Preparation and Characterization of Nanocrystals of Simvastatinfor

PREPARATION AND CHARACTERIZATION OF NANOCRYSTALS OF SIMVASTATINFOR

ENHANCEMENT OFSOLUBILITY ANDBIOAVAILABILITY

M. PHARM DISSERTATION PROTOCOL

SUBMITTED TO THE

RAJIVGANDHIUNIVERSITY OF HEALTH SCIENCES,

KARNATAKA, BENGALURU

BY

ASHWINI.T

B.Pharm.,

UNDER THE GUIDANCE OF

Mr.VENKATESH. J.S.

B.Sc. M.Pharm.,( Ph. D)

PROFESSOR AND HEAD

P. G. DEPARTMENT OF PHARMACEUTICS

S. C. S. COLLEGE OF PHARMACY,

HARAPANAHALLI-583131

2011-12

Rajiv Gandhi University of Health Sciences,

Karnataka, Bengaluru

ANNEXURE – II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

01 / Name and Address of the Candidate / ASHWINI.T
D/O BADARINARAYAN.T
BEHIND KATTI RANGANATH HOUSE MUTTUM STREET
HARAPANAHALLI-583131
Dist.Davangere
02 / Name of the Institution / T. M. A. E. Society’s
S. C. S. College of Pharmacy,
Harapanahalli – 583 131.
(Davangere dist.)
Karnataka.
03 / Course of the Study
Branch / M. Pharm.(Pharmaceutics)
04 / Date of Admission to course / 25/07/2011
05 / Title of the Topic / PREPARATION ANDCHARACTERIZATION OF NANOCRYSTALS OF SIMVASTATIN
FOR ENHANCEMENT OF
SOLUBILITY ANDBIOAVAILABILITY
06 /

Brief resume of the intended work

6.1. Need for the Study /

Enclosure – I

6.2. Review of the Literature / Enclosure – II

6.3. Objective of the Study

/ Enclosure – III
07 /

Materials and Methods

7.1. Source of data /

Enclosure – IV

7.2. Methods of collection of data

/ Enclosure – V
7.3. Does the study require any
Investigations on animals?
If yes give details / Enclosure – VI
7.4. Has ethical clearance been
Obtained from your
institution In case of 7.3. / Yes, Registration No157/199 cpCsea. 25th NOV.1999: (Copies enclosed)
08 /

List of References

/ Enclosure – VII
09 /

Signature of the candidate

/ (ASHWINI.T)
10 /

Remarks of the Guide

/ The present research work is original and not published in any of the journals with best of my knowledge upon extensive literature review. This work will be carried out in the Pharmaceutics laboratory by the above said student under my supervision.
11 /

Name and Designation of

(In Block Letters)
11.1. Guide
( ACA/CDC/PGT-M.Ph/SCS/02/2005-06.Dated on 19.01.09)
11.2.Signature
11.3.Co-Guide
11.4.Signature
11.5. Head of the Department
11.6. Signature / Prof.VENKATESH.J.S.
B.Sc.,M.Pharm.,(Ph. D).
PROFESSOR AND HEAD
P.G. Department of Pharmaceutics
S.C.S.College of Pharmacy.
Harapanahalli-583131, Karnataka
Mr.SHANKRAYYA.M
M.Pharm.,( Ph. D).
ASSISTANTPROFESSOR
P.G. Department of Pharmaceutics
S.C.S.College of Pharmacy.
Harapanahalli-583131, Karnataka
Prof.VENKATESH.J.S.
B.Sc., M.Pharm.,(Ph. D).
12 /

Remark of the Principal

12.1. Signature / The present study is permitted to perform in the Pharmaceutics laboratory of our institution by above said student.
Dr.R.Nagendra Rao

ENCLOSURE-I

Brief resume of the intended work:

6.1 – Need forthe Study:

Aqueous solubility is one of the key determinants in developmentof new chemical entities as successful drugs. However, new drug development technologies, such as combinational chemistry and high throughput screening are based on the basic principles of medicinal chemistry, teaching that the most reliable method to increase in-vitro potency is to add lipophilic moiety at appropriate position of the lead structure. This has led to an increase in the number of lipophilic and poorly soluble molecules being investigated for their therapeutic activity. Various formulation techniques are applied to compensate for their insolubility and consequent slow dissolution rate. These include formulation of the amorphoussolid form, nanoparticles, micro emulsions, solid dispersion, melt extrusion, salt formation and formation of water soluble complexes.1

Therapeutic effectiveness of a drug depends upon the bioavailability and ultimately upon the solubility of drug molecules. Solubility is one of the parameter to achieve desired concentration of drug in systemic circulation for pharmacological response to be shown. Currently only 8% of new drug candidates have both high solubility and permeability. It has been estimated that roughly 40% of all investigational compounds fail development because of poor bioavailability that is often associated with aqueous insolubility (Prentis et al,. 1998). On average, according to the tufts center for the study of drug development, only five out of every 5000 potential drugs are actually tested in clinical trials, and of these only one will eventually be approved for use in patients.2

In the recent years, nanoparticle technology has emerged as a strategy to tackle such formulation problems associated with poorly water soluble and poorly water and lipid soluble drugs. The reduction of drug particle to the nano-scale increases dissolution velocity and saturation solubility, which leads to improved in vivo drug performance.3

Simvastatin (SV) is a cholesterol-lowering agent that is derived synthetically from a fermentation product of Aspergills terreus and widely used to treat hypercholesterolemia. SV, is an inactive lactone, is converted to corresponding b, d-dihydroxy acid in liver by cytochrome P-450 (CYP) 3A after oral administration. SV is a potent inhibitor of 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-Co A) reductase. This enzyme catalyzes the conversion of HMG-Co A to mevalonate, which is an early and rate-limiting step in the biosynthesis of cholesterol. However, it is practically insoluble in water and freely soluble in chloroform, methanol and ethanol. it is poorly absorbed from the gastrointestinal (GI) tract. It is having half-life of 3hr, protein binding 95%, bioavailability of 5% Therefore, it is very important to introduce effective methods to enhance the solubility and dissolution rate of drug this intern increases its bioavailability.4

Hence, improving the aqueous solubility might be able to enhance the bioavailability of this drug. Among the various approaches available to increase theaqueous solubility of drugs, nanocrystals technique has been used extensively.5

Sofar limited work has been reported on application of nanocrystals technique, an attempt will be madeto prepare and evaluatenanocrystals of simvastatin.

ENCLOSURE-II

6.2Review of literature:

Literature survey on nanocrystals is carried out by referring various scientific journals, internet and helinet facility.

Hyeong Jeon Ju,et al., have prepared CAP/PF-127 microspheresincorporating

simvastatin acid by a water-acetone-oil-water (W/A/O/W) triple emulsion process. Devices were then fabricated by pressure-sintering UV-treatedblank and drug-loaded microspheres. Using a multilayered fabrication approach,pulsatile releaseprofiles were obtained. Delivery was varied by changing loading,number of layers, blend ratio, andincubation conditions.6

Rongfeng Hu, et al., have prepared sustained release simvastatin microspheres by the spherical crystallization technique with solid dispersing and release retarding polymers. A differential scanning colorimeter and X-ray diffract meter were used to investigate the dispersion state of simvastatin in the in the microspheres. The shape surface morphology and internal structure of the microspheres were observed using scanning electron microscope. Characterization of the microspheres such as average diameter, size distribution and bulk density of the microsphere was investigated. The release rate of the microsphere was controlled by adjusting the combination ratio of the dispersing agents to the retarding agents.7

Al-Suwayeh SA, et al., have prepared triple drug combination tablet containing aspirin, simvastatin and propranolol HCl to treat the patients suffering from the cardiovascular diseases. The interaction of aspirin with both chosen drugs was studied by using Fourier transformer infrared spectroscopy (FT-IR) and differential scanning calorimetry (DSC). Binary physical mixtures of aspirin with either simvastatin or propranolol HCl were prepared, in different weight ratios, as well as their ternary mixture. FT-IR spectra showed no molecular interaction between aspirin and either simvastatin or propranolol HCl in their physical mixtures. The DSC traces depicted lowering of melting points of aspirin and simvastatin by the action of each one on the other during heating. A single oral tablet containing three drugs was formulated and evaluated. Quantitative detection of the three individual drugs in presence of each other in the prepared tablet was determined by using HPLC method. It was found that HPLC is sensitive, reproducible and valid for determination of aspirin, simvastatin and propranolol HCl in the tablet form. The data revealed promising formula for improved relief patient compliance during treatment or prevention of cardiovascular disease.8

 Hecq. J, et al.,in their studypoorly water-soluble drugs such as nifedipine offer challenging problems in drug formulation as poorsolubility is generally associated to poor dissolution characteristics and thus to poor oral bioavailability. In order to enhance thesecharacteristics, preparation of nifedipine nanoparticles has been achieved using high pressure homogenization. The homogenizationprocedure has first been optimized in regard to particle size and size distribution. Nanoparticles were characterized in terms ofsize, morphology and redispersion characteristics following water-removal. Saturation solubility and dissolution characteristicswere investigated and compared to the un-milled commercial NIF to verify the theoretical hypothesis on the benefit of increasedsurface area.9

P. Kocbek, et al.,adopted melt emulsification method traditionally to prepare solid lipid nanoparticles to producedrug nanosuspensions. The methodwas evaluated in comparison with the well knownsolvent diffusion process for ibuprofen as a model drug. Controlof the preparation variables (stabilizers, drug content, homogenization procedure and cooling conditions) allowed formation of nanosuspensionswith diameters less than 100 nm. The combination of Tween 80 and PVP K25 as stabilizersyields nanosuspensions with the smallest average particle size. The formulation of ibuprofen as a nanosuspension, either in the form of lyophilizedpowder or granules, was very successful in enhancing dissolution rate, more than 65% of the drug being dissolved in the first 10 min comparedto less than 15% of the micronized drug. The increase in in vitro dissolution rate may favorably affect bioavailability and improve safety for thepatient by decreasing gastric irritancy.10

 Hai-Xia Zhang, et al.,successfully prepared Amorphous atorvastatincalcium (AC) ultrafine powder by antisolventprecipitation and spray drying process, in which hydroxypropyl methylcellulose (HPMC) was employedto control the particle size and morphology. The enhancement of drug concentration favored to decrease the particle size from 410 nmto 240 nm. After spray drying process, ultrafine AC powder was obtained, which had good dispersibilityand narrowparticle size distribution. The as-prepared ultrafine ACwas characterized by scanningelectron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy,thermal gravimetric analysis (TG), differential scanning calorimetry (DSC), specific surface area and dissolutiontest. The XRD analyses indicated that the ultrafine AC was amorphous. In the dissolution tests,the amorphous AC ultrafine powder exhibited enhanced dissolution property when compared to the rawmaterial.11

 Abdallah Makhlof,et al.,preparednanocrystals of indomethacin by employing cyclodextrins (CyDs) as protective stabilizers by using the emulsion solvent diffusion method. The effect of changing the type and concentration of CyDs on the formation of IMC Nanocrystals was investigated. Dispersions were freeze-dried to characterize the size, shape, nanoparticle yield, crystallinity, and dissolution behavior of theobtained particles. Submicron-sized particles of IMC with average diameters in the range of 300–500 nm were obtained by incorporating CyD in the outer phase of the primary emulsions. A significant enhancement in the dissolution rate of IMC nanocrystals was observed when compared to the commercial powder.12

Basavaraj K. Nanjwade, et al., preparednanocrystal of lovastatin by using simple precipitationmethod without using stabilizer or surfactant and it was found that formulation at 3 mM concentration of drug with theacetone and methanol as a solvent and at proper dilution (50 times) of drug solution with water, nanocrystals with lessparticle size is possible with slight change in crystallinity. It has also shown that, the drug has enhanced saturation solubility, increased dissolution rate and more bioavailable in biological fluid when drug formulated by using acetoneand methanol as a solvent. Whereas drug formulation with acetonitrile has large particle size, less saturation solubility and low rate of dissolution.13

Nighute A. B, et al., prepared its microcrystals of Cefuroxime Axetil by emulsion solvent diffusion method with various surfactants (HPMC E-5 and E-15; PVP K-30 and K-90; β-cyclodextrin; HP-β-cyclodextrin) to foster its solubility and dissolution rate. Manufacturingof microcrystals implies creation of additional surface area and hence interface. Microcrystals were screened for apparent solubility, dissolution rate, morphology (SEM) and crystallinity (XRD and DSC). Microcrystals prepared with HPMC E-15LV showed highest solubility and dissolution rate than the untreated drug and thecrystals prepared with other surfactants. C-6 crystals (Prepared with HPMC E-15LV)were passed through accelerated stability and wettability studies and observed to be Improve.14

Hua Zhang, et al.,prepared camptothecin nanocrystals with a sonication– precipitation method without additional stabilizing surfactants. Particle characteristics, cellular cytotoxicity, and animal antitumor effect were examined. CPT

nanocrystals were tested to be more potent to MCF-7 cells than CPT solution in-vitro. When tested in MCF-7 xenografted BALB/c mice, the CPT nanocrystals exhibited significant suppression of tumor growth. The drug concentration in the tumor was five times more at 24 h by using the nanocrystal treatment than by using the drug salt solution. Storage stability study indicated that the nanocrystals were stable for at least six months. Overall, CPT nanocrystals were considered to be potentially feasible to overcome formulation challenges for drug delivery and to be used in clinic.15

ENCLOSURE-III

6.3 Objective of study:

The present work is planned with the following objectives:

1.To prepare nanocrystal consisting of Simvastatinby precipitation method employing different solvents.

2.To study the physicochemical properties of Simvastatin nanocrystals.

a) Particle morphology

b) Particle size analysis

c) Crystalline state evaluation

- Powder X-ray diffraction (PXRD)

- Differential scanning calorimetry (DSC)

d) Solubility determination

e) In-vitro release study

f) In-vivoantihyperlipidemic activity

g) Stability studies

ENCLOSURE-IV

7.Materials and Methods

7.1 Source of Data:

References from library - S.C.S. college of pharmacy, harapanahalli-583131.

Textbook and standard reference books.

Internet-

National and international journals and publications.

Medline

Helinet for the RGUHS

ENCLOSURE-V

7.2 - Method of collection of data:

PART-I:

1. Extensive literature survey.

2. Procurement of raw materials and drug.

3. Standardization of raw materials and drugs.

PART-II:

To prepare nanocrystal consisting of Simvastatinby precipitation method employing different solventswith different stabilizers

PART-III: Evaluation of Simvastatin nanocrystals:

a) Particle morphology

b) Particle size analysis

c) Crystalline state evaluation

- Powder X-ray diffraction (PXRD)

- Differential scanning calorimetry (DSC)

d) Solubility determination

e) In vitro release study

PART-IV:

f)In vivoantihyperlipidemic activity.16

Group 1 / Control(receiving vehicle only)
Group 2 / Triton treated
Group 3 / Triton + selected formulation
Group 4 / Triton + standard drug(Simvastatin)

g)Stability studies

PART-V: Statistical Analysis, Data Interpretation and Conclusions.

ENCLOSURE-VI

7.3 Does the study require any investigation or interventions to be conducted on patients or other humans or animals? If so, please describe briefly.

Yes, Albino rats will be used for the evaluation of Anti hyperlipidemic activity.

7.4Has ethical clearance been obtained from your institution in case of 7.3?

Yes, the present study is approved from Institutional Animal Ethics Committee

(SCSCP/583/9/2011-12, Dated-26-11-2011)

(IAEC certificate enclosed.)

ENCLOSURE-VII

8. LIST OF REFERENCES:

1. Loftsson T, Hreinsdottir D (2005) Masson Mar. Evaluation of Cyclodextrin

solubilization of drugs. Int J Pharm 302: 18-28.

1. Tufts center for the study of drug development (2001) Tufts University, Boston Mass USA:

1. Muller RH, Bohm BHL (1998) Nanosuspensions, in Emulsions & Nanosuspensions for the formulation of poorly soluble drugs. Muller R.H., Bentia S., and Bohm B.H.L. Eds. (Medpharm Scientific Publishers), Stuttgart,Germany.

1. Ranjita Shegokar, Rainer H. Müller. Nanocrystals: Industrially feasible multifunctional formulation technology for poorly soluble actives. International Journal of Pharmaceutics. 2010;399:129–139.

1. Hyeong Jeon Ju, Mark Thomas V and David Puleo A. Bioerodible devices for intermittent release of Simvastatin Acid, Int J Pharm. 2007;340(1-2):6–12.

1. Rongfeng Hu, Jiabi Zhu, Guagliang Chen, Yuliang Sun, Kangkang Mei and Shi Li. Preparation of sustained release Simvastatin microspheres by the spherical crystallization technique. Asian Journal of Pharmaceutical Science. 2006;1:47-52.

1. Al-Suwayeh SA, Ahmed MO, Mahrous GM and Taha EI. Preparation and In-vitro Evaluation of Tablets Containing Triple Drug Combination Used for Prevention/treatment of Cardiovascular Diseases.Australian Journal of Basic and Applied Sciences. 2009;3(4):3684-3691.

1. Hecq J, Deleers M, Fanara D, Vranckx H, Amighi K.Preparation & Characterizationof Nanocrystals for Solubility & Dissolution Enhancement of nifidipine. International Journal of Pharmaceutics. 2005;299:167–177.

1. Kocbek P, Baumgartner S, Kristl J. Preparation and evaluation of nanosuspensions for enhancing the dissolution of poorly soluble drugs. International Journal of Pharmaceutics. 2006;312:179–186.

1. Hai-Xia Zhanga, Jie-XinWanga, Zhi-Bing Zhanga, Yuan Lea, Zhi-Gang Shenb, Jian-Feng Chen. Micronization of atorvastatin calcium by antisolvent precipitation process. International Journal of Pharmaceutics. 2009;374:106–113.

1. Abdallah Makhlof, Yuta Miyazaki, Yuichi Tozuka, Hirofumi Takeuchi.Cyclodextrins as stabilizers for the preparation of drug nanocrystals

by the emulsion solvent diffusion method. International Journal of Pharmaceutics. 2008;357:280–285.

1. Basavaraj K. Nanjwade, Ganesh K. Derkar, Hiren M. Bechra, Veerendra K. Nanjwade, Manvi FV.Design and Characterization of Nanocrystals of Lovastatin for Solubility and Dissolution Enhancement. J Nanomedic Nanotechnol. 2011;2(2).

1. Nighute A. B, Bhise S. B.preparation and evaluation of microcrystal of cefuroxime axetil. International Journal of PharmTech Research. 2009;1(3):424-430.

1. Hua Zhang, Christin P. Hollis , Qiang Zhang, Tonglei Li.Preparation and antitumor study of camptothecin nanocrystals. International Journal of Pharmaceutics. 2011;415:293–300.

1. Srikanth Jeyabalan and Muralidharan Palayan.Antihyperlipidemic activity of Sapindus emarginatus in triton WR-1339 induced albino rats.Research J. Pharm. and Tech. 2009,2(2):319-323.