and it channels the active entity solely to the site of
action. At present there is no drug delivery system available for tretinoin that achieves
site specific delivery and control release kinetics of drug in predictable manner.
Paul Ehrlich, in 1909, initiated the era of development for targeted delivery when he
envisaged a drug delivery mechanism that would target directly to diseased cell. Since
then, numbers of carriers were utilized to carry drug at the target organ/tissue, which
include immunoglobulins, serum proteins, synthetic polymers, liposomes,
microspheres, erythrocytes, niosomes etc (Theresa M. Allen, 1998).
Among different carriers liposomes and niosomes are well documented for
transdermal drug delivery.
Drug targeting can be defined as the ability to direct a therapeutic agent specifically to
desired site of action with little or no interaction with nontarget tissue (Dand Speiser
P, 1986).
Niosomes are supposed to give desirable interactions with human skin, when applied
in topical preparations. They are said to especially improve the horny layer
characteristics, both by reducing transepidermal water loss and by increasing
smoothness via replenishing lost skin lipids (Hans E. Junginger et al, 1991)
It is established that transdermal drug delivery with niosomes appears promising for
hydrophobic and amphiphilic drug molecules (Alok Namdeo & N.K.Jain, 1985).
Niosomes loaded with drugs for dermal application are aimed to preferentially show
interactions with inflamed (epidermal) tissue without exerting an immediate or strong
systemic action (Hans E. Junginger et al, 1991).
Niosomes were used for topical delivery of drugs by virtue of the following inherent
advantages compared to conventional topical dosage forms such as creams, gels and
lotions etc. Preparations containing niosomes give significantly higher epidermal and
dermal concentration. Reduced systemic concentration, which is desirable feature for
certain drugs (corticosteroids) in which systemic effect must be avoided. Niosomes
can be excellent solubilizing agents for drugs with low solubility (Dorr R.T. et al
1994).
Protection from degradation acts as sustained release system, which decreases the
irritation and sensitivity of certain drugs on the skin (H. Bouwstra, 1994).
Topical treatment is useful in topical skin infections, that is, those confined to the
stratum corneum, squamous mucosa etc. Such diseases include Acne,
dermatophytosis (ringworm), candidiasis, tinea nigra and fungal keratitis. The most
commonly used anti-acne drugs for local action are benzoyl Peroxide, clindamycin,
clarithromycin, azithromycin, Erythromycin, trerinoin and Isotretinoin.
Topical tretinoin has been a mainstay of acne therapy for nearly 25 years. It is
available in a number of formulations and concentrations, including 0.025%, 0.05%
and 0.1% cream, 0.1% and 0.25% gel, and 0.1% liquid solution. Tretinoin works by
both comedolysis and by normalizing the maturation of follicular epithelium so that
comedo formation ceases. Bernerd et al studied the events in tretinoin-induced
comedolysis in the rhino mouse model. Tretinoin produces irritation on applied skin
so it is hypothesized that niosomal form of Tretinoin can overcome this drawback.
2.1 Aim
The aim of the present investigation was
To study the in-vitro role of the developed products for topical application.
It was hypothesized that incorporation of tretinoin into niosomes will improve the
amount and time of the drug retention within the skin, so as to increase the therapeutic
index of the drug. It was also assumed that, the niosomal form would restrict the entry
of the drug at the site of action. It was also expected that the dose and frequency of
topical application of the drug will reduce and hence toxicity and cost of product.
In-vivo evaluation.
2.3 References
Alok Namdeo & N.K.Jain, Indian J.Pharm. Sci., 1996, 58(2)pg.no. 41-46
Breimer, D. Dand Speiser P. Topics in pharmaceutical Sci. 5 Elsevier Science
Publishers (1986), p.291
Dorr R.T., Von Holf D D, eds. Cancer Chemotherapy hand book 2nd ed. Norwalk,
Application & Lange (1994): 319.
Gregoriadis, G.Targeting of drugs. Nature, 265,1977,p.407-411.
Handjani-Vila, Riber A., Rondot D. and Valerberghe G., Int. J. Cos. Sci.
(1997),1;303-314
Hans E. Junginger, Hans E.J. Hofland and Joke A.Bouwstra, cosmetics and toiletries,
1991, 361, p 45-50.
Kiwada H, Nimura H, Kato Y. Pharm. Bull. (1985), 33:2475-2482.
Saravanan D. and Popli H. J. Pharm. Pharmacol. Communi (1998),4;485-487
Schier, H.Bouwstra, J., J.Controlled Rel. 1994, 30, p. 1-15.
Theresa M. Allen,; Drugs (1998) published by Adis international Ltd.,
Nov:56(5);747-756.
3.1 Niosomes
3.1.1 Introduction
Niosomes or non-ionic surfactant vesicles are microscopic lamellar structures formed
on admixture of non-ionic surfactant and cholesterol with subsequent hydration in
aqueous media (Malhotra M. and Jain N.K., 1994).
They are vesicular systems similar to liposomes that can be used as carriers of
amphiphilic and lipophilic drugs.
In niosomes, the vesicles forming amphiphile is a non-ionic surfactant such as Span
60 which is usually stabilized by addition of cholesterol and small amount of anionic
surfactant such as dicetyl phosphate (Buckton G., Harwood, 1995).
2. They are osmotically active and stable, as well as they increase the stability of
entrapped drug.
6. They improve oral bioavailability of poorly absorbed drugs and enhance skin
penetration of drugs.
7. They can be made to reach the site of action by oral, parenteral as well as topical
routes.
8. They allow their surface for attachment of hydrophilic group and can incorporate
hydrophilic moieties in bilayer to bring about changes in their in-vivo behavior.
10. They improve the therapeutic performance of the drug molecules by delayed
clearance from the circulation, protecting the drug from biological environment
and restricting effects to target cells.
Niosomes are now widely studied as an alternative to liposomes, which exhibit certain
disadvantages such as they are expensive, their ingredients like phospholipids are
chemically unstable because of their predisposition to oxidative degradation, they
require special storage and handling and purity of natural phospholipids is variable.
It has been stated that surfactants can exist as micelles, and these structures are in
dynamic equilibrium with non-associated surfactant molecules, such that the
molecules in the micelles regularly interchange with those, which are free monomers.
Even though, micelles can solubilize drugs, the prospects for using micelles as
delivery system are not too good, as the drug will routinely be able to leave the
micelles. When cholesterol is added to the number of different non-ionic surfactants,
it is possible to form vesicles, which are stable i.e. not in equilibrium in the manner of
micelles.
The first report of non-ionic surfactant vesicles came from the cosmetic applications
devised by LOreal (Handjani-Vila N. et al, 1979).
The application of vesicular (lipid vesicles and non-ionic surfactant vesicles) systems
in cosmetics and for therapeutic purpose may offer several advantages: -
The vesicles may act as a depot, releasing the drug in a controlled manner.
entrapment. They also observed that as HLB value of surfactant decreased, the mean
size was reduced.
two fluidized streams interact at ultra high velocities, in precisely defined micro
channels within the interaction chamber. The impingement of thin liquid sheet along a
common front is arranged such that the energy supplied to the system remains within
the area of niosomes formation. The result is a greater uniformity, smaller size and
better reproducibility of niosomes formed.
are placed in series for up to 8 passages. It is a good method for controlling niosome
size.
The niosomal suspension is centrifuged and the supernatant is separated. The pellet is
washed and then resuspended to obtain a niosomal suspension free from unentrapped
drug.
1. Drug
Entrapment of drug in niosomes increases vesicle size, probably by interaction of
solute with surfactant head groups, increasing the charge and mutual repulsion of the
surfactant bilayers, thereby increasing vesicle size (Stafford S. et al, 1984). In
polyoxyethylene glycol (PEG) coated vesicles; some drug is entrapped in the long
PEG chains, thus reducing the tendency to increase the size (Hu C. and Rhodes D.G.,
1989). The hydrophilic lipophilic balance of the drug affects degree of entrapment.
Doxorubicin niosomes possessing muramic acid and triglycerol surfaces were not
taken up significantly by liver. The triglycerol niosomes accumulated in the tumor
and muramic acid vesicles accumulated in the spleen. Those vesicles with
polyoxyethylene surface were rapidly taken up by the liver and accumulated to a
lesser extent in tumor. Baillie et al investigated the encapsulation and retention of
entrapped solute 5, 6-carboxy fluorescence (CF) in niosomes. They observed that
stable vesicles could not be formed in the absence of cholesterol but were more
permeable to entrapped solute. The physical characteristics of the vesicles were found
to be dependent on the method of production. Gupta P. K. also described various
methods of production and various aspects of characterization of niosomes.
Carter et al reported that multiple dosing with sodium stibogluconate loaded niosomes
was found to be effective against parasites in the liver, spleen and bone marrow as
compared to simple solution of sodium stibogluconate.
3.1.9 Applications
Niosomal drug delivery is potentially applicable to many pharmacological agents for
their action against various diseases. Some of their therapeutic applications are
discussed below.
The cells of RES preferentially take up the vesicles. The uptake of niosomes by
the cells is also by circulating serum factors known as opsonins, which mark them
for clearance. Such localized drug accumulation has, however, been exploited in
treatment of animal tumors known to metastasize to the liver and spleen and in
parasitic infestation of liver (Malhotra M. and Jain N.K., 1994).
It has been suggested that carrier system can be directed to specific sites in the
body by use of antibodies (Gregoriadis G., 1981). Immunoglobulin seems to bind
quite readily to the lipid surface, thus offering a convenient means for targeting of
drug carrier (Weissman G. et al, 1985). Many cells possess the intrinsic ability to
recognize and bind particular carbohydrate determinants and this can be exploited
to direct carriers system to particular cells.
2) Neoplasia
Doxorubicin, the anthracyclic antibiotic with broad spectrum anti tumor activity,
shows a dose dependant irreversible cardio toxic effect. Niosomal delivery of this
drug to mice bearing S-180 tumor increased their life span and decreased the rate
of proliferation of sarcoma (Cummings J., 1984). Niosomal entrapment increased
the half-life of the drug, prolonged its circulation and altered its metabolism.
Intravenous administration of methotrexate entrapped in niosomes to S-180 tumor
bearing mice resulted in total regression of tumor and also higher plasma level and
slower elimination (Suzuki K. and Sokan K., 1990).
3) Leishmaniasis
Niosomes can be used for targeting of drug in the treatment of diseases in which
the infecting organism resides in the organ of reticulo-endothelial system.
Leishmaniasis is such a disease in which parasite invades cells of liver and spleen.
The commonly prescribed drugs are antimonials, which are related to arsenic, and
at high concentration they damage the heart, liver and kidney. The study of
antimony distribution in mice, performed by Hunter et al showed high liver level
after intravenous administration of the carrier forms of the drug. Baillie et al
reported increased sodium stibogluconate efficacy of niosomal formulation and
that the effect of two doses given on successive days was additive.
Niosomes have been used for studying the nature of the immune response
provoked by antigens. Brewer and Alexander have reported niosomes as potent
adjuvant in terms of immunological selectivity, low toxicity and stability.
Slow penetration of drug through skin is the major drawback of transdermal route
of delivery. An increase in the penetration rate has been achieved by transdermal
delivery of drug incorporated in niosomes. Jayraman C.S. et al has studied the
topical delivery of erythromycin from various formulations including niosomes or
hairless mouse. From the studies, and confocal microscopy, it was seen that non-
ionic vesicles could be formulated to target pilosebaceous glands.
a) Sustained release
Azmin et al suggested the role of liver as a depot for methotrexate after niosomes
are taken up by the liver cells. Sustained release action of niosomes can be applied
to drugs with low therapeutic index and low water solubility since those could be
maintained in the circulation via niosomal encapsulation.
drug delivery and being non-ionic; it is less toxic and improves the therapeutic
index of drug by restricting its action to target cells.
Where
ks Partition coefficient for the interfacial partitioning of the penetrant molecule from
a transdermal therapeutic system up to the skin tissues
Dss is the diffusivity for steady state diffusion of the penetrant molecule through the
skin tissue and hs is the total thickness of the skin tissues.
The permeability co-efficient (Ps) for skin penetrant can be considered as a constant,
since Ks, Dss and hs are essentially constant under a fixed condition.
In order to achieve a constant rate of drug permeation it requires a condition being
maintained that the drug concentration on the surface of S. Corneum (Cd) is
consistently and substantially greater than the drug core in the body (Cr) i.e. Cd >>
Cr: therefore Eq-1 can be reduced to
(dq/dt) = Ps.Cd ................................................................................................3
And the rate of skin permeation (dq/dt) becomes constant, if the Cd values remains
fairly constant through out the course of skin permeation. To maintain the Cd at a
constant value it is critical to make the drug to be released at a rate (Rr), which is
always greater than the rate of skin uptake (Ra) i.e. Rr >>Ra.
By doing so, the drug concentration on the skin surface (Cd) is maintained at a level
which is always greater than the equilibrium solubility of the drug in the S. Corneum
(Ces), i.e. Cd>> Ces; and a maximum rate of skin permeation (dq/dt) m, as expressed
by,
(dq/dt) m = Ps Ces ...........................................................................................4
Apparently the magnitude of (dq/dt) m is determined by the skin permeability
coefficient (Ps) of the drug and its equilibrium solubility in the S. Corneum (Ces).
2. The vehicles general influence on the state of the skins hydration and
3. Vehicle effects on the local vesicles, which alter drug clearance and skin
temperature in either case, subtly influencing permeability.
3.4 Acne
3.4.1 Introduction (Kligman AM et al, 1969)
Acne vulgaris is a common and extraordinarily variable disease. Even by using the
strictest definitions, it is possible to state that all persons have some degree of acne at
some time in their lives. Indeed, if we look hard enough, some features of the acne
process can be found throughout life. Unfortunately the most severe disease typically
occurs when patients are least able to deal with cosmetic problems. In addition, acne
accounts for an extraordinary number of visits to dermatologists and generalists and is
a matter of significant economic import, given the millions of prescriptions written for
acne therapy.
perhaps certain sebaceous lipids (Blair C et al, 1970). Lacking a blackhead, the closed
comedo often remains hidden until the skin is stretched. This is a particularly useful
maneuver on relatively lax skin, such as that around the mouth. The earliest
impaction, the microcomedo, is often clinically inapparent, but is the most numerous
type of comedo.
The granular layer becomes more prominent as the process continues. Electron
microscopy reveals lipid-containing inclusions in desquamated keratinous material
and an increase in tonofilaments and lamellar granules as comedones grow, the
associated sebaceous gland becomes progressively smaller. The cause of this atrophy
is not known. Plausible theories include mechanical damage to the gland by comedo
growth and a loss of androgen responsiveness by the sebocytes. Perhaps epithelial
cells elaborate factors that modulate sebaceous gland activity whose levels are altered
by the process of comedo formation. Hairs may be found in most comedones, and
there is evidence to suggest that vellus hair growth is increased in patients with acne.
Closed comedones contain one or two tiny hairs each, whereas open comedones may
bear up to 16 vellus-type hairs (Leyden JJ et al, 1972). The number of hairs appears to
be an indication of the age of the lesion. Interestingly, large hairs are virtually never
found in comedones. It may be that a terminal hair extending to the skin surface
creates a channel for sebum to escape the comedo or that the hair and its motion acts
to break up follicular impactions. The trigger for comedo formation in acne is not
known with any certainty. Many compounds have been shown to produce comedones
in experimental systems, but none is clearly relevant to acne in most patients. There
are two main models for studying comedo formation, the rabbit-ear model and human
skin. In the former, many compounds are capable of producing follicular impaction,
and the system is used for screening pharmaceutic and cosmetic ingredients. In
general, human skin is more resistant to comedo formation than the rabbit ear, but
there is good agreement between the two systems for many compounds. Physical
agents may also trigger comedogenesis. Favre-Racouchaut syndrome and solar or
"senile" comedones are certainly linked to photodamaged skin.
Histologically they are noninflamed giant comedones that are surrounded by marked
solar elastosis. Some animal data also exist to support the role of actinic damage in
comedogenesis. Mills et al. demonstrated that comedogenesis by sulfur, cocoa butter,
sebum, squalene and some sunscreens were UV-enhanced in the rabbit and similar
effects were seen with coal tar and squalene in human subjects. The inflammatory
process itself may also induce comedones. A rat model of chronic inflammation
triggered by Propionibacterium aches cell walls displays follicular plugging in
proportion to the degree of inflammation. Secondary comedones in severe acne may
be caused by a similar mechanism.
secretion seen among adolescent patients with acne. As patients with acne mature, this
difference in population becomes less striking (Burton JL et al, 1971).
a) Molecular details
b) Appearance
(1) Solubility
3.5.2 Identification
A: Infrared absorption
B: Ultraviolet absorption
Solution: 4 g per ml.
Medium: acidified isopropyl alcohol (prepared by diluting 1 ml of 0.01 N
hydrochloric acid with isopropyl alcohol to 1000 ml).
Absorptivities at 352 nm, calculated on the dried basis, do not differ by more than
3.0%.
retinoids induce a slight thinning of the stratum corneum, sunburn might be thought to
be more likely; but this has not been borne out by experience or study. The injunction
to apply tretinoin at night stems from its photolabile propensity; as yet, it is unknown
whether night time application minimizes photodestruction of the compound enough
to alter therapy.
3.6 References
Almira I, Blazek-Walsh and G.Rhodes David Pharm. Res.(2001) 18:5.
Azmin M.N. Florence A.T. Handiani Vila R.M., Stwart J.F. Vanlerberghe G.
Whittakar J.S. J. Pharm. Pharmacol. (1985) 37:237.
B.L. Silver Ed., The Physical Chemistry of Membranes, Alan & Unwin and Soloman
Press. New York (1985) p.209-230.
Baillie A.J. Florence, A.T., Hume. L.R., Muirhead, G.T., Rogerson. A,
J.Pharm.Pharmacol. (1986), 37: 237-242.
Baillie, A.J., Coombs, G.H.,Dolan T.F., Laurie J. J. Pharm. Pharmcol. (1986)
38:502-505.
Baillie, A.J., Florence A.H., Hume L.R., Munhead G.T. and Rogerson A., J. Pharm.
Pharmcol (1985), 37:683-868.
Baillie. A.J; Florence, A.T., Hume L.R, Rogerson.A., Muirhead, G.T., J.Pharm.
Pharmacol. (1984), Supp. 36: 48P.
Brewer J.M., Alexander J.A. Immunology (1992), 75 (4) :570
Buckton G., Harwood, Interfacial phenomena in Drug Delivery and Targeting
Academic Publishers Switzerland, (1995) p. 154-155.
Cable C., and Florence A.T. J.Pharm.Pharmcol (1988) 40: 31p.
Chandraprakash K.S., Udupa N., Umadevi P. Pillai G.K. Ind. J. Pharm. Sci (1992) 54
(5): 197.
Chandraprakash, K.S., Udupa N, Umadevi P. Pillai G.K. (1990), Int. J. Pharma;
61:R1.
Chauhan, S and Luorence, M.J. J. Pharm. Pharmacol.,(1989), 41 : 6p.
Cummings J., Staurt J.F.B., Calman K.C.J. Chromatogr. (1984) 311:125.
D Souza S.A., Ray J., Pandey S., and Udupa N. J. Pharm. Pharmcol (1997), 49 (2)
145-149.
Don A. Van Hal, Joke A. Bouwstra and Hans E. Non ionic surfactant vesicles
containing estradiol for topical application Ph.D. thesis. Centre for drug research P.
330-339.
Gregoriadis G., Lancet (1981), 2: 241
Handjani-Vila N., Ribier, A., Rondot B. Vanlerberghe G. (1979) Int. J. Cos. Sci.
1:303.
Hofland H.F.J; Bouwstra J.A., Spies F.H., Bodde H.E., Cullander, Junginge H.E.,
Prodesses, Inter. Symp. Control.Rel. Bioact Mater Controlled release Society. Inc,
(1992) 19:230.
Hu C. and Rhodes D.G. Int. J. Pharm (1999), 185: 23-35.
Hunter C.A., Dolan T.F., Coombs G.H. Baillie A.J. J.Pharm. Pharmacol. (1988)
44:161.
Jagtap A. and Inamdar D. Ind.J.Pharm. Sci. (2001)63,(1):4954
Jayaraman C.S. J. Pharm. Sci. (1996) 85 (10), 1082-1084.
Khand L., Rogerson A., Halbert G.W., Baillie A.J., Florence A.T.
J.Pharm.Pharmacol (1987). 39 (Supp): 41P.
Khandare, J.N, Madhavi, G., and Tamhankar, B.M., The Eastern Pharmacist (1994)
XXXVII, 61.
KiwadaH. Nimura H. Kato. Y.Chem. Pharm. Bull. (1985), 33:2475-2482.
Malhotra M. and Jain N.K. Indian Drugs (1994), 31 (3) : 81-86
Maver L.D. Bally M.B. Hope. M.J. Cullis P.R. Biochem Biophys. Acta (1985),
816:294-302.
Mc. Cormack B, Gregordias G., Int. J. Pharm (1998), 162:59-69.
Moser P., Arvier M.M., Labrude P., Handjani Vila. R.M., Vignerson C. Pharma.
Acta.Helv. (1989), 64 (7): 192.
Moser P., ArvierM.M., Labrude P., Vignerson C., Pharm Acta Helv. (1990) 65 (3):
82.
Namdeo A. Mishra P.R., Khopade A.J., Jain N.K., Indian Drugs (1999),36 (6):378-
380.
Parthasarthi, G., Udupa N., Umadevi P. and Pillai G.K., J.Drug Target. (1994) 2(2):
173-82.
R.A. Raja Naresh, G.K.Pillai, N.Udupa, Chandrashekhar, Ind.J.Pharmacol (1994),
26:46-48.
Raja Naresh R. A, Udupa N. Uma Devi, J.Pharm. Pharmacol. (1996), 48:1128.
Ramana M.V. and Anil Kumar N. Scientific Abstract 1999 P.371.
Reidman, O.M. and Boger E.,Anal.Chem (1961) 33, 906.
Rogerson, A., Cummings, J., Willmott, N., Florence, A.T. (1988)
J.Pharm.Pharmcol.40: 337.
S. Gayatri Devi, Venkatesh, Udupa N. Inj. J. Pharm. Sci (2000) 12:479.
Sheen I.P., Singh U.V.Kamath R., Uma Devi P., Udupa N., Indian J. Pharm. Sci.
(1998), 1:45-7
Stafford S., Baillie A.J. and Florence A.T. J.Pharm.Pharmacol (1988), 40, 26p.
Suzuki K. and Sokan K., Cosmetic and Toiletries (1990), 105:5
Szoka, F., Papahadyopoulos, D., Ann. Rev. Biophys-Bioeng (1980), 9:467-508.
Vartor K.C., Dolan T.F., Baillie A.J. and Maccolgan C. J.Pharm. Pharmcol (1989),
41:87.
Weissman G., Bloomgarden D., Kaplan R., Cohen C., Hoffstein S., Tucker C.,
Gotlibe A., Nagle P. Proc. Natl. Acad. Sci (1975) 72:88
Yoshida H., Lehr, C.M., Kok W., Jugonger H.E., J.C. and Bouwistra J.A., J.Control
Rel, (1992) 21:145.
Yoshika T and Florence A.T., Int. J.Pharm (1994) 108:117.
Yoshioka T., Stermberg B. and Florence A.T. J. Pharm. Pharmco. Suppl., (1994),
105:1-6.
Yoshioka T., Stermberg B: Moody M., Florence A.T. J.Pharm. Pharmcol.Supp.,
(1992), 44:1044.
4.1.1 Instruments
4.1.2 Materials
Table 3.2: Materials used in investigation
Name of the materials Name of the Suppliers
Tretinoin SOLMAG, Italy
SPAN Croda Chemicals (India) Pvt Ltd.
TWEEN Croda Chemicals (India) Pvt Ltd.
Cholesterol Merck, USA
Dichloromethane Finar Chemicals Limited, Ahmedabad
Ammonium acetate Finar Chemicals Limited, Ahmedabad
Glacial acetic acid Finar Chemicals Limited, Ahmedabad
Ethanol S.D. Fine Chemicals Ltd., Mumbai
Water for injection Inhouse Source
4.1.3 Reagents
B. Solvent blend
Prepare a solvent blend by adding 20% ethanol and 80% acetate buffer solution.
Principle
Procedure
Appropriate aliquots of the stock solution of the drug were taken into 10ml volumetric
flasks. To this, required amount of solvent blend was added to get the desired
strength. It was shaken vigorously for 1 minute. The absorbance was measured at 352
nm against reagent blank with a spectrophotometer. The mean of absorbance values
are recorded in the Table 3 and the regressed calibration curve was plotted in
Figure 4.
1.2
0.8
Absorbance
0.6
0.4
0.2
0
0 5 10 15 20 25
Concentration (mcg/ml)
Tretinoin Tr API
1.
Conditions
Sampling
40C/75 % RH 2 - 8C Tests
schedule
Initial T T
1. Appearance
15 Days T T
2. % Assay
30 Days T T
T = To be tested
Pack details: Amber glass vial with rubber stopper and aluminum seal.
Condition: 2 - 80C
Sr.
Name Ratio Time point Appearance % Assay
No.
Tretinoin 1 Initial No change 101.45
1 15 days No change 100.79
30 days No change 99.89
Tr +chl 1:1 Initial No change 101.79
2 15 days No change 100.94
30 days No change 99.95
Tr + SP40 1 : 10 Initial No change 101.65
3 15 days No change 100.58
30 days No change 99.94
Initial No change 101.32
4 Tr + SP60 1 : 10 15 days No change 100.89
30 days No change 99.74
Initial No change 101.35
5 Tr + SP60 1 : 20 15 days No change 100.49
30 days No change 99.83
Initial No change 101.49
6 Tr + SP85 1 : 10 15 days No change 99.85
30 days No change 98.45
Initial No change 101.36
7 Tr + chl + SP40 1 : 1 : 10 15 days No change 99.46
30 days No change 98.62
Initial No change 101.44
8 Tr + chl + SP60 1 : 1 : 10
15 days No change 100.97
Procedure
Span-60 : Cholesterol at a ratio of 20:1 by weight was dissolved in 20 ml of
Dichloromethane, in a 250 ml round-bottomed-flask. The flask was fitted onto a
rotary flask evaporator and connected to a vacuum pump. The solvent system was
evaporated under vacuum at 50 C using water bath for 10 minutes. The dried
surfactant film was hydrated with 50 ml distilled water for 30 minutes 50 C using
bath sonicator. The hydrated dispersion was kept in refrigerator for 2 hours for sealing
of vesicles.
The optimized conditions were decided on the basis of microscopy and drug
entrapment efficiency, which were determined as follows:
1. Microscopic examination
All the batches of the niosomes were viewed under microscope to observe size, shape
and lamellarity. The representative batches of niosomes were photographed using
Olympus B 201 Microscope at magnification 10x, 40x and 100x.
Niosomes were subjected to particle size analysis using Malvern particle size
analyzer.
In order to obtain niosomes with maximum entrapment and minimum size, the
following formulation and process variables were optimized based on the factors
considered as above.
4. Hydration volume
5. Hydration temperature
6. Hydration time
7. Annealing time
Spans 40, 60, and 85 as well as Tween 80 were used for formulation of niosomes.
Good niosomes were obtained with Span whereas no niosomes were formed with
Tween. Among the Span, niosomes prepared using Span 60 gave maximum drug
entrapment and with an increase in mean particle size the percent entrapment
increased.
Various ratios of Span 60: Cholesterol like 5:1, 10:1, 20:1 by weight were tried.
Increase in proportion of cholesterol in the niosomal membrane, resulted in reduction
in percentage drug entrapment. Thus the batch with surfactant: cholesterol ratio of
20:1 had 53.77% entrapment. This may be due to intercalation of cholesterol in the
bilayer, which has been reported to decrease the entrapment volume and thus
entrapment efficacy. Thus, as the surfactant ratio increases as compared to
cholesterol, the vesicle size increases. This results in an increase in entrapped volume
and percentage drug entrapment. Hence, 20:1 surfactant : cholesterol ratio was
considered optimum.
Various solvent system ratios like 1:1, 1:2, and 1:5 of DCM : Ethanol were tried and
pure DCM was finally selected as the optimized one.
Table 9: Various solvent system ratios of DCM : Ethanol and film formation
4. Hydration volume
Hydration volume was progressively increased from 10 ml, 15 ml, 20 ml, 30 ml, 35
ml and 40 in order to completely hydrate the lipid film formed, as well as to increase
percentage drug entrapment. Finally 40 ml was selected as the optimized one.
35 Insufficient
40 Insufficient
50 Good homogenous dispersion
5. Hydration temperature
6. Hydration time
Various hydration times like 15, 30, 45 & 60 min were used for formation of
niosomes of tretinoin and finally 45 minutes was selected as the optimized one.
7. Annealing time
Various Annealing Times like 1 hour, 2 hours and 3 hours were used for formation of
niosomes of tretinoin and finally 2 hours was selected as the optimized one.
Various film formation times like 5 min, 10 min, 15 min and 20 min were tried for
formation of niosomes of tretinoin and finally 10 min was selected as the optimized
one.
The table below shows optimized process and formulation parameters for
tretinoin niosomes.
4.5.1 Formula
4.5.2 Procedure
120
100
80
% content
60 2 8
25/60 RH
40 40/75 RH
20
0
1 2 3 4 5 6
Time (weeks)
120
100
80
% Content
60 2 8
25/60 RH
40 40/75 RH
RH
20
0
1 2 3 4 5 6
Time (weeks)
4.8 Discussion
Preformulation
The preformulation design was carried out as shown in table 4. The physical
appearance of the Tretinoin is yellow colored powder thus the acceptance criterion
was set as the color change visible by naked eye. The non-ionic surfactants and
cholesterol were mixed in various proportion and the studies were carried out. The
drug is light-sensitive so the amber colored vials were chosen as containers.
The table 5 shows the results obtained after 4 weeks of observation. There was no
color change in any vials, so it can be concluded that there is no visible reaction
between drug and excipients. The sampling was done every week but there was no
sign of color change as well as the physical change in each vial. The samples were
kept at 2 8 C and 40 C / 75 %RH just to test the samples at extreme conditions.
The 60 C condition was not selected because the drug itself is thermo-sensitive.
The results are plotted in separate tables as shown in table 5. Both the tables show no
sign of change in appearance and hence it can be concluded that the preformulation
studies show positive results and by using the excipients along with the drug, further
formulation can be carried out.
DSC study
The drug shows the characteristic peak at 183.66 C as its melting point is
180 183 C reported. The presence of other ingredients may affect the characteristic
peak area due to incompability. The deviation in peak or complete loss of peak
indicates the chemical reaction. The figure 5 shows the characteristic peak of tretinoin
alone. The figure 5 shows the presence of tretinoin and cholesterol at 173.65 C and
145.44 C respectively. The presence of drug peak indicates no significant loss of
tretinoin in presence of cholesterol.
The figure 5 shows the peak of tretinoin and SPAN 60 at 185.28 C and 59.72 C
respectively. The presence of tretinoin peak at 185.28 C indicates the presence of
tretinoin in presence of SPAN 60 and hence there is no significant loss of tretinoin.
The figure 5 shows the combined peaks of cholesterol, SPAN 60 and Tretinoin. The
SPAN 60 indicates its presence at 58.88 C, the cholesterol shows peak at 149.28 C
whereas the tretinoin shows its presence at 168.64 C. All three peaks are present in
graph and hence the tretinoin is not being interfered due to presence of other two
excipients.
Niosome preparation
In preparation of niosomes, various ratios of cholesterol and surfactants were tried but
due to specific reasons some formulas were rejected. The SPAN 85 has HLB value
1.8. By using SPAN 85 the 1 : 10 ratio was applied but due to its very low HLB value
the phase separation was seen after film hydration thus the SPAN 85 was rejected.
The vesicles were not formed properly.
By using SPAN 40, the vesicles were formed but the % entrapment was very low.
After hydration the cholesterol and SPAN 40 particles were also visible. SPAN 40
was used in two ratios 1 : 1 and 1 : 10 but in both the cases, crystals were visible thus
the SPAN 40 was rejected.
Tween 80 is a non-ionic surfactant. It shows good emulsification properties but its
HLB value very high around 15.0 so it not able to form the bilayer vesicles. For
niosome vesicle formation, the HLB value should be between 4.0 8.0. Tween 80
forms emulsion of drug and aqueous part but the vesicles are not being formed. So,
Tween 80 was rejected due to HLB value.
SPAN 60 is chemically sorbitan monostearate having HLB value 4.7. SPAN 60 has
highest phase transition temperature as compare to other SPANs. So once after
formation of vesicles there are less chances of vesicle bursting. The phase transition
temperature is around 50 C for SPAN 60 which is tolerable by the drug and hence the
SPAN 60 shows all the required characteristics. SPAN 60 is highly lipophilic in
nature and hence the gets easily dissolved in dichloromethane.
Process parameters
The surfactant cholesterol ratio is very important to optimize because the cholesterol
acts as stabilizer and itself is lipophilic in nature so increased concentration of
cholesterol may cause reduction in drug entrapment. The optimum concentration is
needed otherwise vesicle stability may decrease. Various ratios were tried for the
preparation purpose ad finally 1 : 20 was selected to prepare all the further batches.
The solvent system plays a major role to in thin film formation because, it is
necessary that the film must be thin enough otherwise there may be lots of problems
in hydration step. Mainly two solvents were tried ethanol and dichloromethane in
various proportions. The ethanol containing film showed stickiness and hence it was
tough to hydrate it, whereas the dichloromethane containing film was easy to hydrate
with warm water. The solubilization capacity of dichloromethane is much higher than
ethanol thus it is also cost effective to use the dichloromethane. Thus, the
dichloromethane was selected as solvent.
The hydration volume must be optimized in order to ensure the complete hydration of
the thin film in rotary flask. The insufficient hydration may lead to decreased
entrapment efficiency. But more hydration volume can lead to dilution of the
dispersion. The hydration volume was finalized as 50 ml at temperature 50 C.
The hydration temperature can affect the drug properties because higher temperature
can lead to degradation of drug. The hydration temperature should be enough higher
than the phase transition temperature of the surfactant. If the temperature has been
kept lower than that then the vesicle formation may not take place as the film
hydration may not occur properly. Various temperatures ware tried to see the
hydration characteristics and finally 50 C was selected as ideal temperature.
After vesicle formation the vesicles are required to be kept for sealing. The dispersion
is thus kept in refrigerator at 2 8 C for sealing. If the vesicles not kept for sealing
they may get break and hence entrapment efficiency decreases. Therefore the
optimization of annealing time is necessary. The optimized time is 2 hours for
niosomal dispersion, less than that the vesicles get ruptured. So after formation of
vesicles, optimized annealing time was 2 hours.
Film formation time plays major role in niosome vesicle formation. If the film is thin
enough then the good vesicles are formed whereas the thick film is not easy to hydrate
thus the vesicles are forming properly. The film should be non-sticky and easy to
hydrate. Various factors affect the film quality like surfactant nature, solvent system,
applied vacuum, flask size etc. The optimized film formation time is 10 minutes for
niosomes. The time less than 10 minutes cannot form a thin film and thus the
hydrations will not occur properly.
Stability studies
The stability studies of niosomes and niosomal gel of Tretinoin were carried out for
six weeks at different accelerated conditions like 2-8 C, 25 C / 60 %RH and 40 C /
75 %RH. The samples were analyzed for assay of the Tretinoin. The result of stability
FTIR study
The tretinoin is chemically all Trans retinoic acid. The figure 15 shows the graph of
tretinoin. Here the COOH group is present in the structure. The acid group shows its
characteristic peak at 1722.49 cm-1. The presence of this bond shows the characteristic
property of the tretinoin.
In figure 15, the FTIR graph of niosomal formulation is seen. The niosomal
dispersion was lyophilized for this purpose. The moisture content was checked by
karl-fisher method and it was found to be less than 3 % thus it cannot interfere the
FTIR studies. The figure 15 shows the peak 1728.28 cm-1 which indicates the
presence of acid group in the structure. The final formulation shows the presence of
the characteristic peak of tretinoin and hence it proves that the excipients used in the
formulation are not interacting with the drug and so the formulation is chemically
stable.
4.9 References
Azmin M. N., Florence A.T. Handjani Vila R.M. Stuart J.F. Vanler Berghe G.
Whittaker J. S. J. Pharm. Pharmacol. (1985), 37;237-242.
Baillie, A. J. Florence A.T., Hume,L. R. Rogersor A., Muirhead, G.P. J. Pharm.
Pharmacol. (1984) Supp. 36;p.48.
Cavrini, V.; Di, Pietra AM.; Raggi, MA. Int. J. Pharm. (1982), 10(feb); 119-124.
Cavrini, V.; Di, Pietra AM.; Raggi, MA. Pharm-Acta-Helv. (1981), 56(6), 163-165.
Cavrini, V.; Di, Pietra AM.; Raggi, MA.Int. J. Pharm. (1982), 10(feb); 119-124.
Chein L.H. and Chem Y.W. Drug Development Ind. Pharm (1994),20 (6) : 935 : 945
Indian Pharmacopoeia (1996) 4th edition, Vol II, published by the controller of
publication, Delhi, Govt. of India. Ministry of Health and Family welfare.
Khandare J.N., Madhavi G. and Tamhankar, B. M. The Eastern pharmacist. (1994)
XXXVII, p.61.
Lemli, J.;Knockaert, I.Pharm. Weekbl, Sci. Ed., 26 Aug. 1983 5(4)142-144(Fr).
Sethi, P. D., Quantitative Analysis of Drugs in Pharmaceutical Formulations (1985),
10, Unique publishers, Delhi.
Zeinab, H., Mohamed, Savasan, M. Amer, Amira M-Elkousasy, J. Pharm. BioMed.
Ana. (1994), 12 (9);1131-1136.
5.1 Introduction
Physiological availability of the drug depends on various factors. In-vitro methods are
valuable as screening procedure and for deducing physicochemical parameters such as
flux, partition co-efficient and diffusion co-efficient. The theoretical disadvantage to
such technique is that, the method is not exactly similar to the behavior of living
tissue insitu, particularly with respect to a capricious blood supply and metabolism
(Chein L.H. and Chem Y.W., 1994).
The diffusion test is the most useful in-vitro method for assuring batch-to-batch
uniformity and bioavailability of the formulation. In present study, a diffusion
apparatus was set up using dialysis sac and the optimized batch was subjected to
diffusion study.
5.2 Experimental
5.2.1.1 Materials
5.2.1.2 Reagents
The pH 7.4 phosphate buffer saline I.P. (PBS) was prepared as per the procedure
described in I.P. 1996. After the diffusion cell was set up as above, 1 ml of sample
from receptor compartment was withdrawn at definite time intervals and equal
volume of phosphate buffer saline was replaced in the receptor compartment. The
amount of Tretinoin present in the sample was determined following the assay
procedure mentioned in sec. 4.1.4
Table 16: Comparison of release study profile using Sigma dialysis membrane
Formulation
Time Plain drug Niosomal
(hrs) Drug solution gel dispersion Niosomal gel
0 0.00 0.00 0.00 0.00
1 36.58 1.77 1.14 0.99
2 56.78 10.74 6.45 5.79
3 68.85 21.75 15.38 12.62
4 78.80 36.33 25.57 20.35
5 86.15 54.04 36.52 25.94
6 92.31 69.08 49.68 32.56
120.00
100.00
80.00
% Release
60.00
40.00
20.00
0.00
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00
-20.00
Time (hours)
. (5)
Where: -
Cr = Concentration of drug in receptor compartment
Vr = Volume of the receptor compartment
Cd = Concentration of drug in donor compartment
Vd = Volume of donor compartment.
Diffusion coefficient was calculated using the formula (Higuchi W.I., 1962).
.. (6)
Where: -
R = Percentage drug release
h = thickness of the membrane (0.04 cm)
T = time (sec)
D = Diffusion co-efficient (cm2/sec)
5.4 Discussion
The release studies show that 92.31 % and 69.08 % drug diffusion occurred within 6
hours from drug solution and plain drug gel respectively, while 49.68 % and 32.56 %
drug diffusion occurred from niosomal dispersion and niosomal gel respectively. The
difference is very large between drug solution and niosomal dispersion as well as the
drug gel and niosomal gel. This indicates the slow release properties of niosomal
formulations. The release rate is retarded due to niosomal barrier. In niosomal gel, the
drug has to pass from two barriers to reach to the receptor component. Thus, the
release rate is slow as compare to conventional formulation.
The figure 18 shows the comparative release profiles of all the formulations together
which makes easy for us to understand the release pattern. The drug solution is highly
diffusible by nature thus the curve shows slight decrease in release rate after 5 hours
of release, whereas in niosomal dispersion, the curve shows slight increase in release
rate as the saturation in receptor component has not been attained yet.
The release rate is slow thus the drug concentration will not be too high in short time
and thus this can prevent the irritation to skin as the Tretinoin is highly irritative by
nature.
Comparing the graphical presentation (see Figure 18 & 19) of percentage release of
drug from free drug formulation and niosomal formulation, it is seen that diffusion
from niosomal formulation follows a sustain release pattern. The diffusion coefficient
values of drug from niosomes as compared to plain drug formulations indicate the
sustained drug release from niosomes, which may be due to diffusion of drug through
niosomal membrane followed by dialysis membrane and release into receptor
compartment.
Since the Tretinoin is entrapped within niosomes, the diffusion of drug will occur
across two membranes, one the niosomal membrane and the other cellophane
membrane. This may be the reason for the sustained release.
The diffusion coefficients were found to be 1.68 x 10-09, 1.26 x 10-09, 9.07 x 10-10 and
5.94 x 10-10 for drug solution, drug gel, niosomal dispersion and niosomal gel
respectively. The diffusion co-efficient is low for the niosomal formulations that
indicates the slow release pattern for the niosomal formulations. The diffusion co-
efficient is highest for the drug solution which indicates the faster release of the drug
across the semi-permeable membrane.
5.5 References
Chein L.H. and Chem Y.W. Drug Development Ind. Pharm (1994),20 (6) : 935 : 945
Chein Y.W., Novel Drug Delivery Systems, Marcel Decker Inc., New York, (1980), p.
11-63
Higuchi W.I. J.Pharm Sci. (1962), 51(8) : 802-804.
Indian Pharmacopoeia (1996) 4th edition, published by the controller of publication,
Delhi, Govt. of India. Ministry of Health and Family welfare pg. 277A.
Shah V.P., Elkins, J.S. and Williams R.I. Pharmacopoeial Forum (1993), 19 (2)
:5048-5060.
Vincent H.L.L. and robinson R.J., Controlled Drug Delivery, Marcel Decker Inc. New
York, 2nd edition (1987), p. 99
6.1 Introduction
It is essential to carry out in-vivo studies of niosomal formulations to see the
difference of effect between plain drug formulation and niosomal formulation. The
animal study was carried out by properly following CPCSEA guidelines under
registration number 197/99/CPCSEA. The project was approved by the
IAEC/SKPCPER/2010-01.
In the present study, total 18 rats were used. They were divided in to two equal
groups. The temperature conditions and relative humidity were maintained properly.
There was no specific diet restriction applied.
6.2 Procedure
Total 18 healthy rats of average weight of 3.5 kg were selected for study. The neck
skin was shaved carefully. The niosomes prepared with optimized parameters, were
incorporated into carbopol 971NF gel base. Simultaneously plain drug was also
incorporated into gel base as control.
Both the formulations of same strength (0.05%) were applied on shaved rat skin for
the determination of irritation characteristics. The applied area was covered by cotton
and bandage. The observations were carried out at regular intervals of 12, 24, 48
hours for various symptoms such as scaling, lesions and erythema by the in charge
pharmacologist.
Where,
A = observation after 12 hours
B = observation after 24 hours
C = observation after 48 hours
8
7
6
5
Score 4
3
2
1
0
1 2 3
Time interval (hours)
8
7
6
5
Score
4
3
2
1
0
1 2 3
Time interval (hours)
From the Table 18, it is seen that the rats treated with plain gel are showing more
irritation characteristics. The erythema signs are continuously increasing at regular
time intervals (initially it was marked as 1 but after 48 hours it was 3). The scaling
was absent with plain gel initially but after 12 hours it appeared on applied skin
portion. The lesions are also increasing as the therapy time increased.
From the Table 18, it is seen that the rats treated with Niosomal gel are showing
comparatively less irritation characteristics. The erythema signs were absent initially
but after 48 hours it showed slight erythema signs. The scaling was completely absent
with niosomal gel initially as well as even after 48 hours. The lesions were also
completely absent initially and even after 48 hours.
So, from the above comparisons of data it can be concluded that niosomal gel is more
effective than plain gel.
Presenting these findings in more illustrative way,
In terms of irritation, the niosomal gel is less irritiative as compare to plain drug
gel.
6.4 References
Cavrini, V.; Di, Pietra AM.; Raggi, MA. Pharm-Acta-Helv. (1981), 56(6), 163-165.
Cavrini, V.; Di, Pietra AM.; Raggi, MA.Int. J. Pharm. (1982), 10(feb); 119-124.
Lemli, J.;Knockaert, I.Pharm. Weekbl, Sci. Ed., 26 Aug. 1983 5(4)142-144(Fr).
PDR Generics (1996), p.2132-2134.
Physicians Generix, (1995), 5th edition, 1334-1335.
Sethi, P. D., Quantitative Analysis of Drugs in Pharmaceutical Formulations (1985),
10, Unique publishers, Delhi.
Therapeutic Drugs, (1991), 1st edition, M190-M194.
7.1 Summary
Tretinoin is one of the widely used anti-acne drug topically.
Niosomes are non-ionic surfactant vesicles obtained on hydration of synthetic
nonionic surfactants of the alkyl or dialkyl polyglycerol ether class, with or without
incorporation of cholesterol or other lipids.
The aim of this investigation was to encapsulate Tretinoin into niosomes and
incorporate these niosomes into suitable dermatological base.
Calibration curve for estimation of Tretinoin was plotted using reported UV-visible
spectroscopy method.
Niosomes were prepared by using thin film hydration technique using rotary flask
evaporator. The optimization of formulation parameters namely composition of
niosomes and that of process variables namely hydration volume, hydration
temperature etc. was carried out with an aim to achieve maximum percent drug
entrapment and uniform niosome size.
Distilled water was used for film hydration; the vacuum of 250 mmHg was used for
evaporation. The percentage drug entrapment was determined by extraction with
suitable solvent. The free drug was separated from niosomes by ultra centrifugation
and was checked for mass balance.
With above optimized parameters percentage drug entrapment was found to be 53.77
% with mean particle diameter of 1.553 m. Photomicrograph was taken of optimized
batch.
The niosome samples were evaluation for the Transmission electron microscopy
(TEM) by negative staining method using Uranyl acetate. The vesicles show good
thick wall and hence we can say that the vesicles are non-leaky.
The FTIR study of the final formulation shows that the peak obtained in a graph of
plain drug remains as such and thus there is not significant change in the chemical
properties of drug after final formulation. This indicates the chemical stability of
tretinoin in presence of SPAN 60 and cholesterol.
Comparative diffusion studies were carried out among plain drug formulations and
the niosomal drug formulations using sigma dialysis membrane. From diffusion study,
it was found that mean flux values of plain drug formulations were higher than that of
the niosomal formulations. It was evident that drug diffusion from plain drug
formulation was significantly more compared to niosomal formulations. The niosomal
formulations showed greater skin retention than plain drug formulations.
In-vivo studies were carried out to evaluate the product for the dermal irritation test.
The tretinoin is highly irritative to skin and hence the plain drug gel causes erythema
when applied for the topical treatment. The healthy rats were selected for the
evaluation purpose and both the formulations were applied on skin for 48 hours. The
skin showed no signs of erythema even after 48 hours with niosomal gel.
7.2 Conclusion
Tretinoin causes erythema when applied on skin for the purpose of acne treatment.
The thin film hydration technique using rotary flask evaporator shows good vesicle
forming properties as well as better efficiency. Tretinoin used in plain gel formation
in strength of 0.05% showed higher release as compare to niosomal gel at the same
time interval. The niosomal gel showed sustained release properties. The niosomal gel
applied on the rat skin showed no signs of erythema whereas the plain drug gel
showed it clearly. Thus, from this research work it can be concluded that the novel
niosomal gel formulation is much better than the conventional gel for topical purpose.