Download Fabrication and Evaluation of Curcumin-loaded

COMPARATIVE EVALUATION OF CURCUMIN NANOPARTICLES
OF CASEIN AND CHITOSAN
Synopsis for M.Pharm Dissertation submitted to the
Rajiv Gandhi University of Health Sciences, Bengaluru, Karnataka.
By
Mr. MAHAVEER SINGH
M.Pharm., Part-I
Under the guidance of
Dr. GANESH N.S, M.Pharm., Ph.D.,
ASSOCIATE PROFFESSOR ,
DEPARTMENT OF PHARMACEUTICS.
SARADA VILAS COLLEGE OF PHARMACY
KRISHNAMURTHYPURAM
MYSORE- 570004
2013-2014
RAJIV GANDHIUNIVERSITY OF HEALTH SCIENCES,
BENGALURU, KARNATAKA.
ANNEXURE II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
1.
NAME OF THE CANDIDATE
MAHAVEER SINGH
AND ADDRESS (IN BLOCK
LETTERS)
M.PHARM., PART–I,
DEPARTMENT OF PHARMACEUTICS,
SARADA VILAS COLLEGE OF PHARMACY,
K.M.PURAM, MYSORE - 04.
2.
NAME OF THE INSTITUTION
SARADA VILAS COLLEGE OF PHARMACY,
MYSORE - 04.
3.
COURSE OF STUDY AND SUBJECT
MASTER OF PHARMACY IN
PHARMACEUTICS.
4.
DATE OF ADMISSION OF COURSE
14-08-2013
5.
TITLE OF TOPIC
COMPARATIVE EVALUATION OF CURCUMIN
NANOPARTICLES OF CASEIN AND CHITOSAN
6.
BRIEF RESUME OF THE
INTENDED WORK
7.
6.1 Need for the study
ENCLOSURE - I
6.2 Review of the literature
ENCLOSURE - II
6.3 Objectives of the study
ENCLOSURE – III
MATERIALS AND METHODS
7.1 Source of data
ENCLOSURE – IV
7.2 Method of collection of data
ENCLOSURE - V
7.3Does studies requireany
ENCLOSURE – VI
investigations or interventions to be
conducted on patients or other
human or animal? If so, please
describe briefly.
7.4 Has ethical clearance been obtained
ENCLOSURE – VI
from your institution in case of 7.3
8.
LIST OF REFERENCES
9.
SIGNATURE OF CANDIDATE
ENCLOSURE – VII
10.
REMARKS OF GUIDE
11.
NAME AND DESIGNATION OF
11.1 Guide
RECOMMENDED
Dr. GANESH N.S, M Pharm, Ph.D.,
ASSOCIATE PROFESSOR,
DEPARTMENT OF PHARMACEUTICS
SARADA VILAS COLLEGE OF PHARMACY
K.M.PURAM, MYSORE – 04,
KARNATAKA.
11.2 Signature
11.3 Co guide (if any)
Not applicable
11.4 Signature
Not applicable
11.5 Head of department
Dr. C. JAYANTHI, M Pharm., Ph.D.,
PROFESSOR AND HEAD,
DEPARTMENT OF PHARMACEUTICS,
SARADA VILAS COLLEGE OF PHARMACY,
K.M.PURAM, MYSORE - 04
KARNATAKA.
11.6 Signature
12.1 Remarks of the Principal
12.2 Name and designation of principal
12.3 Signature
RECOMMENDED & FORWARDED
Dr. K. HANUMANTHACHAR JOSHI
M.Pharm., Ph.D.,
PRINCIPAL,
SARADA VILAS COLLEGE OF PHARMACY,
K.M.PURAM, MYSORE - 04.
6.0 BRIEF RESUME OF THE INTENDED WORK
ENCLOSURE – I
NEED FOR THE STUDY
Nanoparticles are sub-nanosized colloidal structures composed of synthetic or semi
synthetic polymers. Nanospheres are solid core spherical particulates which are nanometric
in size.They contain drug embeded within the matrix or adsorbed on to the surface1.
The major goals in designing nanoparticles as a delivery system are to control particle size,
surface properties and release of pharmacologically active agents in order to achieve the
site-specific action of the drug at the therapeutically optimal rate and dose regimen2.
Nanoscience is an emerging field that deals with interactions between molecules, cells and
engineered substances such as molecular fragments, atoms and molecules. In terms of size
constraints, the National Nanotechnology Initiative (NNI) defines nanotechnology in
dimensions of roughly 1 to 100 nanometers (nm),1 but in boarder range it can be extended
up to 1000 nm. Particles that fall within this range appear to be optimal for achieving a
number of important tasks as nanocarriers, including the alteration of a drug’s reactivity,
strength, electrical properties, and ultimately, its behavior in vivo. There is great interest in
developing new nanodelivery systems for drugs that are already on the market, especially
cancer therapeutics. Ideally, nanodelivery systems will allow for more specific targeting of
the drug, thereby improving efficacy and minimizing side effects. By using nanotechnology
in drug design and delivery, researchers are trying to push nanomedicine to be able to
deliver the drug to the targeted tissue, release the drug at a controlled rate, be a
biodegradable drug delivery system, and to be able to escape from degradation processes of
the body3.
Formulations based on liposomes, misciles, microemulsions, nanocrystals and amorphous
solid dispersions, and gastroretentie floating systems have been developed to improve the
curcumin oral bio availabity however, A considerable interest has recently been paid to the
development of polymeric nanoparticles based drug delivery systems, due to these systems
ability to overcome several problems related to the delivery of hydrophobic drugs dispersed
in aqueous solutions4.
Curcumin, the major yellow pigment is extract from the rhizomes of turmeric Curcuma
longa ). It is a common ingredient in all forms of Indian diet, an effective agent to reduce
the development of tumor and full of antioxidant, anti-inflammatory and anti-carcinogenic
properties. It inhibits cancer in various tissues, including skin, mammary gland, oral cavity,
forestomach, oesophagus, stomach, intestine, colon, lung, and liver . There are difficulties in
the clinical administration of curcumin due to low water solubility and degradation under
physiological conditions. Several methods have been undertaken to enhance the
bioavailability of curcumin. and synthesized the poly(lactic acid-co-glycolic acid)
nanoparticles of curcumin (Nano-CUR) and this formulation found to inhibit the cell
proliferation in chemo/radio-resistant cancer cells. studied glycerol mono oleate (GMO)
nanoparticles to loaded curcumin and improved the systemic bioavailability5.
Although curcumin has shown a wide range of pharmacological activities, there were also
some potential limitations. Photodegradation, short half-life and low bioavailability were
major hurdles for the therapeutic use of curcumin. curcumin, with phenolic group and
conjugated double bounds, which is unstable in the presence of light and basic pH, degrades
within 30 min. In addition, the poor oral bioavailability was due to its low water solubility
under acidic or neutral conditions. Various methods have been tried to overcome these
bioavailability problems. The aim of the present study was to formulate SLNs to increase
photostability and enhance its anticancer activity because it can not only overcome the
membrane stability and drug-leaching, but also reduce the potential toxicity of CUR7.
The aqueous dispersion of nano curcumin aspergillus niger. The results demonstrated that
the water solubility and anti microbial activity of curcumin markedly improved by particle
size reduction upto the nano range. For the selected microorganisms the activity of nano
curcumin was more pronounced against gram positive bacteria than gram negative bacteria.
Further more its antibacterial activity was much better than anti fungal activity, the
mechanism of antibacterial action of curcumin nanopartcles was inestigated by transmission
electron micrograph analysis, which revealed that these particles entered inside the bacterial
cell by completely breaking the cell wall, leading to cell death.
Among the available nanosized drug carrier systems, nanoparticles prepared with proteins
(e.g., albumin, gelatin) are biocompatible, biodegradable, nonantigenic, and relatively easy
to prepare. Further, protein nanoparticles can bind to a large number of drugs in a relatively
nonspecific manner. Because of their surface charge, drugs can physically adsorb onto the
protein surface or can covalently bind to the matrix . Bovine Serum Albumin (BSA) and
Human Serum Albumin (HSA) have been widely used over the past 30 years to prepare
micro and nanoparticles. More than 100 different active or diagnostic molecules have been
incorporated into albumin particles to be administered by different routes including
intravenous, intramuscular, nasal, and ophthalmic route9.
Casein, the major milk protein, is a very practicable biomaterial with good
biocompatibility and biodegradability, easily available in high purity and at low cost, and
therefore it has been utilized extensively for enzyme engineering, pharmaceutical and
medical applications. In contrast, casein, the major milk protein, is a very practicable
biomaterial with good biocompatibility and biodegradability, easily available in high purity
and at low cost, and therefore it has been utilized extensively for enzyme engineering,
pharmaceutical and medical applications . In a way, casein can be thought of as amphiphilic
block copolymers consisting of blocks with hydrophobic or hydrophilic amino acid residues,
exhibiting a strong tendency to self-assemble into casein micelles inaqueous solution . It is a
good nature material for polymer-biomolecule conjugates10.
To increase its aqueous solubility and bioavailability, attempts have been made
through encapsulation in liposome, polymeric nanoparticle, lipid-based nanoparticle,
biodegradable microsphere, cyclodextrin and hydrogels Interest in nanocarriers for cancer
chemotherapy is growing. Nanoparticle-based drug delivery approaches have the potential
for rendering hydrophobic agents like curcumin dispersible in aqueous media, thus
circumventing the pitfalls of poor solubility.
PBCA nanoparticles have been proved to be an effective drug delivery system for the
controlled drug delivery of various pharmacologically active moieties, like anticancer
agents, analgesics, antibiotics and peptide . The PBCA nanoparticles are generally prepared
by emulsion or dispersion polymerization in an acidic aqueous solutions of surfactants
forming a porous structure with high specific area on which various quantities of drugs,
dissolved in the medium during or after polymerization, can be loaded
Surface
characteristics like zeta potential and the solubility property can be modulated to meet the
prerequisites of intravenous administration11.
Polymers derived from natural sources have been used extensively in the pharmaceutical
and food industry. among these polymers, polysaccharides have been widely utilized
because of their bioavailabity
biodegradability, and low toxicity. chitosan, a natural
polymer obtained from the deacetylation of chitin, has been a useful polymer for varius
biomedical applications, sustained drug release and for the enhancement of bioavailabity of
poor water soluble drugs4.
Chitosan is a modified natural carbohydrate polymer prepared by the partial Ndeacetylation of chitin, a natural biopolymer derived from crustacean shells such as
crabs,shrimps and lobsters. chitosan is soluble in most organic acidic solutions at pH less
than6.5 including formic, acetic, tartaric, and citric acid. It is insoluble in phosphoric and
sulfuric acid. Chitosan is available in a wide range of molecular weight and degree of
deacetylation . Molecular weight and degree of deacetylation are the main factors affecting
the particle size, particles formation and aggregation1.
Present study is focussed on the formulation and comparative evaluation of curcumine
nanoparticles of using casein and chitosan.
ENCLOSURE – II
6.2 Review of the literature
1) A.krishna sailaja et al., have studied different techniques used for the preparation of
nanoparticles using Natural polymers and their application and concluded that
Targeting delivery of drugs to the diseased lesions is one of the most important
aspects of drug delivery system. They have been used in vivo to protect the drug
entity in the systemic circulation, restrict access of the drug to the chosen sites and to
deliver the drug at a controlled and sustained rate to the site of action. various
techniques have been discussed in detail for the preparation of nanoparticles using
natural polymers chitosan, alginate and proteins such as albumin, gelatin, legumin.
2) VJ Mohanraj et al., have studied about nanoparticles and concluded that the
nanoparticulate systems have great potentials, being able to convert poorly soluble,
poorly absorbed and labile biologically active substance into promising deliverable
drugs. And also shown To optimize this drug delivery system, greater understanding
of the different mechanisms of biological interactions, and particle engineering, is
still required and also that the nanoparticles have been used in vivo to protect the
drug entity in the systemic circulation, restrict access of the drug to the chosen sites
and to deliver the drug at a controlled and sustained rate to the site of action. Various
polymers have been used in the formulation of nanoparticles for drug delivery
research to increase therapeutic benefit, while minimizing side effects. Here, we
review various aspects of nanoparticle formulation, characterization, effect of their
characteristics and their applications in delivery of drug molecules and
therapeuticgenes.
3)
Dhruba J Bharali et al., have reveived Nanoparticles and cancer therapy; A
concisereview with emphasis on dendrimers and has concluded that The biomedical
application of nanoparticles has experienced exponential growth in the past few
years; however, current knowledge regarding the safety of nanocarriers is insuffi
cient. As these new drug delivery systems are brought to clinical trial we will begin
to be able to identify any negative side effects associated with these compounds.
Preliminary and complementary animal studies should be carried out to identify the
risks associated with nanoparticle use, with particular attention paid to the
elimination processes. and also provides a brief description of nanoparticle
(liposome, quantum dot, and dendrimer) mediated cancer therapy in the last decade
with an emphasis on the development and use of dendrimers in cancer therapeutics.
4)Diana guzman-villanuea
et al., have performed dasign and in in vitro
evaluation of a new nano-microparticulate system for enhanced aqueous-phase solubility
of curcumin and concluded that by taking the advantage of the characteristics of a
natural polymers such as alginate and carrageenan, the drug release can be modulated
by taking into consideration the ability of the polymers to form strong or weak hydrogel
networks.
5) Sathish Sundar Dhilip Kumar et al., have foemulated Curcumin loaded poly(2hydroxyethyl methacrylate) nanoparticles from gelled ionic liquid and also studied In
vitro cytotoxicity and anti-cancer activity in SKOV-3 cells and concluded that novel
method of making PHEMA gels using choline based ionic liquid such as choline
formate C-PHEMA-NPs were prepared. The nanoparticles obtained were spherical in
shape with uniform size distribution. FT-IR, TGA, DSC, XRD studies indicated the
incorporation of curcumin in PHEMA nanoparticles. In vitro release studies confirmed
the sustained drug release characteristics of C-PHEMA-NPs. In vitro Cytotoxicity (MTT
assay) study showed that C-PHEMA-NPs effectively inhibited the proliferation of
cancer cells. and
demonstrates the curcumin loaded PHEMA nanoparticles have
potential therapeutic values in the treatment of cancer.
6) R. Shelma et al., have studied In vitro and in vivo evaluation of curcumin loaded
lauroyl sulphated chitosan for enhancing oral bioavailability and concluded that
encapsulation of curcumin in the lauroyl sulphated chitosan with a view to improve its
bioavailability. And had demonstrate that the curcumin loaded matrix has shown a
superior pharmacological availability in vivo over curcumin.
7) J. Chen et al., have performed fabrication and evaluation of curcumin loaded
nanoparticles based on solid lipid as a new type of colloidal drug delivary system and
concluded that curcumin-loaded solid lipid nanoparticles could be prepared successfully
with high drug entrapment efficiency and loading capacity. Curcumin-loaded solid lipid
nanoparticles may be a promising drug delivery system to control drug release and
improve bioavailability.and also shown The TEM technique was performed in order to
confirm the formation and morphology of lipid nanoparticles. The measurements
showed that nanoparticles were prepared successfully in the shape of similar spherical
structure. The diameter of nanoparticles was about 100 nm.
8) Sarbari Acharya et al.,have studied PLGA nanoparticles containing various
anticancer agents and tumour delivery by EpR effect and concleded
that Nanotechnology has become an enabling technology for personalised oncology in
which cancer detection, diagnosis and therapy are tailored to each individual's tumour
molecular profile, and also for predictive oncology in which genetic/molecular markers
are used to predict disease development, progression and clinical outcomes. In this
article, we have explored PLGA NPs serving as agents of novel antineoplastic
treatments in various aspects of cancer therapies. the use of drug-loaded PLGA
nanoparticulate technology in developing a new generation of more effective cancer
therapies capable of overcoming the many biological, biophysical and biomedical
barriers that the body stages against conventional cancer therapies.
9) B. Yedomon et al., have done Preparation of Bovine Serum Albumin (BSA)
nanoparticles by desolvation using a membrane contactor: A new tool for large scale
production and concluded that the membrane contactor technique could be a suitable
method for the preparation of albumin nanoparticles at large scale with properties
similar to that obtained at small scale. And have investigated the preparation of BSA
nanoparticles at large scale using a membrane contactor. The technique is based on the
desolvation method: ethanol forms small jets at the outlet of the membrane pores, and
albumin coacervates are formed in the aqueous BSA solution flowing tangentially to the
membrane surface. BSA nanoparticles are then, obtained by reticulation of BSA
coacervates with glutaraldehyde.
10) Zhifeng Cao et al.,have studied novel Temperature- and pH-responsivePolymerbiomolecule Conjugate Composed of Caseinand Poly(N-isopropylacrylamide).and
concluded that A novel double-responsive water-soluble copolymerthat consists of
temperature-sensitive segment andpH-sensitive segment was successfully synthesizedby
the facile method, which was metal ion-free andorganic reagent-free.
11) Jinghua Duan et al.,have doneSynthesis and in vitro/in vivo anti-cancer evaluation
of curcumin-loaded poly(butyl cyanoacrylate) nanoparticles and concluded that the
bioavailability of curcumin is, however, poor. The prepared chitosan stabilized PBCA
nanoparticles can entrap the curcumin effectively and circumvent this problem by
permitting intravenous administration. And demonstrates that curcumin-PBCA
nanoparticles provide an opportunity to expand the clinical repertoire of this efficacious
agent by enabling ready aqueous dispersion.
12) Anindita mukherji et al.,have performed Formulation, Characterization and
Evaluation of Curcumin-loaded PLGA Nanospheres for Cancer Therapy and concluded
thaturcumin-loaded PLGA nanospheres were prepared by using a s/o/w emulsion
solvent evaporation technique. Our studies showed that smooth, spherical PLGA
nanospheres were formed and exhibited high yield and drug entrapment efficiency, with
a narrow size range of 35 nm to 100 nm and mean particle diameter of 45 nm. The in
vitro curcumin release studies from the nanospheres showed that curcumin was released
in a sustained manner over a prolonged period of time. Intracellular uptake and cell
viability assays also demonstrated efficient uptake and action of the curcumin
nanospheres in prostate cancer cell lines. It is therefore concluded that PLGA
nanospheres are capable of delivering curcumin over a prolonged period achieving a
sustained delivery of curcumin, thus making it a potential candidate for cancer therapy.
13) Bhawana, et al., developed a method for the preparation of nanoparticles of
curcumin with a view to improve its aqueous-phase solubility and examine the effect on
its antimicrobial properties. Nanoparticles of curcumin (nanocurcumin) were prepared
by a process based on a wet-milling technique and were found to have a narrow particle
size distribution in the range of 2−40 nm and mechanism of antibacterial action of
curcumin nanoparticles was investigated by transmission electron micrograph (TEM)
analysis, which revealed that these particles entered inside the bacterial cell by
completely breaking the cell wall, leading to cell death.
14) Suman Ramteke and N.K.Jain has prepared nanoparticles of clarithromycin by
desolvation technique using gliadin and they observed that the clarithromycin containing
gliadin nanoparticles provided greater antibacterial activity than the plain drug
formulation.
ENCLOSURE – III
6.3 Objectives of the study
1.
To prepare curcumin nanoparticles using casein and chitosan
2.
To optimize the procedure for the preparation of nanoparticles
3.
To study the effect of excipient and process variables on the characteristics of
nanoparticles.
4.
To evaluate the prepared nanoparticles.
5.
To study the drug polymer compatibility and surface morphology of the
prepared nanoparticles.
6.
To carry out the in vitro release, stability and bioavailability studies prepared
MATERIALS AND METHODS:
DRUG:
Curcumin.
POLYMERS: Casein ,chitosan.
Piperine,cyclodextrin,alginate,carragenan,polycaprolactone,PLGA etc..
SOLVENTS: Acetone, Ethyl acetate, Triacetin, Benzyl alcohol. Ethanol, Acetonitrile,
sodium alginate etc…..
Methods
Preparation of nanoparticles by:
1. Solvent emulsification method.
2. Polymerization method
3. Co-precipitation/coacervation method.
4. Nano encapsulation method.
5. Ionic gelation method etc..
Evaluation
 Compatibility study of curcumin and casein by Fourier Transform Infra-Red
Spectroscopy (FTIR).
 Surface Morphology15.
 by Scanning Electron Microscopy (SEM).
 Particle size distribution studies
 Drug entrapment efficiency.
 In vitro release/dissolution studies.
 Solid state by Differential Scanning Calorimetry (DSC)
 Evaluation of curcumin release from nanospheres
 Surface morphology of nanospheres.
 Sufercritical fluid technology.
ENCLOSURE – IV
7.1. Source of data
a. Library: Sarada Vilas College of Pharmacy.
b. E-library: Sarada Vilas College of Pharmacy.
ENCLOSURE-V
7.2. Method of collection of data
1. Literature search.
2. Laboratory experiments.
A) Preparation:
Preparation of curcumin nanoparticles of casein and chitosan by suitable method.
B) Characterization:
Characterization of nanoparticles, compatibility study by FTIR, surface morphology by
SEM, particle size distribution, drug entrapment efficiency, in vitro release study, solid state
by DSC/XRD, bioavailability study.
ENCLOSURE – VI
7.3. Does the study require any investigation or intervention to be conducted on patients or
other humans or animals? If so, please mention briefly.
- Yes -
7.4. Has ethical clearance been obtained from your institution in case of 7.3?
- In process-
ENCLOSURE – VII
LIST OF REFERENCES:
1. A.Krishna Sailaja, P.Amareshwar, P.Chakravarty. Different techniques used for the
preparation of nanoparticles using Natural polymers and their application. Int J pharm
pharm sci, 2011;3(2):45-50.
2. VJ Mohanraj and Y Chen. Nanoparticles – A Review. Trop J Pharm Res, 2006;5(1).
3. Dhruba J Bharali Marianne Khalil Mujgan Gurbuz Tessa M Simone Shaker A Mousa
Nanoparticles and cancer therapy;A concise review
International Journal of
Nanomedicine 2009:4:1–7.
4. Diana guzman-illanuea, IbrahimM.el-sherbiny, dea Herrera-ruiz and hugh D.C smyth
design and iniVtro evaluation of a new nano-microparticulate system for enhanced
aqueous phase solubility of curcumin creative commons attribution license 2013;1.
5. Feng-lin yen, tzu-hui wu , cheng-wei tzeng , liang-tzung lin and chun-ching lin.
Curcumin nanoparticles improve the physicochemical properties of curcumin and
effectively enhance its antioxidant and antihepatoma activities. Journal of Agricultural
and Food Chemistry. 2012;60(21):5388-99.
6. R. Shelma, Chandra P. Sharma. In vitro and in vivo evaluation of curcumin loaded
lauroyl sulphated chitosan for enhancing oral bioavailability. 2013;95(1):441–48.
7. J. Chen, W. T. Dai, Z. M. He, L. Gao, X. Huang, J. M. Gong, H. Y. Xing, and W.
D. Chen. Fabrication and Evaluation of Curcumin-loaded Nanoparticles Based on Solid
Lipid as a New Type of Colloidal Drug Delivery System. Indian J Pharm Sci.
2013;75(2):178–84.
8. Sarbari Acharya, Sanjeeb K. Sahoo. PLGA nanoparticles containing various
anticancer agents and tumour delivery by EPR effect. 2011;63(3):170-83.
9. B. Yedomon, H. Fessi, C. Charcosset. Preparation of Bovine Serum Albumin
(BSA) nanoparticles by desolvation using a membrane contactor: A new tool for large
scale production. 2013;85(3):398-405.
10. Zhifeng Cao, Yong Jin1, Biao Zhang, Qing Miao, and Chunyan Ma1. Anovel
Temperature and ph-responsive Polymer-biomolecule Conjugate Composed of Casein
and Poly(N-isopropylacrylamide). Iranian Polymer Journal 2010;19(9):689-98.
11. Jinghua Duan, Yangde Zhang, Shiwei Han, Yuxiang Chen, Bo Li, Mingmei Liao,
Wei Chen, Xingming Deng, Jinfeng Zhao. Synthesis and in vitro/in vivo anti-cancer
evaluation of curcumin-loaded poly(butyl cyanoacrylate) nanoparticles. 2010;400(12)211-20.
12. Anindita Mukerjee and Jamboor k. Vishwanatha. Formulation, Characterization and
Evaluation of Curcumin-loaded PLGA Nanospheres for Cancer Therapy. 2009;29:10
3867-3875.
.
13. Bhawana, Rupesh Kumar Basniwal, Harpreet Singh Buttar, Jain V. K., and Nidhi
Jain. Curcumin Nanoparticles: Preparation, Characterization, and Antimicrobial Study.
J. Agric. Food Chem 2011; 59: 2056–61.
14. Suman R, Uma Maheswari RB, Jain NK. Clarithromycin based oral sustained
release nanoparticulate drug delivery system. Indian J Pharm Sci 2006; 68(4):479-84.
.