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Formulation, Development, Optimization and

Evaluation of Novel Topical Anti-acne Drug Delivery


System
A
Dissertation
Submitted for the degree of
MASTER OF PHARMACY
IN
THE FACULTY OF PHARMACY
(INDUSTRIAL PHARMACY)
TO
GANPAT UNIVERSITY
YEAR: 2009-10

Research Guide: Industrial Research Guide: Submitted By:


Dr. R. P. Patel Dr. Hamsaraj Karanth Kushal A. Modi
M. Pharm., Ph.D. M. Pharm., Ph.D. B. Pharm.

SHREE S. K. PATEL COLLEGE OF PHARMACEUTICAL


EDUCATION AND RESEARCH, GANPAT UNIVERSITY,
KHERVA-382711, GUJARAT, INDIA.
CERTIFICATE
I hereby certify that Mr. Kushal A. Modi has completed his thesis for Master of
Pharmacy on the topic Formulation, Development, Optimization and Evaluation of
Novel Topical Anti-acne Drug Delivery System. I further certify that the work done by
him is of his own and original and tends to the general advancement of knowledge. The
work was carried out under my supervision and guidance at Department of Industrial
Pharmacy, Shree S. K. Patel College of Pharmaceutical Education and Research,
Ganpat University and Cadila Pharmaceuticals Limited, Dholka, Ahmedabad during
the academic year 2009-2010. This work is up to my satisfaction.

Institutional Research Guide:

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.

Place: Ganpat University Kushal A. Modi


Date: B. Pharm.
ACKNOWLEDGEMENT
First, I would like to express my salutation to GOD for giving me the strength,
confidence and moral boost to successful completion of this project.

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.

It is very great pleasure and profound sense of reverence that I express my


gratitude and thank to my esteemed research guide Dr. R. P. Patel, Head of
Department of Pharmaceutics & Pharmaceutical Technology, Shree S. K. Patel
College of pharmaceutical Education and Research, Ganpat University, Kherva, for
his inestimable guidance, valuable suggestions and constant encouragement during
the course of this investigation.

My special thanks to my industrial research guide Dr. Hamsaraj Karanth, Research


Scientist, Cadila Pharmaceutical Limited, Dholka, Ahmedabad, for his valuable
guidance, inspiration and also for providing the excellent facilities for my work.

I am very thankful to Dr. Shrenik Shah for providing me excellent guidance


regarding thesis compilation.

I am grateful to our Principal Dr. N. J. Patel for providing the facilities in the
institute for completion of this work.

It gives me immense pleasure in expressing my sincere thanks to H.R. and F&D


Department of Cadila Pharmaceutical Limited, Dholka, Ahmedabad, who provided
me this opportunity to work in such a prestigious organization.

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.

I sincerely thanks to Mr. P. I. Patel for providing me library facilities at S. K. College


of pharmaceutical Education and Research, Ganpat University, Kherva as well as
thanks to Mr. Manojbhai, Mr. Sanjaybhai for providing me library facilities at
Cadila Pharmaceuticals Limited.

I would like to express my thanks to non-teaching staff especially, Mr. Manishbhai


Patel (Lab Assistant), Mrs. Kiranben Patel (Lab Assistant), Mr. Dineshbhai Patel
(Store Incharge), Mr. Bharatbhai Patel (Lab Assistant), Ms. Chaulaben Patel
(Computer lab assistant), and also peons of the college for their untiring help and
support through out my M.Pharm course.

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.

I am thankful to my parents and my aunty Ms. Vandana Mody (GM, Cadila)


who led me from darkness to light, ignorance to enlighten and confusion to clarity
throughout my life.

I am also thankful to all my classmates, seniors as well as my dearest juniors


for their support and helpful nature.

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.

GANPAT UNIVERSITY Kushal A. Modi


2009-10 B.Pharm
DEDICATED TO MY DEAREST

GRAND FATHER

Mr. DASHARATHLAL L. MODI

&

GRAND MOTHER

(LATE) Mrs. PUSHPABEN D. MODI


INDEX
Sr. No. Topic Page no.
1.0 ABSTRACT 1
2.0 INTRODUCTION 2
2.1 Aim 4
2.2 Plan of work 4
2.3 References 5
3.0 LITERATURE SURVEY 6
3.1 Niosomes 6
3.2 Skin structure 18
3.3 Drug delivery from topical formulations 20
3.4 Acne as a disease 21
3.5 Drug Profile 28
3.6 References 31
4.0 EXPERIMENTAL SETUP 34
4.1 Materials and methods 34
4.2 Preformulation studies 36
4.3 Preparation of niosomes 42
4.4 Optimization of formulation parameters 44
4.5 Preparation of gel 51
4.6 Accelerated stability studies 52
4.7 FTIR studies 54
4.8 Discussion 55
4.9 References 59
5.0 IN-VITRO RELEASE STUDIES 60
5.1 Introduction 60
5.2 Experimental 60
5.3 Data analysis 63
5.4 Discussion 64
5.5 References 65
6.0 IN-VIVO STUDIES 66
6.1 Introduction 66
6.2 Procedure 66
6.3 Results and discussion 69
6.4 References 71
7.0 SUMMARY AND CONCLUSION 72
7.1 Summary 72
7.2 Conclusion 74
Chapter 1 Abstract

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.

S.K.P.C.P.E.R. M.Pharm Thesis 1


Chapter 2 Introduction

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 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 (Saravanan D, 1997).

Handjani-Vila et al first reported the formation of vesicles on hydration of mixture of


nonionic and nontoxic surfactants termed as niosomes, which can entrap solute and
are osmotically active and stable. Since then, number of nonionic surfactants have
been used to prepare vesicles viz polyglycerol alkyl ether, glucosyl dialkyl ethers,
ester linked surfactants, polyoxy ethylene alkyl ethers, Brij and series of Spans and
Tweens (Gregoriadis et al, 1977).

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)

S.K.P.C.P.E.R. M.Pharm Thesis 2


Chapter 2 Introduction

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.

S.K.P.C.P.E.R. M.Pharm Thesis 3


Chapter 2 Introduction

2.1 Aim
The aim of the present investigation was

To encapsulate Tretinoin into niosomes and incorporate these niosomes into


suitable dermatological base.

To optimize the process and formulation variables of niosomal preparation

To study the in-vitro role of the developed products for topical application.

To decrease the erythema caused by tretinoin at the sight of 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.

2.2 Plan of work

Preparation of niosomes containing tretinoin method selection.

Optimization of process parameters such as type of surfactants, surfactant : drug


ratio, drug : cholesterol ratio, hydration temperature, hydration volume etc.

Characterization of prepared niosomes in terms of size, entrapment efficiency,


lamellarity etc.

In-vitro evaluation of optimized batch.

Stability study of optimized batch.

In-vivo evaluation.

S.K.P.C.P.E.R. M.Pharm Thesis 4


Chapter 2 Introduction

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.

S.K.P.C.P.E.R. M.Pharm Thesis 5


Chapter 3 Literature survey

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).

3.1.2 Advantages of niosomes


Niosomes possess several advantages like:

1. They entrap solute in a manner analogous to liposomes.

2. They are osmotically active and stable, as well as they increase the stability of
entrapped drug.

3. Handling and storage of surfactants requires no special conditions.

4. They possess an infrastructure consisting of hydrophobic and hydrophilic moieties


together and as a result can accommodate drug molecules with a wide range of
solubilities.

5. They exhibit flexibility in their structural characteristics (composition, fluidity,


and size) and can be designed according to desired application.

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.

9. The surfactants are biodegradable, biocompatible and non-immunogenic.

S.K.P.C.P.E.R. M.Pharm Thesis 6


Chapter 3 Literature survey

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.

11. Niosomal dispersion in an aqueous phase can be emulsified in a non-aqueous


phase to regulate the delivery rate of drug and administer normal vesicle in
external non-aqueous phase.

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 vesicle suspension is waterbased vehicle. This offers high patient


compliance in comparison with oily dosage forms.

The vesicle allows the incorporation of hydrophilic, amphiphilic and lipophilic


drugs. The vesicles supply both a lipophilic and an aqueous environment for the
encapsulation of drugs.

The characteristics of the vesicle formulation are variable and controllable.


Altering vesicle composition, size, lamellarity, tapped volume, surface charge and
concentration can control the vesicle characteristics.

The vesicles may act as a depot, releasing the drug in a controlled manner.

S.K.P.C.P.E.R. M.Pharm Thesis 7


Chapter 3 Literature survey

Difference in characteristics exists between liposomes and non-ionic surfactant


vesicles (NSV), especially since NSVs are prepared from uncharged single-chain
surfactant and cholesterol whereas liposomes are prepared from double-chain
phospholipids (neutral or charged). Handjani-Vila et al were first to report the
formation of vesicular system on hydration of mixture of cholesterol and a single-
alkyl chain non-ionic surfactant.
Niosomes behave in-vivo like liposomes, prolonging the circulation of entrapped drug
and altering its organ distribution and metabolic stability (Azmin M.N. et al, 1985).
Encapsulation of various anti neoplastic agents in these carrier vesicles has been
shown to decrease drug induced toxic side effects, while maintaining, or in some
instances, increasing the anti-tumor efficacy (Raja Naresh R. et al, 1996). Such
vesicular drug carrier systems alter the plasma clearance kinetics, tissue distribution,
metabolism and cellular interaction of the drug (Mc. Cormack B, Gregordias G.,
1998). They can be expected to target the drug to its desired site of action and/or to
control its release (Baillie. A. J., 1984). In addition, handling and storage of niosomes
requires no special conditions. In mice, the distribution of drugs incorporated in NSVs
has proved to be similar to that obtained after administration of drug encapsulated in
liposomes (Kiwada H. et al, 1985). As with liposomes, the properties of niosomes
depends both on the composition of the bilayer and on method of their production
(Szoka F., 1980).
It was observed by Baillie et al that the intercalation of cholesterol in the bilayers
decreases the entrapment volume during formulation and thus entrapment efficiency.
As the concentration of cholesterol increases, entrapment efficiency decreases.
It was reported that the entrapment efficiency increases with increase in the
concentration and lipophilicity of surfactant (Chandraprakash K.S, 1991).
Azmin et al used non-ionic hydrophilic surfactants like Tween 80 for making
niosomes entrapped with methotrexate. They used hand shaking method for
preparation of niosomes, with surfactant:cholesterol:dicetylphosphate at ratio of
60:30:5 and hydration of film at 70 C. They reported that niosomes improved the
absorption of Methotraxate in blood after oral and intravenous administration in mice.
Chandraprakash et al made Methotrexate loaded non-ionic surfactant vesicles using
lipophilic surfactants like Span 40, Span 60 and Span 80 and found that Span 60
(HLB = 4.7) gave highest percent entrapment while Span 85 (HLB = 1.8) gave least

S.K.P.C.P.E.R. M.Pharm Thesis 8


Chapter 3 Literature survey

entrapment. They also observed that as HLB value of surfactant decreased, the mean
size was reduced.

3.1.3 Method of preparation


A. Ether injection method (Florence, A.T. et al, 1988)
This method provides a means of making niosomes by slowly introducing a solution
of surfactant dissolved in diethyl ether into warm water maintained at 60 C. The
surfactant mixture in ether is injected through 14-gauge needle into an aqueous
solution of material. Vaporization of ether leads to formation of single layered
vesicles. Depending upon the conditions used the diameter of the vesicle range from
50 to 1000 nm.

B. Hand shaking method (Thin film hydration technique)


The mixture of vesicles forming ingredients like surfactant and cholesterol are
dissolved in a volatile organic solvent (diethyl ether, chloroform or methanol) in a
round bottom flask. The organic solvent is removed at room temperature (20 C)
using rotary evaporator leaving a thin layer of solid mixture deposited on the wall of
the flask. The dried surfactant film can be rehydrated with aqueous phase at 60 C
with gentle agitation. This process forms typical multilamellar niosomes.
Thermo sensitive niosomes were prepared by Raja Naresh et al by evaporating the
organic solvent at 60 C and leaving a thin film of lipid on the wall of rotary flash
evaporator. The aqueous phase containing drug was added slowly with intermittent
shaking of flask at room temperature followed by sonication.

C. Sonication (Baillie A. J. et al, 1986)


A typical method of production of the vesicles is by sonication of solution as
described by Florence and Cable. In this method an aliquot of drug solution in buffer
is added to the surfactant/cholesterol mixture in a 10-ml glass vial. The mixture is
probe sonicated at 60 C for 3 minutes using a sonicator with a titanium probe to yield
niosomes.

D. Micro fluidisation (Khandare, J. N. et al, 1994)


Micro fluidization is a recent technique used to prepare unilamellar vesicles of
defined size distribution. This method is based on submerged jet principle in which

S.K.P.C.P.E.R. M.Pharm Thesis 9


Chapter 3 Literature survey

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.

E. Multiple membrane extrusion method (Yoshida H. et al, 1992)


Mixture of surfactant, cholesterol and dicetyl phosphate in chloroform is made into
thin film by evaporation. The film is hydrated with aqueous drug solution and the

are placed in series for up to 8 passages. It is a good method for controlling niosome
size.

F. Reverse phase evaporation technique (REV) (R.A. Raja Naresh et al 1994)


Cholesterol and surfactant (1:1) are dissolved in a mixture of ether and chloroform.
An aqueous phase containing drug is added to this and the resulting two phases are
sonicated at 4-5 C. The clear gel formed is further sonicated after the addition of a
small amount of phosphate buffered saline (PBS). The organic phase is removed at
40 C under low pressure. The resulting viscous niosome suspension is diluted with
PBS and heated on a water bath at 60 C for 10 min to yield niosomes.
Raja Naresh et al have reported the preparation of Diclofenac sodium niosomes using
Tween 85 by this method.

G. Trans membrane pH gradient (inside acidic) drug uptake process (remote


loading) (Maver L. D. et al, 1985)
Surfactant and cholesterol are dissolved in chloroform. The solvent is then evaporated
under reduced pressure to get a thin film on the wall of the round bottom flask. The
film is hydrated with 300 mM citric acid (pH 4.0) by vortex mixing. The
multilamellar vesicles are frozen and thawed 3 times and later sonicated. To this
niosomal suspension, aqueous solution containing 10 mg/ml of drug is added and
vortexed. The pH of the sample is then raised to 7.0-7.2 with 1M disodium phosphate.
This mixture is later heated at 60C for 10 minutes to give niosomes.

S.K.P.C.P.E.R. M.Pharm Thesis 10


Chapter 3 Literature survey

H. The Bubble method (Chauhan S and Luorence, 1989)


It is novel technique for the one step preparation of liposomes and niosomes without
the use of organic solvents. The bubbling unit consists of round-bottomed flask with
three necks positioned in water bath to control the temperature. Water-cooled reflux
and thermometer is positioned in the first and second neck and nitrogen supply
through the third neck. Cholesterol and surfactant are dispersed together in Tris buffer
(pH 7.4) at 70 C, the dispersion mixed for 15 seconds with high shear homogenizer
and immediately afterwards bubbled at 70 C using nitrogen gas.

I. Formation of niosomes from proniosomes (Almira I et al, 2001)


Another method of producing niosomes is to coat a water-soluble carrier such as
sorbitol with surfactant. The result of the coating process is a dry formulation. In
which each water-soluble particle is covered with a thin film of dry surfactant. This
preparation is termed Proniosomes. The niosomes are recognized by the addition of
aqueous phase at T > Tm and brief agitation.
T = Temperature.
Tm = mean phase transition temperature.

Almira I. et al have reported the formulation of niosomes from maltodextrin based


proniosomes. This provides rapid reconstitution of niosomes with minimal residual
carrier. Slurry of maltodextrin and surfactant was dried to form a free flowing
powder, which could be rehydrated by addition of warm water. Table 1 shows in
brief, example of some drugs incorporated into niosomes using different methods.

S.K.P.C.P.E.R. M.Pharm Thesis 11


Chapter 3 Literature survey

Table 1: Drugs incorporated into niosomes by various methods

Method of preparation Drug incorporated


Ether Injection Sodium stibogluconate
Doxorubicin
Hand Shaking Methotrexate
Doxorubicin
Sonication 9-desglycinamide
8-arginine
Vasopressin
Oestradiol

3.1.4 Separation of unentrapped drug


The removal of unentrapped solute from the vesicles can be accomplished by various
techniques, which include: -

1. Dialysis (Chauhan S and Luorence 1989)

The aqueous niosomal dispersion is dialyzed in dialysis tubing against phosphate


buffer or normal saline or glucose solution.

2. Gel filtration (Blazek-Walsh and G. Rhodes David, 2001)

The unentrapped drug is removed by gel filtration of niosomal dispersion through a


Sephadex-G-50 column and elution with phosphate buffered saline or normal saline.

3. Centrifugation (Hu C. and Rhodes D.G., 1999)

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.

S.K.P.C.P.E.R. M.Pharm Thesis 12


Chapter 3 Literature survey

3.1.5 Characterization of niosomes


1. Entrapment efficiency
After preparing niosomal dispersion, unentrapped drug is separated by dialysis,
centrifugation, or gel filtration (Szoka, F. et al, 1980) as described above and the drug
remained entrapped in niosomes is determined by complete vesicle disruption using
50% n-propanol or 0.1% Triton X-100 and analyzing the resultant solution by
appropriate assay method for the drug. Where,
Entrapment efficiency (EF) = (Amount entrapped / total amount) x 100
2. Vesicle diameter
Niosomes, similar to liposomes, assume spherical shape and so their diameter can be
determined using light microscopy, photon correlation microscopy and freeze fracture
electron microscopy. Freeze thawing (keeping niosomal suspension at 20 C for 24
hrs and then heating to ambient temperature) of niosomes increases the vesicle
diameter, which might be attributed to fusion of vesicles during the cycle.
3. In-vitro release
A method of in-vitro release rate study includes the use of dialysis tubing. A dialysis
sac is washed and soaked in distilled water. The vesicle suspension is pipetted into a
bag made up of the tubing and sealed. The bag containing the vesicles is placed in 200
ml of buffer solution in a 250 ml beaker with constant shaking at 25 C or 37 C. At
various time intervals, the buffer is analyzed for the drug content by an appropriate
assay method (Yoshioka T. et al, 1994).

3.1.6 Factors affecting vesicles size, entrapment efficiency and release


characteristics

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.

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2. Amount and type of surfactant


The mean size of niosomes increases proportionally with increase in the HLB of
surfactants like Span 85 (HLB 1.8) to Span 20 (HLB 8.6) because the surface free
energy decreases with an increase in hydrophobicity of surfactant (Yoshika T and
Florence A.T., 1994).
The bilayers of the vesicles are either in the so-called liquid state or in gel state,
depending on the temperature, the type of lipid or surfactant and the presence of other
components such as cholesterol. In the gel state, alkyl chains are present in a well-
ordered structure, and in the liquid state, the structure of the bilayers is more
disordered. The surfactants and lipids are characterized by the gel-liquid phase
transition temperature (TC). Phase transition temperature (TC) of surfactant also
effects entrapment efficiency i.e. Span 60 having higher TC, provides better
entrapment.
3. Cholesterol content and charge
Inclusion of cholesterol in niosomes increases its hydrodynamic diameter and
entrapment efficiency (Yoshioka T. et al, 1994). In general, the action of cholesterol
is two folds; on one hand, cholesterol increases the chain order of liquid-state bilayers
and on the other, cholesterol decreases the chain order of gel state bilayers. At a high
cholesterol concentration, the gel state is transformed to a liquid-ordered phase (B. L.
Silver, 1985).
An increase in cholesterol content of the bilayers resulted in a decrease in the release
rate of encapsulated material and therefore an increase of the rigidity of the bilayers
obtained. Presence of charge tends to increase the interlamellar distance between
successive bilayers in multilamellar vesicle structure and leads to greater overall
entrapped volume.

3.1.7 Niosomes as drug carriers


A number of workers have reported the preparation, characterization and use of
niosomes as drug carriers.
Niosomes containing anti-cancer drugs, if suitably designed, will be expected to
accumulate within tumors in a similar manner to liposomes. The niosomal
encapsulation of methotrexate and Doxorubicin increases drug delivery to the tumor
and tumoricidal activity of the drug.

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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.

Azmin et al reported the preparation and oral as well as intravenous administration of


methotrexate loaded niosomes in mice. They observed significant prolongation of
plasma levels and high uptake of methotrexate in liver from niosomes as compared to
free drug solution.

Chandraprakash et al reported the formation and pharmacokinetic evaluation of


methotrexate niosomes in tumor bearing mice.
Cable et al modified the surface of niosomes by incorporating polyethylene alkyl
ether in the bilayered structure. They compared the release pattern and plasma level of
doxorubucin in niosomes and doxorubucin mixed with empty niosomes and observed
a sustained and higher plasma level of doxorubicin from niosomes in mice.

D Souza et al studied absorption of Ciprofloxacin and Norfloxacin when


administered as niosome encapsulated inclusion complexes.
Jain N. K. and Khopade A. J. reported the formulation and evaluation of
Indomethacin loaded niosomes and showed that therapeutic effectiveness increased
and simultaneously toxic side effect reduced as compared with free Indomethacin in
paw edema bearing rats.

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Parthasarthi et al prepared niosomes of vincristine sulfate which had lesser toxicity


and improved anticancer activity. Jagtap and Inamdar prepared niosomes of
pentoxifylline and studied the in-vivo bronchodilatory activity in guinea pigs. The
entrapment efficiency was found to be 9.26 1.93% giving a sustained release of
drug over a period of 24 hrs.

Raja Naresh et al reported the anti-inflammatory activity of niosome encapsulated


diclofenac sodium in arthritic rats. It was found that the niosomal formulation
prepared by employing a 1:1 combination of Tween 85 elicited a better consistent
anti-inflammatory activity for more that 72 hrs after administration of single dose.
Anil Kumar N. prepared niosomes containing Ibuprofen with 6 different surfactants of
Tweens and Spans and maximum entrapment was obtained with Span 60 with size
range 0.1-9.5 microns.

3.1.8 Route of administration


During experimentation, niosomes have been administered to the laboratory animals
orally, transdermally and most usually intravenously through caudal vein.

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.

1) Targeting of bioactive agents

a) To reticulo-endothelial system (RES)

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).

b) To organs other than RES

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

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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.

4) Delivery of peptide drugs

Yoshida et al investigated oral delivery of 9-desglycinamide, 8-arginine


vasopressin entrapped in niosomes in an in-vitro intestinal loop model and
reported that stability of peptide increased significantly.

5) Immunological application of niosomes

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.

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6) Niosomes as carriers for hemoglobin.

Niosomes can be used as a carrier for hemoglobin. Niosomal suspension shows a


visible spectrum super imposable onto that of free hemoglobin. Vesicles are
permeable to oxygen and hemoglobin dissociation curve can be modified similarly
to non-encapsulated hemoglobin (Moser P. et al, 1989)

7) Transdermal delivery of drugs by niosomes

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.

8) Other applications (Reidman, O.M. and Boger E., 1981)

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.

b) Localized drug action

Drug delivery through niosomes is one of the approaches to achieve localized


drug action, since their size and low penetrability through epithelium and
connective tissue keeps the drug localized at the site of administration. Localized
drug action results in enhancement of efficacy of potency of the drug and at the
same time reduces its systemic toxic effects e.g. Antimonials encapsulated within
niosomes are taken up by mononuclear cells resulting in localization of drug,
increase in potency and hence decrease both in dose and toxicity (Hunter C.A. et
al, 1994). The evolution of niosomal drug delivery technology is still at an infancy
stage, but this type of drug delivery system has shown promise in cancer
chemotherapy and anti-leishmanial therapy. Niosomes are promising vehicle for

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drug delivery and being non-ionic; it is less toxic and improves the therapeutic
index of drug by restricting its action to target cells.

3.2 Skin structure


3.2.1 Introduction
The Skin is a multilayered organ that is complex both in structure and function.
Microscopically, two distinct layers are apparent, the outer epidermis and the inner
dermis. The epidermis comprises of viable epidermis and the S. corneum. The S.
Corneum is breached by hair follicles and sweat ducts, which could provide a shunt
diffusion pathway across the skin. Thus the major source of resistance to the
penetration and permeation of the skin is the S. corneum. The coherent membrane,
which is 15-20 mm thick over much of the human body primarily, consists of blocks
of cytoplasmic protein matrices embedded in extra cellular lipid. The protein matrices
containing cells are arranged in an interlocking structure similar to bricks and motor.
In human, the motor consists of structured complex containing several cells or the
horny layer the corneocytes, which containing very little lipid. The Stratum corneum
can offer two possible routes of penetration. The route through which permeation
occurs is largely dependent on the penetrants. Physico- chemical characteristics, the
most important being the relative ability to partition into each skin phase.

3.2.2 Fundamentals of skin permeation


For a systemically active drug, to reach a target tissue remote from the site of drug
administration on the skin surface, it has to possess some physicochemical properties,
which are capable of facilitating the absorption of drug by the S.Corneum. Hence the
penetration of drug through various skin tissue, and also the uptake of the drug by the
capillary network in the dermal papillary layers.
The rate of permeation (dq/dt) across a skin can be expressed, mathematically, by the
following relationship.
(dq/dt) = Ps (cd - cr) ........................................................................................1
Where cd and cr are, respectively the concentration of a skin penetrant in the donor
compartment i.e. the drug concentration on the surface of S. Corneum and in the
receptor compartment i.e. in the body and Ps is thepermeability coefficient of the skin
as defined by,
Ps = (ks. Dss)/hs ................................................................................................2

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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).

3.2.3 Parameters affecting skin permeability


1. Physico chemical properties of the penetrants like partition coefficient, pH
conditions and penetrant concentration.

2. Physico chemical properties of the drug delivery system like release


characteristic, composition of the drug delivery system and enhancement of
transdermal permeation.

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3. Physiological and pathological conditions of the skin like reservoir effect of


horny layer, lipid film and skin hydration, skin temperature regional tissues
pathological injury and cutaneous metabolism of skin.

3.3 Drug delivery from topical formulations


Drug delivery from topical formulations for local or systemic effect essentially
involves passive diffusion of the drug through the skin.
The release of a drug molecule from a vehicle into the skin and diffusion across the
skin are controlled by physico chemical factors sensitive to the molecular properties
of the permeant, the vehicle and the skin.
Katz and Poulsen have identified four categories of interactions with bearing on the
delivery process. Rate-influencing interactions are possible between,

1. Drug and skin

2. Vehicle and skin

3. Drug and vehicle and

4. Drug, vehicle and skin.


Example of drugskin interactions include alteration of the surface structure of the
skin by the drug components of formulations as we as the binding of drugs to
constituents of the skin as they diffuse through the issue field.
A vehicle-skin interaction occurs when one or another of the vehicles main
components of the formula, effects a change in the physical state of the skin, in turn,
affecting the skins permeability.
Vehicle-skin interactions can be divided into following three categories.

1. Penetration enhancement effects brought about by solvent action on the skin,

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.

Drug-vehicle interactions are those in which physico chemical interactions between


the drug and the vehicle kinetically or thermodynamically govern the release of the
drug into the skin.

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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.

The comedo and its formation


The pathogenesis of acne is multifactorial, involving disturbances of keratinization,
hormonal regulation, and immunity, but if there is one central defect in acne, it is the
formation of the comedo. The site of disease is the sebaceous follicle of the head and
upper trunk, which consists of a follicular lumen, a rudimentary hair and a large
sebaceous gland. Like the skin to which it attaches, the normal epithelial lining of the
follicle desquamates as microscopic keratinous debris that is carried to the skin
surface with the flow of sebum. When this process is disturbed, follicular impaction
occurs and is the initial event in the development of acne (Kligman AM et al, 1969).

Figure 1: Scarring nodular acne


There are a variety of noninflammatory acne lesions. Clinically, comedones are
described as open if they have a visible plug in the follicular, orifice and closed if the
opening has not been distended enough to be visible without magnification. The tip of
an open comedo is usually dark brown or black. Contrary to the conclusions of most
mothers, this does not result from dirt but from the oxidation of melanin pigment and

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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.

Figure 2: Scarring nodular acne with conglobate lesions


Marks and Dawber described a valuable technique whereby microcomedones may be
harvested noninvasively by using cyanoacrylate glue. By this method,
microcomedones may be seen to some degree on the skin of patients without acne and
their numbers increase significantly in patients with acne. Close observation of
patients with acne over time suggests that microcomedones may be the forerunners of
most inflammatory lesions. Secondary comedones, as defined by Kligman, are the
result of reencapsulation of ruptured and inflamed primary lesions and may have
several interconnected open comedones. Epithelial cysts may be part of the spectrum
of secondary comedones in some patients with acne, but in general, the relation of
true cysts to the acne process is not certain. Comedo formation begins with a faulty
desquamation of the follicular lining. Rather than desquamation as tiny keratinous
particles, aggregates of cells are shed that cannot pass through the acroinfundibulum,
resulting in a plug at the opening of the follicle. This material becomes layered, much
like an onion, in concentric lamina of keratinous squamae in the lumen, and the
follicle becomes distended. The abnormal desquamation is first detectable at the
junction of the follicular epithelium and the sebaceous duct and involves distal cells.

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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.

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3.4.2 Microbiology of acne


The skin provides several distinct microbial habitats that are determined mainly by the
availability of three factors: moisture, nutrients and the degree of oxygenation. These
ecologic factors are present in various combinations on different parts of the skin
surface and determine the microbial flora of each region. The pilosebaceous unit after
puberty is a deep (from a bacterial point of view) invagination that is filled with
sebum and keratin that oxygen does not readily penetrate. The surface of sebaceous
regions has large populations of aerobic grampositive cocci and yeast of the
Pityrosporum genus. The inner reaches of the postpubertal follicle are normally
inhabited by significant populations of P. acnes, an anaerobic gram-positive
diphtheroid that also produces large amounts of extracellular lipase. Other
propionibacteria, such as P. avidum and P. granulosum, may be isolated from human
skin but not in numbers approaching those of P. aches, P. acnes populations are
tremendous, reaching tens of millions per square centimeter on the normal face, yet
true infection with the organism is rare. The most common P. acnes infections result
from craniotomies, colonization of prostheses or heart valves and occur far less
frequently than those caused by the oral flora or Staphylococcus epidermidis. There
are two serotypes and many bacteriophage types of P. acnes, but none is found in
increased proportion among P. acnes strains recovered from internal infections,
suggesting that all P. acnes strains are of equally low pathogenicity. It is also difficult
to establish P. acnes infections experimentally. Injection of the organism into the skin
or into steatocystomas results in inflammation but not in infection. Taken together,
these observations suggest that P. acnes is among the most benign of commensals.
P. acnes is dependent on sebum secretion for nutrition and populations are linked to
the amount of triglycerides in sebum. Animals with triglyceride-deficient sebum do
not carry P. acnes on their skin, just as prepubertal children who secrete trivial
amounts of sebum also do not support the organism. When the pubertal surge in
androgens stimulates sebum production, stable P. acnes populations develop in
regions bearing follicles with large, active sebaceous glands, mainly the head and
upper trunk. Sebum-deficient areas, such as the distal extremities, carry no stable
numbers of P. acnes and thus are devoid of inflammatory acne lesions. Teenage
patients with acne have substantially more numerous P. acnes populations than do
age-matched control subjects. This difference is proportional to the increased sebum

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secretion seen among adolescent patients with acne. As patients with acne mature, this
difference in population becomes less striking (Burton JL et al, 1971).

3.4.3 Treatment approaches


The first step in treating a patient with acne is to determine the extent and severity of
disease. Often the severity is perceived to be less by the physician than by the patient.
Acne occurs at a most sensitive time when identity and personality are being forged
and the disease can have effects that extend well beyond the dermis. Every
dermatologist is familiar with the tearful patient with acne. Teenagers with even mild
acne feel stigmatized and frustrated; severe acne can be seen as a curse. Before
beginning treatment, most patients need to be disabused of some common myths. First
and foremost, it must be made clear to patient and parent that ache is not caused by
dirt and that washing does not help. In fact, the vigorous cleansing used by some may
actually create more lesions and scars through damage to intact comedones. Gentle
washing is sufficient. Picking and popping of pimples is also forbidden for similar
reasons. If the compulsion to manipulate lesions cannot be resisted, I have patients try
to substitute the spot application of benzoyl peroxide for their destructive habit.

Figure 3: Stepwise treatment approaches

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Table 2: Treatment approaches


Comedo formation Topical Tretinoin
Adapatene
Azeleic acid
Benzoyl peroxide
Salicylic acid
Propylene glycol
Systemic Isotretinoin
Inflammatory lesions Topical Erythromycin
Clindamycin
Benzoyl peroxide
Azeleic acid
Systemic Erythromycin
Tetracycline
Doxycycline
Minocycline
Trimethoprim/sulfamethoxazole
Ciprofloxacin
Isotretinoin

3.5 Drug profile


3.5.1 Description
Chemical name: all-trans retinoic acid

a) Molecular details

Drug Molecular formula Molecular weight


Tretinoin C20H28O2 300.44 g/mol

b) Appearance

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i) Color and odor

A yellow, odorless or almost odorless, crystalline powder

ii) Physical properties

(1) Solubility

Practically insoluble in water, 1 gm in 500 ml of alcohol, 1 in 150 ml of


DMSO, 1 in 20 ml of DCM.

(2) Melting point: About 180

(3) Dissociation constant (Pka): 6.0

(4) Stability pH : 4.5 - 5.5

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%.

3.5.3 Mechanism of action


Acne therapy may be directed against comedo formation, androgen production,
sebaceous activity, P. acnes, or the inflammatory/immune response (Darley CR,
1984). Often, the most successful approach is directed against 2 or more targets, 1 of
which is usually the comedo. For more than 25 years, topical vitamin A acid or
tretinoin has been the mainstay for comedolytic therapy (Kligman AM, 1969). Its use
was first reported by Stuttgen, who showed that oral vitamin A was beneficial in acne.
Later work by Kligman et al documented the efficacy of topical tretinoin in both
comedonal and inflammatory acne. 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. Initially there was an inflammatory phase in which mast cells degranulated,
and lymphocytes and Langerhans cells were activated. Epidermal hyperplasia and

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hypergranulosis with an increase in membrane-coating granules followed in the


second week. Finally the horny layer became disorganized, and comedones were
broken up. A similar sequence probably occurs in human acne treated with tretinoin,
in that existing impactions are loosened and are eventually expressed. Topical
tretinoin has no significant anti-inflammatory, antihormonal or antisebaceous effects.

3.5.4 The safety of topical tretinoin


Topical tretinoin is an exceedingly safe drug. There are essentially no systemic side
effects because little of the drug that is applied to the face is absorbed (1%) and the
amount that does reach the circulation does not alter plasma vitamin A levels.
Although classified as pregnancy category C, the lack of effect on plasma levels
argues that topical tretinoin is a safe drug for use in pregnancy; a fact that has been
borne out by epidemiologic data. This study of a group of women given prescriptions
for topical tretinoin during the first trimester found that major congenital
abnormalities occurred in 1.9% of these patients versus 2.6% in women who were not
exposed to tretinoin. In spite of these reassuring data, many doctors elect to defer
tretinoin therapy until after delivery to minimize any patient concerns. The adverse
effects of topical tretinoin are limited to the skin. Many patients experience a pustular
flare after 1 to 2 weeks of use. This exacerbation is usually mild, but patients are best
forewarned. It is rarely so severe as to warrant corticosteroid therapy. The major
disadvantage of topical tretinoin is the irritation that often begins after a week or 2 of
therapy. Fair-skinned and especially atopic patients are most sensitive, and therapy is
typically begun with 0.025% or 0.05% creams in conjunction with light emollients.
The chin and melolabial folds are typically the most sensitive areas in these patients.
Often the drug may be used everywhere else on their faces with little irritation.
Differentiation between tretinoin preparations is often difficult because most result in
irritation in the classical 21-Day Cumulative Irritation Test. A recent investigation
was conducted to determine whether a Modified Cumulative Irritation Test would
distinguish between the irritation potential of tretinoin formulations and certain
nontretinoin acne treatments. The irritation profile of 0.05% tretinoin cream was
higher than 0.025% tretinoin cream and 0.05% tretinoin emollient cream. Finally,
20% azelaic acid cream was found to be more irritating than all of the other tested
tretinoin formulations (P < 0.001). Much has been made of the photosensitizing
potential of topical retinoids, but there are few data to support the contention. Because

S.K.P.C.P.E.R. M.Pharm Thesis 29


Chapter 3 Literature survey

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.

S.K.P.C.P.E.R. M.Pharm Thesis 30


Chapter 3 Literature survey

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.

S.K.P.C.P.E.R. M.Pharm Thesis 31


Chapter 3 Literature survey

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.

S.K.P.C.P.E.R. M.Pharm Thesis 32


Chapter 3 Literature survey

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.

S.K.P.C.P.E.R. M.Pharm Thesis 33


Chapter 4 Experimental setup

4.1 Materials and methods

4.1.1 Instruments

Table 3.1: Instruments used in investigation

Name of the Instruments Manufacturer Model


Digital weighing balance Sartorious CP2245
Contech, Mumbai CB-Series AP
Sartorious GE 1302
Magnetic Stirrer Eltek MS 205
pH meter Lab India, Baroda Eutech pH 5
Rotary flask evaporator Buchi -
Microscope Olympus -
zeta sizer Malvern -
Bath sonicator PCI -
Transmission electron microscope Philips -
DSC Shimadzu TA 60 WS
FTIR Shimadzu 8400S
UV Spectrophotometer Shimadzu UV 1700

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

S.K.P.C.P.E.R. M.Pharm Thesis 34


Chapter 4 Experimental setup

4.1.3 Reagents

A. Acetate buffer solution (pH 4.5 - 5.5)

Add an accurately weighed quantity of 77.1 gm of Ammonium acetate in distilled


water and add 57 ml glacial acetic acid and dilute with water upto 1000 ml.

B. Solvent blend

Prepare a solvent blend by adding 20% ethanol and 80% acetate buffer solution.

C. Stock solution of tretinoin

A stock solution of 100 mcg/ml was prepared in solvent blend.

4.1.4 Calibration curve

Principle

UV-SP method was selected for estimation of tretinoin. absorption spectrum of


tretinoin solution (20 g/ml) showed that maximum absorption occurs at the
wavelength of 352 nm (max=352 nm).

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.

S.K.P.C.P.E.R. M.Pharm Thesis 35


Chapter 4 Experimental setup

Table 3: Calibration curve for estimation of tretinoin

Sr. Concentration Absorbance Average


No (g/ml) I II III absorbance
1 0 0.000 0.000 0.000 0.000
2 1 0.066 0.067 0.068 0.067
3 3 0.152 0.151 0.151 0.151
4 5 0.261 0.262 0.261 0.261
5 8 0.363 0.361 0.359 0.361
6 10 0.452 0.451 0.450 0.451
7 13 0.592 0.591 0.590 0.591
8 15 0.698 0.698 0.698 0.698

Equation of regression line: y = 0.0467x + 0.0057


Correlation co-efficient R2 = 0.9973

1.2

0.8
Absorbance

0.6

0.4

0.2

0
0 5 10 15 20 25
Concentration (mcg/ml)

Figure 4: Calibration curve of tretinoin

S.K.P.C.P.E.R. M.Pharm Thesis 36


Chapter 4 Experimental setup

4.2 Preformulation studies:


4.2.1 Preformulation study protocol
4.2.1.1 Name of the product: Tretinoin Niosomal Gel.
4.2.1.2 Objective: To carry out drug-excipient compatibility studies.
4.2.1.3 Strength: 0.05%
4.2.1.4 Sample size at each station: One vial each.
4.2.1.5 Packaging details: The stability study will be carried out in amber glass
vials with rubber stopper and aluminum seal
4.2.1.5 Materials to be used for the study:
Sr. No Name of drug/excipients Short form Category

Tretinoin Tr API
1.

2. Cholesterol Chl Stabilizer

3. SPAN 40 SP40 Surfactant

4. SPAN 60 SP60 Surfactant

5. SPAN 85 SP85 Surfactant

4.2.1.6 Container storage orientations: All containers shall be stored up-right


through out the stability study.

4.3 Testing parameters and acceptance criteria:

Tests Acceptance criteria


Appearance No change
% Assay Between 98 102 %

S.K.P.C.P.E.R. M.Pharm Thesis 37


Chapter 4 Experimental setup

Table 4 Preformulation design


Sr. No. Ingredients Ratio
1 Tretinoin 1
2 Tr +chl 1:1
3 Tr + SP40 1 : 10
4 Tr + SP60 1 : 10
5 Tr + SP60 1 : 20
6 Tr + SP85 1 : 10
7 Tr + chl + SP40 1 : 1 : 10
8 Tr + chl + SP60 1 : 1 : 10
9 Tr + chl + SP60 1 : 1 : 20
10 Tr + chl + SP85 1 : 1 : 10

4.4 Testing time points / Testing storage conditions:

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.

S.K.P.C.P.E.R. M.Pharm Thesis 38


Chapter 4 Experimental setup

4.2.2 Data compilation

Table 5 Data compilation of preformulation studies of drug + excipient

Condition: 40 0C/ 75 %RH.


Sr.
Name Ratio Time point Appearance % Assay
No.
Tretinoin 1 Initial No change 101.15
1 15 days No change 99.22
30 days No change 98.62
Tr +chl 1:1 Initial No change 101.21
2 15 days No change 99.69
30 days No change 98.25
Tr + SP40 1 : 10 Initial No change 101.23
3 15 days No change 99.56
30 days No change 98.92
Initial No change 101.59
4 Tr + SP60 1 : 10 15 days No change 99.78
30 days No change 98.54
Initial No change 101.36
5 Tr + SP60 1 : 20 15 days No change 100.05
30 days No change 99.97
Initial No change 101.85
6 Tr + SP85 1 : 10 15 days No change 99.73
30 days No change 98.49
Initial No change 101.45
7 Tr + chl + SP40 1 : 1 : 10 15 days No change 99.59
30 days No change 97.26
Initial No change 100.97
8 Tr + chl + SP60 1 : 1 : 10 15 days No change 99.54
30 days No change 98.12
Initial No change 101.94
9 Tr + chl + SP60 1 : 1 : 20
15 days No change 99.72

S.K.P.C.P.E.R. M.Pharm Thesis 39


Chapter 4 Experimental setup

30 days No change 98.34


Initial No change 101.65
10 Tr + chl + SP85 1 : 1 : 10 15 days No change 99.85
30 days No change 97.93

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

S.K.P.C.P.E.R. M.Pharm Thesis 40


Chapter 4 Experimental setup

30 days No change 99.36


Initial No change 101.37
9 Tr + chl + SP60 1 : 1 : 20 15 days No change 100.56
30 days No change 99.67
Initial No change 101.38
10 Tr + chl + SP85 1 : 1 : 10 15 days No change 100.49
30 days No change 99.22

4.2.3 DSC study:

Figure 5 DSC study

S.K.P.C.P.E.R. M.Pharm Thesis 41


Chapter 4 Experimental setup

4.3 Preparation of niosomes

4.3.1 Preparation of placebo niosomes


Niosomes can be prepared by various methods like hand shaking method film
hydration technique (Azmin M. N et al, 1985), sonication (Zeinab, H. et al, 1994),
micro-fluidization (Baillie, A. J., 1984), reverse phase technique (Khandare J.N.,
1994) etc. Niosomes prepared by hand shaking method showed good physical
stability and no breakage even after a week. Therefore, in this study, hand shaking
(film hydration technique) was used for preparation of niosomes.

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.

Figure 6: Flow diagram of preparation of placebo niosomes

S.K.P.C.P.E.R. M.Pharm Thesis 42


Chapter 4 Experimental setup

4.3.2 Preparation of niosomes containing drug

Span-60 : Cholesterol at a ratio of 20:1 by weight and 50 mg of Tretinoin were


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.
While preparing niosomes factors considered for optimization were:

Stability of drug itself at the temperature of preparation

Drug entrapment efficiency

Particle size of prepared niosomes

Figure 7: Flow diagram of preparation of drug containing niosomes

S.K.P.C.P.E.R. M.Pharm Thesis 43


Chapter 4 Experimental setup

4.4 Optimization of formulation parameters


From the initial observations based on microscopic examination and physical stability
of the preparation, the niosomes prepared by film hydration technique gave more
promising results. Several process variables were studied for optimization of the
batch.

4.4.1 Factors considered

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.

Figure 8: Optimized niosomes under 40x magnification

S.K.P.C.P.E.R. M.Pharm Thesis 44


Chapter 4 Experimental setup

Figure 9: Optimized niosomes under 100x magnification

Figure 10: Transmission electron microscopy photograph under 5500x zoom

S.K.P.C.P.E.R. M.Pharm Thesis 45


Chapter 4 Experimental setup

Figure 11: Transmission electron microscopy photograph under 7000x zoom

2. Particle size analysis

Niosomes were subjected to particle size analysis using Malvern particle size
analyzer.

Figure 12: Malvern particles size report of optimized batch

S.K.P.C.P.E.R. M.Pharm Thesis 46


Chapter 4 Experimental setup

4.4.2 Parameters optimized

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.

1. Type of non-ionic surfactant

2. Surfactant: Cholesterol ratio

3. Solvent system ratio

4. Hydration volume

5. Hydration temperature

6. Hydration time

7. Annealing time

8. Film formation time

1. Type of non-ionic surfactant

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.

Table 6: Data of various non-ionic surfactants

Brand name HLB value Molecular weight


Sorbitan Monoesters
Sorbitan Monopalmitate Span 40 6.7 402.56
Sorbitan MonoStearate Span 60 4.7 430.6
Sorbitan Trioleate Span 85 1.8 957.5
Polysorbates
Polysorbate 80 Tween 80 15.0 1308.9

S.K.P.C.P.E.R. M.Pharm Thesis 47


Chapter 4 Experimental setup

Table 7: Percentage entrapment particle size analysis and microscopic


examination of niosomes prepared using different surfactants

% Drug Mean diameter


Surfactant Microscopic examination
entrapment (m)
Spherical, Multilamellar,
Span 40 27.30 2.92 Cholesterol particles in
background
Span 60 53.77 1.553 Spherical, Multilamellar
Span 85 Not formed - -
Tween 80 Not formed - -

2. Surfactant : Cholesterol ratio

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.

Table 8: Various Surfactant:Cholesterol ratios and percentage drug entrapment

Surfactant : Cholesterol ratio by weight % Entrapment


5:1 10.23
10:1 26.35
20:1 53.77

S.K.P.C.P.E.R. M.Pharm Thesis 48


Chapter 4 Experimental setup

3. Solvent system ratio

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

DCM : Ethanol ratio Film formation


1:0 Thin non-sticky film
1:1 Sticky film
1:5 Thick sticky film

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.

Table 10: Various hydration volumes and hydration

Hydration volume (ml) Hydration


10 Insufficient, particles visible
15 Insufficient, particles visible

20 Insufficient, particles visible

30 Insufficient, particles visible

35 Insufficient
40 Insufficient
50 Good homogenous dispersion

5. Hydration temperature

Various hydration temperatures like 35 C, 40 C, 50 C, 60 C and 70 C were tried


for formation of niosomes and finally 50 C was selected as the optimized one.
Increase in hydration temperature from 35 C, 40 C and 50 C increased the
percentage entrapment, but when temperature was increased to 70 C, it was found

S.K.P.C.P.E.R. M.Pharm Thesis 49


Chapter 4 Experimental setup

that there was decrement in percentage drug entrapment. Highest percentage of


entrapment was found at 50 C, so it was considered as optimum.

Table 11: Various hydration temperatures and hydration

Hydration temp. C Hydration


35 Improper, particles visible
40 Improper.
50 Good homogenous dispersion
60 Stickiness on wall
70 Changes in drug physical properties

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.

Table 12: Various hydration times and hydration

Hydration time (min) Hydration


15 Insufficient, film remained on wall
30 Insufficient, particles visible
45 Sufficient
60 Sufficient

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.

Table 13: Various annealing times and dispersion characteristics

Annealing time (hr) Dispersion characteristics


1 Phase separation
2 Good homogenous dispersion
3 Good homogenous dispersion

S.K.P.C.P.E.R. M.Pharm Thesis 50


Chapter 4 Experimental setup

8. Film formation time

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.

Table 14: Optimized process and formulation parameters

Parameter Optimized value


Surfactant SPAN 60
Sur:Chol. ratio 20:1 by weight
Hydration volume 50 ml
Hydration temp. 50 C
Hydration time 45 minutes
Annealing time 2 hour
Film-formation time 10 minutes
% Entrapment 53.77 %
Mean diameter 1.553 microns

4.5 Preparation of niosomal gel

4.5.1 Formula

Carbopol 971 NF......................................................... 1 gm


Niosomal Dispersion ................................................... 50 gm.
Distilled water upto ..................................................... 100 gm.

4.5.2 Procedure

1.0 gm of Carbropol 971 NF was dispersed in 20 ml distilled water to form a thick


gel. 50 ml Niosomal dispersion was added to the prepared gel. The final weight of gel
was adjusted to 100 gm with distilled water to get the 0.05% final strength of the gel.
The neutralization was done with 10% NaOH solution to adjust the pH between 5.0-
5.5. The gel was transferred to collapsible tubes and stores at 2- 8 C in refrigerator.

S.K.P.C.P.E.R. M.Pharm Thesis 51


Chapter 4 Experimental setup

4.6 Accelerated stability studies


The stability studies of niosomes and niosomal gel and creams were carried out at
different temperatures.

Table 15: Percentage assay at various conditions at specific time intervals

Formulation Condition Percent drug retained (Time in weeks)


Initial 1 2 3 4 6
2 8 C 102.56 100.11 98.89 97.12 96.97 94.26
25 C /60 %RH 102.56 99.31 97.24 96.95 94.65 92.11
Niosomes 40 C /75 %RH 102.56 96.94 91.23 85.14 80.26 71.45

2 8 C 101.44 100.64 98.69 97.52 96.13 93.12


25C /60 %RH 101.44 99.16 98.14 96.29 94.13 91.94
Niosomal gel 40C /75 %RH 101.44 95.44 89.56 82.76 75.39 63.54

S.K.P.C.P.E.R. M.Pharm Thesis 52


Chapter 4 Experimental setup

120

100

80

% content
60 2 8
25/60 RH
40 40/75 RH

20

0
1 2 3 4 5 6
Time (weeks)

Figure 13: Accelerated stability studies of niosomal dispersion

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)

Figure 14: Accelerated stability studies of niosomal gel

S.K.P.C.P.E.R. M.Pharm Thesis 53


Chapter 4 Experimental setup

4.7 FTIR studies:

Figure 15: FTIR spectra

S.K.P.C.P.E.R. M.Pharm Thesis 54


Chapter 4 Experimental setup

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

S.K.P.C.P.E.R. M.Pharm Thesis 55


Chapter 4 Experimental setup

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

S.K.P.C.P.E.R. M.Pharm Thesis 56


Chapter 4 Experimental setup

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

S.K.P.C.P.E.R. M.Pharm Thesis 57


Chapter 4 Experimental setup

studies showed steady decrease in percentage drug retained in samples of niosomal


dispersion and gel (see Table 15 and Figure 13 and 14).
Percentage drug retained in niosomes stored at 2-8 C showed 100.11 at first week
which was reduced gradually to 94.26 at sixth week, in niosomes stored at 25 C / 60
% RH showed 99.31 at first week which was reduced gradually to 92.11 at sixth
week, whereas, in niosomes stored at 40 C / 75 %RH showed 96.94 at first week
which was reduced gradually to 71.45 at sixth week.
Percentage drug retained in niosomal gel stored at 2-8 C showed 100.64 at first week
which was reduced gradually to 93.12 at sixth week, in niosomes stored at 25 C / 60
%RH showed 99.16 at first week which was reduced gradually to 91.94 at sixth week,
whereas, in niosomes stored at 40 C / 75 %RH showed 95.44 at first week which was
reduced gradually to 63.54 at sixth week.
From above results, it can be concluded that the niosomes and niosomal gel showed
maximum stability at 2 to 8 C and hence such formulations should be stored at
refrigerated conditions.

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.

S.K.P.C.P.E.R. M.Pharm Thesis 58


Chapter 4 Experimental setup

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.

S.K.P.C.P.E.R. M.Pharm Thesis 59


Chapter 5 In-vitro release studies

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 Preparation of the membrane for in-vitro studies

1. Artificial membrane (AFM)

Sigma dialysis membrane, 200 m in thickness was used as an artificial membrane


for preliminary in vitro studies because of simplicity, homogeneity and uniformity.
This membrane was hydrated in pH 7.4 phosphate buffer saline for 24 hours prior to a
permission run.

5.2.1.1 Materials

Sigma dialysis membrane


Disodium hydrogen phosphate
Potassium dihydrogen phosphate
Sodium chloride

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

S.K.P.C.P.E.R. M.Pharm Thesis 60


Chapter 5 In-vitro release studies

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)

Drug solution Plain drug gel Niosomal dispersion Niosomal gel

Figure 18: Graphical representations of release profiles

S.K.P.C.P.E.R. M.Pharm Thesis 61


Chapter 5 In-vitro release studies

Figure 19: Graphical representations of release profiles

S.K.P.C.P.E.R. M.Pharm Thesis 62


Chapter 5 In-vitro release studies

5.3 Data analysis

5.3.1 Percentage drug release

The percentage drug diffusion was determined by (Shah V.P et al),

. (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.

5.3.2 Diffusion coefficient

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)

Table 17: Diffusion co-efficient values across the dialysis membrane

Formulation Diffusion co-efficient (cm2/sec)


Drug solution 1.68 x 10-09
Plain drug gel 1.26 x 10-09
Niosomal dispersion 9.07 x 10-10
Niosomal gel 5.94 x 10-10

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Chapter 5 In-vitro release studies

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.

S.K.P.C.P.E.R. M.Pharm Thesis 64


Chapter 5 In-vitro release studies

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

S.K.P.C.P.E.R. M.Pharm Thesis 65


Chapter 6 In-vivo studies

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.

The symptoms, lesions and erythema were graded as


3 = severe
2 = moderate
1 = mild and
0 = absent
And scaling as
1 = present
0 = absent

S.K.P.C.P.E.R. M.Pharm Thesis 66


Chapter 6 In-vivo studies

Figure 20: Initial observation of skin

Figure 21: Observation of skin after 24 hours

S.K.P.C.P.E.R. M.Pharm Thesis 67


Chapter 6 In-vivo studies

Figure 22: Observation of skin after 48 hours

Table 18: Observations and calculations of in-vivo studies

Plain drug gel Niosomal gel


Symptoms A B C A B C
Scaling 0 1 1 0 0 0
Lesions 1 2 3 0 0 0
Erythema 1 2 3 0 0 1
Total score 4 4 7 0 0 1

Where,
A = observation after 12 hours
B = observation after 24 hours
C = observation after 48 hours

S.K.P.C.P.E.R. M.Pharm Thesis 68


Chapter 6 In-vivo studies

8
7
6
5
Score 4
3
2
1
0
1 2 3
Time interval (hours)

Scaling Lesions Erythema Total score

Figure 23: Graphical presentation of in-vivo studies of plain drug gel

8
7
6
5
Score

4
3
2
1
0
1 2 3
Time interval (hours)

Scaling Lesions Erythema Total score

Figure 24: Graphical presentation of in-vivo studies of niosomal gel

6.3 Results and discussions


From the result of in-vitro studies, the beneficial role of niosomal formulations over
the plain drug formulations was indicated with regard to higher skin permeation and
retention. Hence, it was thought worthwhile to investigate the effect of Tretinoin
niosomal formulations.

S.K.P.C.P.E.R. M.Pharm Thesis 69


Chapter 6 In-vivo studies

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.

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Chapter 6 In-vivo studies

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.

S.K.P.C.P.E.R. M.Pharm Thesis 71


Chapter 7 Summary and conclusion

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.

Parameters were optimized as under,

Parameter Optimized value


Surfactant SPAN 60
Sur:Chol. ratio 20:1 by weight
Hydration volume 50 ml
Hydration temp. 50 C
Hydration time 45 minutes
Annealing time 2 hour
Film-formation time 10 minutes
% Entrapment 53.77 %
Mean diameter 1.553 microns

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Chapter 7 Summary and conclusion

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 optimized batches of tretinoin niosomes after incorporation into different


dermatological bases were subjected to stability studies at 2-8 C and 40 C / 75 %RH
for 6 weeks and percentage drug retained was evaluated at definite time intervals.
From the result of the stability study it was seen that both the formulations viz.
niosomes and niosomal gel were stored at 2-8 C showed maximum % drug retained
after 6 weeks i.e. 94.26% and 93.12% respectively. Hence such formulations should
be stored at refrigerated conditions.

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.

S.K.P.C.P.E.R. M.Pharm Thesis 73


Chapter 7 Summary and conclusion

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.

S.K.P.C.P.E.R. M.Pharm Thesis 74

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