Formulation and In-Vitro Evaluation of Nanoparticulate Drug Delivery System Containing

FORMULATION AND IN-VITRO EVALUATION OF NANOPARTICULATE DRUG DELIVERY SYSTEM CONTAINING RIFAMPICIN

M.Pharm Dissertation Protocol Submitted to

Rajiv Gandhi University of Health Sciences, Karnataka

Bangalore– 560 041

By

Mr. RABI SANKAR CHAKRABORTYB.Pharm.

Under the Guidance of

Dr.ROOPA KARKIM.PHARM,Ph. D

Professor& HOD of Industrial Pharmacy

Department of Industrial Pharmacy,

Acharya & B.M. Reddy College of Pharmacy,

Acharya Dr.Sarvepalli Radhakrishnan road,

Soldevanahalli,Achitnagar(Post)

Hesaraghatta, Main Road, Bangalore – 560107.

2012-2013

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

KARNATAKA, BANGALORE.

ANNEXURE - II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1. / Name of the Candidate
and Address / RABI SANKAR CHAKRABORTY
S/O- MR.RAMESH CHAKRABORTY, JOYNAGAR, NETAJI SUBHASH LANE, ROAD NO-6, DASHAMIGHAT MAIN ROAD, P.O- AGARTALA, DIST- WEST TRIPURA, PIN-799001, AGARTALA, TRIPURA, INDIA
2. / Name of the Institution / ACHARYA & B.M. REDDY COLLEGE OF PHARMACY
ACHARYA DR. SARVEPALLI RADHAKRISHNAN ROAD,SOLDEVANAHALLI, HESARAGHATTA MAIN ROAD,CHIKKABANAVARA POST.
BANGALORE-560107
3. / Course of Study and Subject / M. Pharm
(INDUSTRIAL PHARMACY)
4. / Date of Admission / 15-01-2013
5. TITLE OF PROJECT:-
“FORMULATION AND IN-VITRO EVALUATION OF NANOPARTICULATE DRUG DELIVERY SYSTEM CONTAINING RIFAMPICIN”
6.
6.1
6.2 / BRIEF RESUME OF THE INTENDED WORK:
NEED FOR THE STUDY:
Tuberculosis has historically been a significant life-threatening disease and is still a world -wide health threat with an increasing incidence of multiple-drug-resistant (MDR) clinical strains of Mycobacterium tuberculosis.Approximately one-third of the world population is infected with Mycobacterium Tuberculosis, resulting in more than eight million new cases and two million deaths annually.One of the major problems is non-compliance to prescribed regimens, primarily because treatment of TB involves continuous, frequent multiple-drug dosing. Adherence to treatment and the outcome of therapy could be improved with the introduction of long-duration drug formulations releasing the antimicrobial agents in a slow and sustained manner, which would allow reduction in frequency and dosing numbers.1
The word ‘Nano’ is derived from Latin word, which means dwarf. Nanotechnology can be termed as the synthesis, characterization, exploration and application of Nanosized (1-100nm) materials for the development of science. It deals with the materials whose structures exhibit significantly novel and improved physical, chemical, and biological properties, phenomena, and functionality due to their Nano scaled size.2 As 40% or more of the new chemical entities are coming, they are having the problems of their physical/chemical /pharmaceutical/pharmacological parameters. Nanoparticles can solve this problem as it.3
(i) Increases surface area.
(ii) Enhances solubility.
(iii) Increases rate of dissolution.
(iv) Increases oral bioavailability.
(v) More rapid onset of therapeutic action.
(vi) Less amount of dose required.
A Nano-suspension is a submicron colloidal dispersion of drug particles. A pharmaceutical Nano-suspension is defined as very finely colloid, biphasic,dispersed, solid drug particles in an aqueous vehicle, size below 1µm ,without any matrix material , stabilized by surfactants and polymers, prepared by suitable methods for drug delivery applications, through various routes of administration like oral,topical, parenteral,ocular and pulmonary routes.
Advantages :
(1) Physical Long-term Stability.
(2) Increases Saturation Solubility.
(3) Targeted drug delivery.
(4) Bioavailability enhancement.4
DRUG PROFILE
RIFAMPICIN 5

Fig. Rifampicin
DESCRIPTION
i) Physical Appearance: Orange-brown to red-brown powder.
ii) Molecular formula: C43H58N4O12
iii) Molecular weight: 822.94 g/mol.
iv) Melting point: 183-188˚ C
v) Solubility: Soluble-Dimethylsulfoxide, Dimethylformamide, Methanol, 0.1 N HCl,
Chloroform, Ethyl Acetate, Acetone and Phosphate Buffer pH 7.4
Slightly Soluble- Water, Ethanol.
vi) Half-life-: 1.5- 3h 6.
vii)MOA - Rifampicin inhibits bacterial DNA-dependent RNA synthesis by inhibiting bacterial DNA dependentRNA polymerase.
viii) Site of action- Entire Gastrointestinal tract.
ix) Pharmacokinetics- Orally administered rifampicin results in peak plasma concentrations in about two to four hours. Rifampicin is easily absorbed from thegastrointestinal tract. Only about 7% of the administered drug will be excreted unchanged through the urine, though urinary elimination accounts for only about 30% of the drug excretion. About 60% to 65% is excreted through the faeces.7
Rifampicin is the primary drug for the treatment of tuberculosis. It is having less side effects compared to other drugs which are used for the treatment of tuberculosis and it is having more therapeutic effects. The present purpose of my research work is to reduce the dosing frequency of the drug , increase the aqueous solubility, to reduce hepatotoxicity, to increase bioavailability as it undergo rapid 1st pass metabolism which reduces its bioavailability and to formulate and evaluate Nano-suspension which can overcome earlier problems of Rifampicin.
REVIEW OF LITERATURE:

1. Mohanraj VJ and Chen Y., had worked on particulate systems like nanoparticles have been used as a physical approach to alter andimprove the pharmacokinetics and pharmacodynamicsof various types of drugmolecules. 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 andsustained rate to the site of action.8
2. Venkatesh T et al., had worked on, preparation methods, advantages of such methods,

characterization of Nano-suspensions, considerations and their applications have been reviewed
hoping to make easy the future research in this area. Apart from the advancements till now, it
scopes for further investigations in terms of pharmacokinetic and pharmacological correlations,
in vivo bioavailability studies and integration of physical chemistry, bio-informatics and
biochemistry to study the effects of Nano-suspensions.9

1. Patravale BV et al., had worked on Nano-suspensions that emerged as a promising strategy for the efficient delivery of hydrophobic drugs because of their versatile features and unique advantages. Techniques such as media milling and high pressure homogenization have been used commercially for producing Nano-suspensions. Recently, the engineering of Nano-suspensions employing emulsions and micro emulsions as templates has been addressed in the literature. The unique features of Nano-suspensions have enabled their use in various dosage forms, including specialized delivery systems such as muco-adhesive hydrogels. Rapid strides have been made in the delivery of Nano-suspensions by parenteral, oral, ocular and pulmonary routes. Currently, efforts are being directed to extending their applications in site-specific drug delivery.10
2. Nagaraju P et al., had observed one of the critical problems associated with poorly soluble drugs is low bioavailability and or erratic absorption. The problem is even more complex for drugs such as itraconazole and Carbamazepine (belonging to BCS CLASS II) as they are poorly soluble in both aqueous and organic media .There are number of formulation approaches to resolve the problems of low solubility and low bioavailability. But all those have some limitations and hence have limited utility in solubility enhancement. Nanotechnology can be used to resolve these problems associated with conventional approaches. Nanotechnology is defined as the science and engineering carried out in the Nano scale that is 10-9 nm. Nano suspensions consist of the pure poorly water-soluble drug without any matrix material suspended in dispersion. A Nano-suspension not only solves the problems of poor solubility and bioavailability but also alters the pharmacokinetics of drug and thus improves drug safety and efficacy.11
3. Tamizhrasi S and co-authors, had formulated and evaluated Lamivudine loaded poly meth- acrylic acid nanoparticles. The aim of this study was to prepare and evaluate poly meth-acrylic acid nanoparticles containing lamivudine in different drug to polymer ratio by Nano-precipitation method. The developed formulation overcome and alleviates the drawbacks and limitations of lamivudine sustained release formulations and could possibility beadvantageous in terms of increased bioavailability of lamivudine12.
4. Singh A et al., had developed losartan potassium loaded chitosan nanoparticles by ionic gelation of chitosan with tripolyphosphate anions with different core: coat ratio and evaluated for drug content, loading efficiency, particles size, zeta potential, in vitro drug release and stability studies. The developed formulation overcomes and could possibility be advantageous in terms of sustained release dosage forms of losartan potassium.13
5. Rajan M and Raj V., made an attempt to encapsulate anti-tuberculosis drug, Rifampicin (RIF) with a model drug delivery system. The designed carrier Chitosan (CS) and polyethylene glycol 600 (PEG) nanoparticles were prepared by Ionic gelation technology, and then used for entrapping RIF. The PEG binding with CS-RIF changed the character and the surface of the nanoparticles and slightly increased its particle size, while the drug encapsulation was also increased. PEG bind with CS-RIF achieved a significantly prolonged retention compared with non-coated CS-RIF. Various parameters and methodologies such as loading capacity, encapsulation efficiency, SEM, FTIR and in vitro release have been utilized for characterization of nanoparticles. The release of drugs was influenced by their initial drug concentration, indicating that the release of drugs could be controlled by varying the initial drug concentration. All results suggested that CS and CS-PEG nanoparticles are promising system for delivering RIF in treatment of tuberculosis.14
6. Booysen L et al., determined the effect that PLGA (coated/ uncoated with PEG/Pluronic F127) Nano-encapsulation of rifampicin (RIF) and isoniazid (INH) has on plasma protein binding of these drugs invitro. The bio distribution of Rhodamine 6G labelled PEG-coated and Pluronic F127- coated nanoparticles was evaluated. Poly-lactic-glycolic-acid (PLGA) nanoparticles were prepared by the double emulsion spray drying technique. This study concludes that poloxamer coating of polymer nanoparticles presents a longer circulation time due to decreased protein binding with a subsequent increase of nanoparticles accumulation in tissues, primarily plasma and spleen.15
7. Arunkumar N et al., had prepared & evaluated atorvastatin Nano-suspension using homogenisation method. Through the various studies it was found that crystalline atorvastatin was converted to amorphous form and exhibit improved dissolution and higher solubility. The increase in drug dissolution rate and solubility can be expected to have significant impact in the oral bioavailability of the drug.16
8. Suganeswari M et al., had prepared and characterized nanoparticles containing atorvastatin calcium: D1N amlodipine besylate: D2N loaded by Nano-precipitation method using tribloere (Pluronic F68). The amlodipine has potency to promote the activity of atorvastatin. Therefore, atorvastatin and amlodipine combination were used for this research. This research showed a new way towards the formulation of nanoparticles of atorvastatin & amlodipine.17

1. Clisson ME et al., had developed ciprofloxacin-loaded nanoparticles for improved intracellular therapy by employing polyalkylcyanoacrylate as the polymer. The entrapment efficiency, in vitro drug release and the drug-polymer interactions were investigated. Finally a model describing the association of ciprofloxacin with the polymer was proposed.18
2. Matsumoto J et al., had described the preparation and evaluation of biodegradable PLA-PEG-PLAnanoparticles containing progesterone as a model drug. Solvent evaporation method was used to prepare the nanoparticles. The drug release from nanoparticles was controlled by the PEG content.19
3. Hiroyuki et al.,reported in article that Nifedipine (NI) is a poorly water-soluble drug and its oral bioavailability is very low. To improve water solubility, NI-lipid nanoparticle suspension were prepared by a combination of co-grinding by a roll mill and high pressure homogenization without any organic solvent.It was found however, that the addition of sugars to the suspension before freeze drying inhibited the aggregation of NPs, suggesting that long term stability storage of freeze dried NI-lipid nanoparticle after reconstitution would be overcomed.20
4. Agnihotri SMet al., had found thatpolymeric nanoparticles suspensions were prepared from poly (lactide-co-glycolide) and poly (lactide-co-glycolide-leucine) {poly [Lac (Glc-Leu)]} biodegradable polymers and loaded with diclofenac sodium to improve the ocular availability of the drug. Nanoparticle suspensions were prepared by emulsion and solvent evaporation technique and system showed a better size distribution for ophthalmic application. In vitro release tests showed an extended-release profile of diclofenac sodium from the nanoparticles suspensions.21
5. Vijayan Vet al.,had prepared solid lipid nanoparticles by the hot homogenization using bio acceptable lipids such as Cephalin and lecithin and Tween 80 as a emulsifier. Drug loaded SLNs showed average diameters in the colloidal size range, a good loading capacity and drug release. 22

6.3
7.0
7.1
7.2
7.3
7.4
8.0 / OBJECTIVES OF THE STUDY:
Following are the objectives of the present study

1. To carry out identification and preformulation study of drug and selected polymers.
2. To formulate Nano-suspension by high pressure homogenization/ultra sonication/ precipitation/media milling or any suitable / developed method.
3. Evaluation of formulated Nano-suspension for various parameters like:

Particle size analysis and shape morphology.
Entrapment efficiency.
Drug content estimation.
Zeta potential.
4. To carry out in vitro dissolution studies of Rifampicin Nano-suspension.
5. To carry out stability studies on the most satisfactory formulation.
MATERIALS AND METHODS:-
SOURCE OF DATA:-
Review of literature from:

• Journals – such as

• Indian Journal of Pharmaceutical Sciences
• European Journal of Pharmaceutical Sciences

• International Journal of Pharmaceutics
• International Journal of Pharmacy and Pharmaceutical Sciences
• African Journal of pharmaceutics
• European Journal of Pharmaceutics and Biopharmaceutics

• Pub Med
• Science Direct
• J-Gate@Helinet
• Library: Acharya & B. M. Reddy College Of Pharmacy

Materials
Drug : Rifampicin
Polymer:PVP, Eudragit RS100, Eudragit RL100, Ethyl cellulose, PEG etc.
Stabiliser:Methyl cellulose, Lecithin, Poloxamer etc.
Surfactant:Polysorbates, Oleic acid Etc.
Solvent:Ethanol, Chloroform, Acetone, Ether, Water Etc.
Method of collection of data:
1. Preformulation studies of drug & selected polymer
a. Solubility studies.
b. Melting point determination.
c. Purity of the drug
d. Compatibility studies by FTIR/DSC.
2. Preparations of Nano-suspension by high shear homogenization or any suitable / developed method.

• Procedure for Homogenisation:Homogenization involves the forcing of the suspension under pressure through a valve having a narrow aperture. The instrument can be operated at pressures varying from 100 to 1500 bars. In some instruments, a maximum pressure of 2000 bars can be reached. Most of the cases require multiple passes or cycles through the homogenizer, which depends on the hardness of the drug, the desired mean particle size, and required homogeneity. High‐pressure homogenizers are available with different capacities ranging from 40ml (for laboratory purposes) to a few thousand litres (for large‐scale production).Before subjecting the drug to the homogenization process, it is essential to form a presuspension of the micronized drug in a surfactant solution using high‐speed stirrers.23

3. The resultant product will be freeze dried using lyophilizer and final product thus obtained will be subjected to evaluation parameters.
4. Evaluation of the various properties of Rifampicin Nano-suspension.
Entrapment efficiency.
 Drug content estimation.
 Particle size and shape morphology by SEM.
 Zeta potential by using zeta seizer
5. To carry out in vitro dissolution study of formulated Nano-suspension.
6. To carry out stability studies on the most satisfactory formulation

DOES THE STUDY REQUIRE ANY INVESTIGATION TO BE CONDUCTED ON PATIENT OR OTHER HUMANS OR ANIMALS?
“NO”

HAS ETHICAL CLEARANCE BEEN OBTAINED FROM YOUR INSTITUTION IN CASE OF 7.3?
“NOT APPLICABLE”
REFERENCES:-
1)Gelperina S, Kisich K, Iseman MD, Heifets L. The Potential Advantage of Nanoparticle Drug Delivery System in Chemotherapy of Tuberculosis. Am. J. Respir. Crit. Care. Med. 2005 Dec 15; 172(12):1487-90.
2)Jain NK. Pharmaceutical Nanotechnology. National. Sci. Dig. Lib. 2008:02-19.
3)Vishvajit K, Jagdale A, Deepali M, Vilasrao KJ. Nano suspension: A novel drug delivery system. Int. J. Pharm. Biosci. 2010; 1(4): 352-60.
4)Krishna BK, Prabhakar Ch. A Review onNano suspensions In Drug Delivery. Int. J.Pharm and Bio Sci. 2011; 2(1): 549-58.
5)Rifampicin, [online] [cited 05-09-13]; available from http// www. En. Wikipedia. Org / wiki / Rifampicin.
6)Sigma Aldrich: Rifampicin Product information [cited 05-09-13] available from: www. SigmaAldrich. Com/ Rifampicin/ Product information/ PDF.
7)G Curci, A Ninni, A.D Aleccio. Atti Tavola Rotonda Rifampicina. Lepetit, Milano: Edizioni Rassegna Medica; 1969: 19.
8)Mohanraj VJ and Chen Y. Nanoparticles- A Review. Trop. J. Pharm. Res. 2006; 5 (1): 561-73.
9)Venkatesh. T, Reddy KA, Maheswari UJ, Dalith DM, Kumar ACK. Nano suspensions: Ideal Approach for the Drug Delivery of Poorly Water Soluble Drugs. Der Pharmacia Lettre. 2011; 3(2): 203-13.
10)Patravale BV, Date AA, Kulkarni MR. Nano suspensions: a promising drug delivery strategy. J. Pharm. Pharmacol. 2004; 56: 827-40.
11)Nagaraju. P, Krishnachaithanya. K, Srinivas. V.D.N and Padma. S.V.N. Nano suspensions: A Promising Drug Delivery Systems. Int. J. Pharm. Sci. Nanotech. 2010; 2(4): 679-84.
12)Tamizhrasi S, Shukla A, Shivkumar T, Ravi V, Rathi JC. Formulation and evaluation of lamivudine loaded polymethacrylic acid nanoparticles. Int. J. Pharm. Tech. Res. 2009; 1(3): 411-15.
13)Singh A, Deep A. Formulation and evaluation of nanoparticles containing losartan potassium. Int. J. Pharm. Res. Tech. 2011; 1(1): 17-20.
14)Rajan. M and Raj. V. Encapsulation, Characterisation and In-Vitro Release Of Anti- Tuberculosis Drug Using Chitosan- Poly Ethylene Glycol Nanoparticles. Int. J. Pharm. Pharm. Sci. 2012; 4(4): 255-59.
15)L Booysen, B Semete-Makokotlela, L Kalombo, H Swai, AF Kotze. In vitro characterisation of PLGA nanoparticles encapsulating rifampicin and isoniazid- Towards IVIVC. IN: Proceedings of the CSIR 3rd Biennial Conference; 30 August-01 September 2010, Science CSIR International Convent ion Centre, Pretoria: South Africa; 2010.
16)Arunkumar N, Deecaraman M, Rani C, Mohanraj KP, Venkates KK. Preparation and solid state characterization of atorvastatin Nano suspensions for enhanced solubility and dissolution. Int. J. Pharm. Tech. Res; 2009; 1(4): 1725-30.
17)Suganeswari M, Shering A, Azhagesh RK, Bharathi P, Sathish B. Preparation, characterization and evaluation of nanoparticles containing hypolipidemic drug and antihypertensive drug. Int. J. Pharm. Bio. Archives. 2011; 2(3): 949-53.