FORMULATION AND EVALUATION OF PRONIOSOMES OF ANTIVIRAL
AGENT ACYCLOVIR
M. PHARM DISSERTATION PROTOCOL
Submitted to
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES KARNATAKA,
BENGALURU
By
Mr. JAYADEV. N HIREMATH
Under the guidance of
Dr. ANITA R. DESAI, M.Pharm, Ph.D
Asst. Professor
DEPARTMENT OF PHARMACEUTICS
H.S.K COLLEGE PHARMACY
BAGALKOT-587 101
(2011-12)
1
ANNEXURE II
PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION
1
Name of the Candidate and Address
JAYADEV N. HIREMATH
H.S.K COLLEGE OF PHARMACY
BVVS Campus
Bagalkot-587101.
PERMANENT ADDRESS
JAYADEV HIREMATH
C/o B.R Salimath
Near Nagappana Katti
Extn Area, Bagalkot -587101.
2
Name of the Institution
3
Course of the Study and Subject
4
Date of Admission
5
Title of the project:- “FORMULATION AND EVALUATION OF PRONIOSOMES OF
ANTIVIRAL AGENT ACYCLOVIR.”
H.S.K COLLEGE OF PHARMACY
H.S.K COLLEGE OF PHARMACY
BVVS Campus
Bagalkot-587101.
M. PHARMACY
(PHARMACEUTICS)
13 JULY - 2011
2
6
BRIEF RESUME OF INTENDED WORK
6.1 Need for the study
Drug delivery systems using colloidal particulate carriers such as liposomes1 or niosomes2
have distinct advantages over conventional dosage forms because the particles can act as drug
containing reservoirs, and modification of the particle composition or surface can adjust the drug
release rate and or the affinity for the target site. Liposomes or niosomes in dispersion can carry
hydrophilic drugs by encapsulation or hydrophobic drugs by partitioning of these drugs into
hydrophobic domains. Liposomes are unilamellar or multilamellar spheroid structures composed
of lipid molecules, often phospholipids, assembled into bilayers. Because of their ability to carry
variety of drugs, liposomes have been extensively investigated for their potential application in
pharmaceutics, such as drug delivery3-4 for drug targeting5 for controlled release6 or for increasing
solubility3. However, there remain significant problems in the general application of liposomes for
drug delivery. In a dispersed aqueous system, liposomes have problems associated with
degradation by hydrolysis or oxidation7 and sedimentation, aggregation, or fusion of liposomes
during storage8. Other problems associated with the clinical application of liposomes include
difficulties in sterilization and large-scale production to obtain a product with adequate physical
and chemical stability9.
One alternative involves formation of liposome like vesicles from the hydrated mixtures
of cholesterol and nonionic surfactant such as monoalkyl or dialkyl polyoxyethylene ether
niosomes10. Niosomes are unilamellar or multilamellar vesicles capable of entrapping hydrophilic
and hydrophobic solutes11. From a technical point of view, niosomes are promising drug carriers
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as they possess greater stability and lack of many disadvantages associated with liposomes, such
as high cost and the variable purity problems of phospholipids12. Another advantage is the simple
method for the routine and large scale production of niosomes without the use of unacceptable
solvents. However, even though niosomes exhibit good chemical stability during storage, there
may be problems of physical instability in niosome dispersions. Like liposomes, aqueous
suspensions of niosomes may exhibit aggregation, fusion, leaking of entrapped drugs or hydrolysis
of encapsulated drugs, thus limiting the shelf life of the dispersion.
A dry niosome (proniosomes) which could be hydrated immediately before use would
avoid many of the problems associated with aqueous niosome dispersions and problems of
physical stability (aggregation, fusion, leaking) could be minimized. The additional convenience
of the transportation, distribution, storage and dosing would make proniosomes a promising
industrial product. Proniosomes are dry, free flowing, granular product which, upon addition of
water, disperses or dissolves to form a multilamellar niosome suspension suitable for
administration by oral or other routes.
Acyclovir is a synthetic acyclic purine nucleoside analog known for its antiviral activity
against Varicella zoster virus(VZV),Epstein barr virus, Cytomegallo virus, Herpes simplex virus
(HSV) and human Herpes virus 613. Chemically Acyclovir is 2-Amino-1,9-[(2-hydroxy ethoxy)
methyl] – 1H purine6 – (9H) one. The oral bioavailability of acyclovir is poor & variable (15% 30%)14 with a terminal half-life 2- 3 h for adults15 which requires frequent dosing.
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In the present study proniosome concept was developed to encapsulate acyclovir in
surfactant vesicles and evaluate for their in vitro characteristics and an attempt to improve the oral
bioavailability of the drug.
6.2 Review of literature
Hengjiu et al16 described procedure for producing a dry product which may be hydrated
immediately before use to yield aqueous niosome dispersions similar to those produced by more
cumbersome conventional methods. This report describes the preparation of dispersions of
proniosome derived niosomes, comparison of these niosomes to conventional niosomes, and
optimization of proniosome formulations. In addition, conventional and proniosome derived
niosomes are compared in terms of their morphology, particle size, particle size distribution, and
drug release performance in synthetic gastric or intestinal fluid. In all comparisons, proniosome
derived niosomes are as good as or better than conventional niosomes. Further they suggest that
these prepared proniosomes minimize problems of niosome physical stability such as aggregation,
fusion and leaking and provide additional convenience in transportation, distribution, storage and
dosing.
Almira et al17 reported a novel method for rapid preparation of proniosomes with a wide range of
surfactant loading. They developed slurry method to produce proniosomes using maltodextrin as
the carrier. The time required to produce proniosomes by this simple method is independent of the
ratio of surfactant solution to carrier material and appears to be scalable. The flexibility of the
proniosome preparation method would allow for the optimization of drug encapsulation in the
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final formulation based on the type and amount of maltodextrin. This formulation of proniosomes
is a practical and simple method of producing niosomes at the point of use for drug delivery.
Tamizharasi et al18 describes the preparation of indomethacin loaded maltodextrin based
proniosome by slurry method with different surfactant to cholesterol ratio. Prepared proniosomes
were optimized for highest percentage drug entrapment. They confirm all particles are uniform in
size and shape through microscopy and entrapment efficiency was determined by separating the
untrapped drug using dialysis. The in vitro release studies of drug from niosomes exhibited a
prolonged release as studied over a period of 24 h. On the basis of in vitro characterization, the
niosome showing maximum entrapment and suitable release rate were selected for in vivo
performance evaluation. They conclude that the niosomal formulation could be a promising
delivery system for indomethacin with improved bioavailability and prolonged drug release
profile.
Mahmoud et al19 developed proniosomal gels or solutions of flurbiprofen based on span 20 ,span
40, span 60 and span 80 with and without cholesterol. Nonionic surfactant vesicles (niosomes)
formed immediately upon hydrating proniosomal formulae. They studied influence of different
processing and formulation variables such as surfactant chain length, cholesterol content, drug
concentration, total lipid concentration, negatively or positively charging lipids and pH of the
dispersion medium on flurbiprofen percentage encapsulation efficiency and also, they studied
release of the prepared niosomes in phosphate buffer (pH 7.4). Results indicated that the
percentage encapsulation efficiency followed the trend Sp 60 >Sp 40 >Sp 20 >Sp 80. Cholesterol
increased or decreased the percentage encapsulation efficiency depending on either the type of the
surfactant or its concentration within the formulae. The maximum loading efficiency was 94.61%
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when the hydrating medium was adjusted to pH 5.5. Increasing total lipid or drug concentration
also increased the percentage encapsulation efficiency of flurbiprofen into niosomes. However,
incorporation of either dicetyl phosphate (DCP) which induces negative charge or stearyl amine
(SA) which induces positive charge decreased the percentage encapsulation efficiency of
flurbiprofen into niosomal vesicles. Finally, they suggest in vitro release data for niosomes of Sp
40 and Sp 60 showed release profiles of flurbiprofen from niosomes of different cholesterol
contents is an apparently biphasic release process. As a result, this study suggested the potential of
proniosomes as stable precursors for the immediate preparation of niosomal carrier systems.
Ajay et al20 investigated the combined influence of 3 independent variables in the preparation of
piroxicam proniosomes by the slurry method. They used a 3-factor, 3-level Box-Behnken design
to derive a second order polynomial equation and construct contour plots to predict responses. The
independent variables selected were molar ratio of Span 60: cholesterol (X1), surfactant loading
(X2), and amount of drug (X3). They prepared fifteen batches by slurry method and evaluated for
percentage drug entrapment (PDE) and vesicle size. The transformed values of the independent
variables and the PDE (dependent variable) were subjected to multiple regression to establish a
full-model second-order polynomial equation. F was calculated to confirm the omission of
insignificant terms from the full-model equation to derive a reduced model polynomial equation to
predict the PDE of proniosome derived niosomes. Contour plots were constructed to show the
effects of X1, X2 and X3 on the PDE. A model was validated for accurate prediction of the PDE
by performing checkpoint analysis. The computer optimization process and contour plots
predicted the levels of independent variables X1, X2, and X3 (0, -0.158 and –0.158 respectively),
for maximized response of PDE with constraints on vesicle size. The Box-Behnken design
demonstrated the role of the derived equation and contour plots in predicting the values of
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dependent variables for the preparation and optimization of piroxicam proniosomes.
Ajay et al21 characterize and optimize aceclofenac proniosomes using central composite design
and carry out stability studies. They selected three independent variables molar ratio of drug to
lipid (X1), surfactant loading (X2) and volume of hydration (X3). Based on central composite
design, they prepared 16 batches of proniosomes by slurry method and evaluated for the
percentage drug entrapment and mean volume diameter. The percentage drug entrapment and
mean volume diameter (dependent variables) and the transformed values of independent variables
were subjected to multiple regressions to establish a second order polynomial equation. Contour
plots were constructed to further elucidate the relationship between the independent and dependent
variables. The conformity of the polynomial equations was checked by preparing three checkpoint
batches. From the computer optimization process and contour plots, predicted levels of
independent variables X1, X2, and X3(-0.77, -0.8 and 0 respectively), for an optimum response of
percentage drug entrapment with constraints on mean volume diameter were determined. The
optimized batch was subjected to stability studies. The polynomial equations and contour plots
developed using central composite design suggested preparation of proniosomes with optimum
responses.
Chintankumar et al22 prepared aceclofenac loaded maltodextrin based proniosome by slurry
method with different surfactant to cholesterol ratio. The proniosome formulations were evaluated
for FT-IR study, angle of repose and scanning electron microscopy. The niosomal suspensions
were further evaluated for entrapment efficiency, in vitro release study, kinetic data analysis,
stability study, in vivo anti-inflammatory study. The result from SEM analyses has showed smooth
surface of proniosome. The formulation F4 which showed higher entrapment efficiency of 83.24 ±
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1.34 and in-vitro releases of 97.122% at the end of 24hr was found to be best among the all 7
formulation. Release was best explained by the zero order kinetics. Kinetic analysis shows that the
drug release follows super case II transport diffusion. Proniosome formulation has showed
appropriate stability for 90 days by storing the formulation at refrigerator condition.
Chandra et al23 prepared piroxicam proniosomes by conventional technique and employing
maltodextrin and sorbitol as base. The prepared lipid vesicles were evaluated for entrapment
efficiency and vesicle size of niosomes formed. The morphology of the proniosomes was studied
by scanning electron microscopy. The results reveals that span 60 based formulations produced
vesicles of smallest size and higher entrapment efficiency while those of span 80 produced
vesicles of least entrapment efficiency. Incorporation of lecithin further enhanced entrapment
efficiency. Further they investigate permeation of piroxicam from proniosome based reservoir
type transdermal gel formulation across excised rat abdominal skin Keshery Chein diffusion cell.
There was considerable improvement in flux over the control gel formulation. Proniosomes were
prepared Maximum flux achieved was 35.61g/cm2/h, an enhancement of 7.39 times was achieved
for transdermal system based on proniosomal gel as compared to control gel. The in-vivo antiinflammatory studies revealed that proniosome based transdermal drug delivery system of
piroxicam were promising carriers for delivery of piroxicam. There was significant reduction in
carrageenan induced rat paw inflammation compared to control.
Ibrahim et al24 prepared ketorolac proniosomes using spans and tweens for transdermal delivery.
The encapsulation efficiency and size of niosomal vesicles formed by proniosome hydration were
also
characterized by specific high performance liquid chromatography method and scanning
electron microscopy. Each of the prepared niosomes achieved about 99% drug encapsulation.
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Vesicle size was markedly dependent on the composition of the proniosomal formulations. Further
they investigated permeation of a potent nonsteroidal anti-inflammatory ketorolac, across excised
rabbit skin from various proniosome gel formulations was investigated using Franz diffusion cells.
Each of the prepared proniosomes significantly improved drug permeation and reduced the lag
time (P>0.05). Proniosomes prepared with span 60 provided a higher ketorolac flux across the
skin than did those prepared with Tween 20 (7- and 4-fold the control, respectively). A change in
the cholesterol content did not affect the efficiency of the proniosomes, and the reduction in the
lecithin content did not significantly decrease the flux (P>0.05).
6.3 Objective of the study
The present work is planned with the following objectives.
1. To prepare proniosomes containing Valsartan by different methods using surfactants,
cholesterol, lecithin and other ingredients.
2. To characterize proniosomes by UV, FTIR, SEM, encapsulation efficiency.
3. To study in vitro release of drug from prepared proniosomes using modified diffusion cell
apparatus.
4. Statistical interpretation of the results.
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MATERIALS AND METHODS
7.1 Source of data
The primary data will be collected by performing various tests and investigations in the
laboratory. The secondary data will be collected by referring various national and international
journals, books, helinet, pubmed, pharmacopeias and websites etc.
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7.2 Method of collection of data
The data is planned to collect from laboratory experiments which includes,
1. Slurry method, Vora’s method with some modifications, Perrett’s modified method was
used to prepare proniosomes.
2. Drug content estimation, percentage entrapment efficiency, interaction studies, stability
studies and in vitro release studies will be used to collect above data.
3. Instruments like over head stirrer, dissolution/diffusion apparatus, scanning electron
microscopy, UV, IR spectroscopy and stability chamber were also used to collect the data.
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REFERENCES
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7. Hunt C, Tsang S. Tocopherol retards auto-oxidation and prolongs the shelf-life of
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niosomes-nonionic surfactant vesicles. J Pharm Pharmacol. 1985;37:863-868.
11. Yoshioka T, Sternberg B, Florence AT. Preparation and properties of vesicles (niosomes)
of sorbitan monoesters (Span 20, 40, 60, and80) and a sorbitan triester (Span 85). Int J
Pharm. 1994;105:1-6.
12. Vora B, Khopade AJ, Jain NK. Proniosome based transdermal delivery of levonorgestrel
for effective contraception. J Control Release. 1998;54:149-165.
13. Wagstaff AJ, Faulds D, Goa KL. Acyclovir: a reappraisal of its antiviral activity,
pharmacokinetic properties and therapeutic efficacy. Drugs. 1994;47:153–205.
14. Miranda P, Krasny HC, Page DA, Elion CB. Species differences in the disposition of
acyclovir. Ame J. Med. 1982; 73:31-35.
15. Thummel KE, Shen DD. Design and optimization of dosage regimens: Pharmacokinetic
data. In: Hardman JG, Limbird LE, editors. Goodman & Gilman’s the pharmacological
basis of therapeutics. 10th ed. New York: McGraw-Hill Co Inc; 2001. 1925.
16. Hengjiu H, David GR. Proniosomes: A Novel Drug Carrier Preparation. Int J Pharm.
2000;206:110-122.
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17. Almira IBW, David GR. Maltodextrin-Based Proniosomes. AAPS Pharmsci Tech.
2001;3(1):1-8.
18. Tamizharasi S, Sunil B, Vaishali R, Jagdish Chandra R. Formulation and evaluation of
Maltodextrin based proniosomes loaded with indomethacin. International Journal of Pharm
Tech Research. 2009;1(3):517-523.
19. Mahmoud M, Omaima AS, Mohammed AH, Nagia AM. Effect of some formulation
parameters on flurbiprofen encapsulation and release rates of niosomes prepared from
proniosomes. Int J Pharm. 2008 ;361:104-111.
20. Ajay BS, Jolly RP, Rajesh HP. Formulation and Optimization of Piroxicam Proniosomes
by 3-Factor, 3-Level Box-Behnken Design. AAPS Pharm Sci Tech. 2007;8(4):1-7.
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Studies of Aceclofenac Proniosomes.
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DP, Ghanshyam RP. Formulation and evaluation of aceclofenac loaded maltodextrin based
proniosome. International Journal of Chem Tech Research. 2009;1(30):567-573.
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Signature of the Candidate
(JAYADEV HIREMATH)
10
Remarks of the Guide:
The
topic
selected
for
dissertation
is
satisfactory and feasible.
11
Name and Designation :
11.1. Guide:
Dr. ANITA R. DESAI
HEAD OF THE DEPARTMENT OF
PHARMACEUTICS
H.S.K COLLEGE OF PHARMACY,
BAGALKOT-587101
11.2. Signature of Guide
(Dr. ANITA R. DESAI)
-------
11.3. Co-Guide
11.4. Signature of Co- Guide
-------
11.5. Head of the Department:
Dr. ANITA R. DESAI
HEAD OF THE DEPARTMENT OF
PHARMACEUTICS
H.S.K COLLEGE OF PHARMACY,
BAGALKOT-587101
11.6. Signature of HOD
(Dr .ANITA R. DESAI)
14
12
12.1. Remarks of the principal
Recommended for Approval
12.2. Principal
Dr. I. S. MUCHCHANDI
PRINCIPAL
H.S.K COLLEGE OF PHARMACY
BAGALKOT-587101
12.3. Signature of the Principal
(Dr. I. S. MUCHCHANDI)
15