Formulation and Validation of Pilocarpine Nanoparticles Forthe Treatment of Glucoma

FORMULATION AND VALIDATION OF PILOCARPINE NANOPARTICLES FORTHE TREATMENT OF GLUCOMA

M. PHARM DISSERTATION PROTOCOL

SUBMITTED TO THE

RAJIVGANDHIUNIVERSITY OF HEALTH

SCIENCES, KARNATAKA, BANGALORE

BY

SUCHETHA REDDYALETI

B.Pharm.

UNDER THE GUIDANCE OF

Dr.R.NAGENDRA RAO

M.Pharm., Ph.D.

PROFESSOR & HEAD

.

P. G. DEPARTMENT OF QUALITY ASSURANCE

S. C. S. COLLEGE OF PHARMACY,

HARAPANAHALLI-583131

2009-10

RajivGandhiUniversity of Health Sciences, Karnataka, Bangalore

Annexure – II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

01 / Name and Address of the Candidate / SUCHETHA REDDY ALETI
1-20/7, BEHINDVIJAYPUBLICSCHOOL, VIDHYANAGAR COLONY
ARMOOR-503224,DIST-NIZAMABAD
ANDRAPRADESH
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.,
Quality Assurance
04 / Date of Admission to course / 28/10/2009
05 / Title of the Topic / FORMULATION AND VALIDATION OF PILOCARPINE NANOPARTICLES FORTHE TREATMENT OF GLUCOMA
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 / NO
7.4. Has ethical clearance been
obtained from your institution
In case of 7.3. / NOT APPLICABLE
08 /

List of References

/ Enclosure – VI
09 /

Signature of the candidate

/ (SUCHETHA REDDY ALETI)
10 / Remarks of the Guide / The present work is well designed and the outcome of the present work may be helpful in overcoming frequent dosing of drug.
11 / Name and Designation of
(In Block Letters)
11.1. Guide reference no. of RGUHS
ACA/CDC/PGT-
M.Ph/SCS/02/2005-06/19.01.09
11.2.Signature
11.3.Co-Guide (if any)
11.4.Signature
11.5. Head of the Department
11.6.Signature / Dr.R.NAGENDRA RAO
M.Pharm, Ph.D
PROFFESSOR & HEAD OF QUALITY ASSURANCE,
------
Dr.C.NAGESH
M.Pharm, Ph.D
PROFFESSOR & HEAD OF PHARMACEUTICS,
------
Dr.R.NAGENDRA RAO
M.Pharm, Ph.D
PROFFESSOR & HEAD OF QUALITY ASSURANCE,
------
12 / Remarks of the Principal
12.1. Signature / The present study is permitted to perform in the Quality assurance laboratory of our institution by above said student.
------

ENCLOSURE-I

06. Brief resume of Intended Work

6.1 Need for the study.

The need to provide therapy for multifactorial eye diseases such as glaucoma, retinal diseases, cataract and for surgical complications such as ocular inflammation and infection represents growing opportunities for ophthalmic drug delivery. Among these, glaucoma is one of the most prevalent eye disease1. Almost 3 million people in the United States and 14 million people worldwide are reported to be suffering from glaucoma. Glaucoma is the third leading cause of blindness worldwide and the second leading cause of blindness in the United States, where it is the leading cause of blindness among blacks and Hispanics.

Glaucoma occurs when an imbalance in production and drainage of fluid in the eye (aqueous humors) increases eye pressure to unhealthy levels. Normally, the aqueous fluid, which nourishes the eye, is produced by the ciliary body behind the iris (in the posterior chamber) and flows through the pupil to the front of the eye (anterior chamber), where it exits into drainage canals between the iris and cornea (the “angle”). When functioning properly, the system works like a faucet (ciliary body) and sink (drainage canals). Balance between fluid production and drainage between an open faucet and a properly draining sink keeps the fluid flowing freely and prevents pressure in the eye from building up2.

Various drugs are used for treatment of glaucoma. Among them Pilocarpine is one of the oldest and widely used drug for treatment of glaucoma. Pilocarpine is an alkaloid obtained from the Pilocarpus microphyllus. It is a cholinergic agonist drug. It has prominent muscarinic action and also stimulates the ganglia-mainly through ganglionic muscarinic receptors. Small dose of pilocarpine generally causes fall in blood pressure but higher dose elicit rise in blood pressure and tachycardia which is probably due to ganglionic stimulation3.

Pilocarpine belongs to Class-2 of BCS classification system. Because of its low solubility and high permeability it exhibits shorter half-life. Hence, the persons who are advised for Pilocarpine medications are recommended for repeated dose administrations. Thus patients using ophthalmic drops containing pilocarpine are faced with frequent dosing schedules and difficulty in drop instillation. This has provided a good opportunity for formulation scientist to work towards development of ocular formulation containing Pilocarpine for single dose administration. Many such attempts are reported in literature. Recently progress has made to use the nanoparticles of biodegradable or non-degradable polymeric nanoparticles as carriers for development of formulation of various categories of drugs. Nanoparticles are defined as particulate dispersions or solid particles with a size in the range of 10-1000nm. The drug is dissolved, entrapped, encapsulated or attached to a nanoparticles matrix. Depending upon the method of preparation, nanoparticles, nanospheres or nanocapsules can be obtained4-5.

The major goal 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 regimen.

Process validation is an integral part of development of novel formulations. Process validation is the means of ensuring and providing documentary evidence that processes (within their specified design parameters) are capable of consistently producing a finished product of the required quality. Process validation is carried out in every step of development of novel formulation to ensure the quality of finished product. In the present work also, the concept of process validation will be utilized for selecting an ideal formulation6.

Inspired by clinical significance of Pilocarpine, opportunity for the development of suitable once-a-day formulation of pilocarpine and significance of nanoparticles as carriers for active pharmaceutical ingredients, in the present work an attempt will be made to prepare Pilocarpine nanoparticles and thus prepared nanoparticles will be formulated into formulations for ocular delivery. Further, the method of preparation of Pilocarpine nanoparticles and formulation of Pilocarpine nanoparticles will be validated by using various physicochemical and analytical parameters.

ENCLOSURE-II

6.2 Review of Literature:

Pilocarpine is one of the most widely used drug in the treatment of glaucoma. Pilocarpine comes under the class-2 of BCS classification so it has a low solubility. To overcome this solubility problem and to reduce the dose frequencies various attempts have been made. Some of them are mentioned below.

1)A long-lasting pilocarpine-loaded chitosan (CS)/Carbopol nanoparticles ophthalmic formulation was developed. The physicochemical properties of the prepared nanoparticles were investigated using dynamic light scattering, zeta-potential, transmission electron microscopy, Fourier transform infrared ray spectroscopy (FT-IR) and differential scanning calorimetry (DSC). The sustained-release effects of pilocarpine-loaded nanoparticles were evaluated using in-vitro release and in-vivo miotic tests and compared with pilocarpine in solution, gel and liposome7.

2)The regional pharmacokinetics as well as the pharmacodynamics of pilocarpine-loaded nanoparticles for the treatment of glaucoma were investigated and compared to a solution of this drug. Polybutylcyanoacrylate nanoparticles were prepared by an emulsion polymerization process. Formulations with different drug concentrations (2-6%) as well as different particle concentrations were investigated and analyzed for size and drug loading. Drug binding to the particles was achieved at a level of 10-18% of the total drug content. The colloidal nanoparticles were sufficiently small (diameter: 100-300 nm) for a non-irritating application to the eye8.

3)Nanoparticles with pilocarpine and timolol were produced by micelle polymerization. The influence of manufacturing temperature, type of monomer, drug concentration and ethylcyanoacrylate concentration on the particle size were investigated with a Coulter Nano Sizer. The nanoencapsulation succeeded only with the pilocarpine or timolol base, not with the salts of these drugs. Different micelle formation in aqueous medium compared with organic solvents was evidently responsible for this phenomenon. The particle formation was affected both by the manufacturing temperature and the type of monomer. The smallest particles were produced at lower temperatures and with the most lipophilic monomers. The monomer and drug concentration had very little influence on the particle size. The nanoparticles made with pilocarpine were, in general, much bigger than those made with timolol9.

4)Pilocarpine nitrate loaded egg albumin microspheres were prepared by thermal denaturation process in the size range of 1-12 µm. A series of batches were prepared to study factors, which may affect the size and entrapment efficiency of drug in microspheres and optimized the process. Drug loaded microspheres so obtained were evaluated for their size, entrapment efficiency, release rate and biological response. Electron photomicrographs were taken (8000X) to study the morphological characteristics of microspheres. The entrapment and encapsulation of pilocarpine after process optimization was found to be 82.63% and 62.5% respectively. In vitro dissolution rate studies revealed that the release of drug from the microspheres followed spherical matrix mechanism. Biological response of microspheric suspension was measured by reduction in intraocular pressure in albino rabbit eyes and compared with marketed eye drops. Various pharmacokinetic parameters viz. onset of action, duration of action, T-max and AUC were studied. A measurable difference was found in the mean miotic response, duration and AUC of pilocarpine nitrate microspheric suspension10.

5)There are reports which describes the efficacy ofmucoadhesive co polymeric micelle nanoparticlesmade of N-isopropylacrylamide (NIPAAM), Nvinylpyrrolidone(VP) and acrylic acid (AA) havingcross-linkage with N, N’ Methylene bis acrylamide(MBA) and containing non-steroidal anti-inflammatory drugs (NSAIDs) into the polymericnetwork as ocular drug carriers for enhancedbioavailability. The drug loaded nanoparticles were characterized by various physicochemical methods like DLS, TEM, SEM, FT-IR, 1H-NMR, XRD, UV/visible and fluorescence spectroscopic studies. The Drug release studies from the nanoparticles were done in different pH buffers at 25 and 37oC.The prepared nanoparticles were examined for in vitro corneal permeability11.

6)Fluorescent chitosan nanoparticles were prepared by ionotropic gelation. The stability of the particles in the presence of lysozomes was investigated by determining the size and their interaction with mucin, by measuring the viscosity of the mucin dispersion. The in vivo interaction of CS-FL nanoparticles with rabbit cornea and conjunctiva was analyzed by spectroflurometry and confocal microscopy. Their potential toxicity was assessed in human conjuctival cell line by determining cell survival and viability12.

ENCLOSURE -III

6.3 Objectives of the study:

In the present work,first Pilocarpine Nanoparticles will be prepared by employing suitable literature methods. Thus prepared Pilocarpine nanoparticles will be evaluated by various analytical techniques. The process employed for preparation of Pilocarpine nanoparticles will be validated (if required) by modification of employed method for generation of ideal nanoparticles. Further, Pilocarpinenanoparticles will be formulated into various ocular drug delivery systems. Thus prepared formulations will be validated for selecting an ideal formulation by employing various parameters. The steps which are intended to be carried out are as follows.

Step-1: Preparation of pilocarpine Nanoparticles.

Step-2: Characterization of pilocarpine Nanoparticles

Step-3: Validation of preparation methods employed for formation of pilocarpine

nanoparticles.

Step-4: Formulation of pilocarpine Nanoparticles into various ocular drug delivery

systems.

Step-5: Validation of prepared formulation by using various physico-chemical

parameters13.

ENCLOSURE – IV

7. Material & methods:

7.1 Source of data:

The primary data required for designing the work will be collected from

1. Various national and international journals available in college library,
2. From various open access journals available in internet.
3. From helinet service of RGUHS, Bangalore.
4. From various reference books available in college library.
5. From various search engines like google.com, ask.com etc.,
6. By referring various journals from libraries of Indian Institute of Science, Bangalore, Libraries of various Universities liker KuvempuUniversity, Shankargatta, KarnatakaUniversity, Dharwad.
7. For validation of preparation methods for Nanoparticles, the data collected from various analytical techniques like DSC,SEM, etc.,
8. For validation of formulation, various data collected from evaluation of prepared formulations like % drug loading, % encapsulation efficiency, particle size distribution, SEM, in-vitro dissolution studies, etc.

ENCLOSURE – V

7.2 Method of collection of data:

1. By using suitable literature methods, Nanoparticles of pilocarpine will be

prepared.

2. Thus prepared Nanoparticles will be characterized by various analytical

techniques like SEM, DSC, etc.,

3. Based on data obtained from analytical methods, if required validation of the

preparation method employed for preparing Nanoparticles will be carried out.

4. Thus prepared pilocarpine Nanoparticles will be formulated into various

categories of ocular drug delivery systems.

5. Thus prepared formulations will be evaluated for

1. Percentage drug loading by UV spectroscopy.
2. Percentage encapsulation efficiency by UV spectroscopy.
3. Particle size distribution by Scanning Electron Microscopy.
4. Compatibility studies between various ingredients by using DSC.
5. In-vitro dissolution study by USP protocol.

6. Validation for identification of ideal formula by using the data obtained from

the above methods (Step-5).

ENCLOSURE – VI

8.0List of references:

1)Patel Geeta M., Patel Madhabhai M.,India Recentadvances and challenges in

ocular drug delivery system, Pharma times, January-2007;vol-39(1);21-25.

2)Glaucoma Eye Disorders Merck Manual Home Edition.mht

3)TripathiKD,Essentials of medical pharmacology, 6th edition page no: 97-
98,147.

4)RathoreKS,SisodiaSS, Ranawat MS, NemaRK, Ophthalmic
nanoparticles drug delivery systems, free on line article base directory, article base.com

5)Das Swarnali, Preeti K. Suresh,Drug delivery to eye: Special reference to;

nanoparticles, International Journal of Drug Delivery,(2010);2:12-21.

6)Agallow Jams, Carleton Frederic, Validation of pharmaceutical processes, Third
edition.

7)Kao HJ, Lin HR, Lo YL, Yu SP. Department of Pharmacy, ChiaNanUniversity
of Pharmacy and Science, Tainan, Taiwan.Characterization of pilocarpine-
loaded chitosan/Carbopol nanoparticles.J. Pham. Pharmacol.Feb 2006;
58(2):179-86.

8)Zimmer A, Mutschler E, Lambrecht G, Mayer D, Kreuter J.Institut für
Pharmazeutische Technologie, Pharmacokinetic and pharmacodynamic
aspects of an ophthalmic pilocarpine nanoparticles-delivery-system. Pham.
Res. Oct1994; 11(10):1435-42.

9)Tuulikki Harmia-Pulkkinen, Manufacture of polyalkylcyanoacrylate
nanoparticles with pilocarpine and timolol by micelle polymerization:
Factors influencing particle formation, Journal of Microencapsulation,
January 1989;Volume 6: Issue 1: Pages 87 – 93

10)Rathod S, Deshpande SG. Albumin microspheres as an ocular delivery system
for pilocarpine nitrate. Indian J Pham Sci.2008;70:193-7

11) GuptaAK, Gupta M & Maitra AN, Polymeric Nanoparticles Encapsulating
NSAIDs for Ocular Delivery: Corneal Penetration and Polymorph nuclear
Leukocyte Migration Studies U.S Patent: 6,579,519 European Cells and
Materials2003;Vol. 6: Suppl. 2:Page 40 ISSN:1473-2262

12)AngelaM.de Campos, YolandaDiebold, EdisonLSCarvalho, AlejandroSánchez and MariaJosé Alonso, Chitosan Nanoparticles as New Ocular Drug Delivery Systems: in Vitro Stability, in Vivo Fate, and Cellular Toxicity, pharmaceutical research.

13) Kalimuthu Selvakumar, YadavAV, Formulation and evaluation of carvedilol
loaded eudragite 100 nanoparticles, International journal of Pharmtech
research,April-June 2009;vol.1, no.2:Page 179-183, coden (USA): ijprif
ISSN: 0974-4304,