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3.Theoretical Analysis

3.1.Oral Controlled Release Drug Delivery System

Definition:

It provides the continuous oral delivery of drugs at predictable and

reproducible kinetics for predetermined period throughout the course of

GIT.140

Advantages:

1.) Reduction in plasma drug level fluctuations.

2.) Reduction in adverse effects and health care cost.138

Polymers used:

These are broken down into biologically acceptable molecules that are

metabolized and removed from the body.

Poly (2-hydroxy ethyl methacrylate)

PolyN vinyl pyrrolidine

Polyvinyl alcohol

Polyacrylic acid

Polyacrylamide

Polyethylene glycol

Additional Polymers3

Polylactides

Polyglycolides

Polyanhydrides

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Natural Polymers

Albumin

Gelatin

Chitosan

Cellulose

Collagen

Continuous Release Systems

These release the drug for prolonged time with normal transit of dosage

form. Various systems are –

1) Dissolution Controlled Release Systems :

In this drug, particle is coated with polymer layer which controls the rate

of release of drug.

(i) Encapsulated Dissolution Control: The individual particles or

granules are coated with slowly dissolving material, compressed

directly into tablets as in spansules.141

(ii) Matrix Dissolution Control: Drug is homogenously dispersed

throughout a rate controlling medium which controls the drug

dissolution by controlling rate of dissolution fluid penetration into

matrix.

2) Diffusion Controlled Release Systems :

In this, diffusion of dissolved drug through a polymeric barrier occurs.

The two types are –

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Reservoir Devices (Laminated Matrix Devices) :

In this, water insoluble polymeric material encloses a core of drug,

partitions into membrane and exchange with fluid surrounding

tablet and additional drug will enter the membrane diffuse to

periphery and exchange with surrounding media.141

Matrix Devices :

In this, solid drug is dispersed in a insoluble matrix and the drug

release is dependent on rate of drug diffusion and not on solid

dissolution rate.

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3) Diffusion and Dissolution Controlled Release Systems:

Drug core is with partially soluble membrane where dissolution part of

membrane allows for diffusion of contained drug through pores in

polymer coat.

4) Ion Exchange Resins:

Resins are special granules passes ion active site for acidic/cationic

drugs.

5) pH Independent Formulation :

By mixing basic/acidic drug with buffering agents and finally coating

with GI fluid permeable film forming polymer.141

6) Osmotic Pressure Controlled Systems:

A drug fabricated by semi-permeable membrane is delivered by osmosis

through delivery orifice.140

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The hydrodynamic pressure created squeezes the collapsible drug

reservoir to release medicament through delivery orifice.141

Delayed Transit and Continuous Release Systems:

These detain residence time in stomach by prolonging residence time in

GIT.

Altered Density Systems:

By altering density, the residence time of drug is prolonged.

Mucoadhesive Systems:

The bioadhesive polymer continuously releases a fraction of drug into

intestine over prolonged periods of time.

Size Based System:

Based on size, the gastric emptying is delayed allowing once daily dosing

Intestinal Release Systems:

Drugs designed to prevent destabilization in gastric pH.

Colonic Release Systems:

These are used in ulcerative colitis to deliver drug in colon.140

Currently Marketed Oral Controlled Release Systems:

1.) Coating tablets, granules and non pereil sugar beads.

2.) Matrix systems made of swellable or non-swellable polymers.

3.) Slowly eroding devices.

4.) Osmotically controlled devices.

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3.2. Methods of Preparation of Nanoparticles

Definition of Nanoparticles:

Nanoparticles are defined as particulate dispersions or solid

particles with a size range of 10-1000 nm.

These are more reactive because of their greater surface area per

unit weight than larger particles.

Methods of Preparation:

1.) Preparation from natural macromolecules

2.) Emulsion Based method

3.) Coacervation method

4.) Salting-out methods

5.) Controlled Gelatin process

6.) Direct Precipitation method

Preparation of Nanoparticles from Natural Macromolecules:

By using proteins such as albumin, gelatin, leguminor vicillin and

polysaccharides such as alginates and agarose, a water in oil emulsion is

prepared and is subjected to subsequent heat denaturation or chemical

cross linkng of macromolecules. These proteins and polysaccharides

have a wide interest because of their properties of biodegradability and

biocompatibility.

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Emulsion based Method:

In this process, emulsification of an aqueous solution of albumin

in vegetable oil142 (cottonseed oil) is carried at room temperature and is

homogenized. This highly dispersed emulsion is added to a large volume

of preheated oil at about 7120C under constant stirring.144 This leads to

coagulation of solid nanospheres of albumin by denaturation due to

immediate vaporization of water. Then, the suspension is allowed to cool

down at room temperature in an ice bath.

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Coacervation Method :

This is a controlled desolvation method in which, nanoparticles are

produced by phase separation process in an aqueous medium and is

subsequently stabilized by crosslinking with Glutaraldehyde. This

method is mainly to overcome the utilization of large amount of organic

solvent to obtain nanoparticles.144

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Salting-out Methods :

An aqueous phase of saturated electrolytes like Magnesium acetate

and Magnesium chloride with PVA as stabilizing and viscosity increasing

agent is added to acetone solution of polymer under vigorous stirring.

Electrolytes in aqueous phase prevent the miscibility of aqueous and oil

phases. The addition of aqueous phase is carried until a phase inversion

occurs to form o/w emulsion. Then, the equilibrium between the two

phases is disrupted by adding pure water sufficiently to allow complete

diffusion of acetone into water thereby, spherical nanospheres are

formed by polymer precipitation.145 This techniques permits the usage of

water miscible solvents like acetone and tetrahdyrofuran instead of

traditional chlorinated solvents and also avoids the usage of surfactants

to produce nanospheres. A broad spectrum of polymers including PLA,

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methacrylic acid and co-polymers such as cellulose derivatives can be

employed in this technique.

Controlled Gelation Process :

By changing the pH, temperature or by addition of appropriate

counteriors in the protein or polysaccharides, phase separation is

induced in aqueous solution. In this, nanospheres are produced by

induction of divalent cation, calcium chloride and are stabilized by

polyelectrolytic complexation with a polyamine, poly (L-lysine).

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Direct Precipitation Method :

In this, direct polymer precipitation is induced in an aqueous

medium with or without surfactant by progressive addition of polymer

solution under stirring without emulsification. The solvent is vaporized

under reduced pressure after nanoparticle formation. But completely

miscible solvents with aqueous phase, mainly acetone and also ethanol

and methanol should be only used for precipitation.

3.3. Standardization of Nanoparticles

Particle Size:

Particle size determines the in-Vivo distribution, biological fate,

tonicity and the targeting ability of nanoparticle system and also

influences drug loading, drug release and stability of nanoparticles. Due

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to their smaller size and relative mobility, nanoparticles have relatively

higher intracellular uptake compared to the microparticles.

Desai et al. 146 found that 100 nm particles had a 2.5 fold greater

uptake that 1µm particles and 6 fold greater uptake than 10µm

microparticles in a caco 2 cell line.

Smaller particles have larger surface area where drug associated

would be at or near the particle surface leading to fast drug release.

Hence, the challenge is always to formulate the nanoparticles with

smallest size possible with maximum stability.

Currently, the fastest and most routine method of determination of

particle size is by Photon Correlation Spectroscopy or Dynamic Light

Scattering which requires the viscosity of medium to be known and is the

diameter of the particle determined by Brownian motion147 and Light

Scattering properties.

The results are usually verified by Scanning or Transmission Electron

Microscopy(SEM/TEM)

Scanning Electron Microscopy:

SEM is an instrument that produces largely magnified image by

using electrons instead of light to form an image. Electron gun produces

a beam of electrons which follows the vertical path through the

microscope between electromagnetic fields and lenses towards the

sample due to which electrons and X-rays are ejected from sample.

97

Detectors collect the X-rays, backscatter electrons and secondary

electrons and convert them to signal and produce a final image on

screen.

The water must be removed from sample before the water would

vaporize in the vaccum.

Surface properties of Nanoparticles:

Zeta potential of a nanoparticle reflects the electric potential of particles

and is used to characterize the surface charge properties and to

determine whether the charged particle is encapsulated within the centre

or adsorbed on to the surface of nanocapsule.

Drug loading:

2 methods – 1) Incorporation method

2) Adsorption/Absorption method

It depends on solid state drug solubility in matrix material or polymer.

Macromolecules of protein show greatest loading efficiency when loaded

at a point nearer to its isoelectric point.12 when it has minimum

solubility and maximum absorption.

Drug Release:

The drug release rate depends on:

1) Solubility of drug

2) Desorption of surface bound/adsorbed drug

3) Drug diffusion through matrix

4) Matrix erosion/degradation149

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5) Combination erosion and diffusion process

Methods to study the in-Vitro release of drug are:

a. Side by side diffusion cells

b. Dialysis bag diffusion

c. Reverse dialysis bag technique150

d. Agitation followed by centrifugation

e. Ultrafiltration or centrifugal ultrafiltration technique

3.4. Evaluation of Bio-pharmaceutical Parameters

Pharmacokinetics is defined as kinetics of drug adsorption,

distribution, metabolism and excretion and their relationship with the

pharmacologic, therapeutic or toxicologic response in man and animals.

Pharmacokinetic Parameters:

1) Peak Plasma Concentration Cmax140: Expressed in mcg/ml

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It is also known as Maximum drug concentration or peak height

concentration.

When Cmax is attained by a drug in the plasma, the absorption rate is

equal to the elimination rate of the drug.

We can calculate plasma concentration by using equation,

2) Time of Peak Concentration (Tmax)151: Expressed in hrs. It is

defined as the time taken for a drug to reach the peak concentration in

plasma.

Useful in estimating the rate of absorption.

tpeak = Ιn (Ka/Ke) / (Ka-Ke)

Cp = [ F*Dose*Ka (e-Ket – e-Kat) ] / V* (Ka – Ke)

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3) Area Under the Curve (AUC): Expressed in mg/ml hr. It represents

the total integrated area under the plasma level-time profile and

expresses the total amount of drug that comes into the systemic

circulation after its administration.

Estimation of AUC : 1) By using trapezoidal rule

2) By integration method

4.) Half-Life (140,152): Defined as the time period required for the

concentration of drug to decrease by one half.

Ct = Coe-Kt

Co/2 = Coe-K/2 log Co/2 = log Co – Kt1/2/2.303

For Zero order : t1/2 = Co/2Ko

For First order: t1/2 = 0.693/K

Relationship between the elimination rate constant and t1/2 given by,

K=0.693/t1/2

Other equations used for calculating are, t1/2 = 0.693/K,

T1/2 = 0.693Vd/cl

5.) Absorption Rate Constant: Differential Equations, Drug amount to

be absorbed Xg

Drug in GIT → Drug in patient → Drug eliminated

Xg = F.Dose.e-Kat

Ka is calculated by using,

i) Method of residuals and

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ii) Wagner Nelson Method

6.) Bioavailability16 : It is Defined as the extent or rate at which active

moiety enters systemic circulation thereby, accessing the site of action.

The most reliable measure of a drug‟s bioavailability is by measuring

AUC.

AUC α the total amount of unchanged drug that reaches systemic

circulation

Method of assessment of bioavailability :

Indirect/Pharmacokinetic Methods :

(i) Plasma data

a.) tmax

b.) Cmax

c.) AUC

(ii) Urine data

a.) dxu/dt

b.) xu

c.) tu

F → absolute bioavailability

Fr = [AUC]test * [dose]std / [AUC]std * [dose]test

F relative bioavailability

7.) Apparent Volume of Distribution152 : Expressed in litres

F = [AUC]oral * [dose]iv / [AUC]iv * [dose]oral

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Apparent volume of distribution is the theoretical volume of fluid

into which the total drug administered would have to be diluted to

produce the concentration in plasma.

Given by Vd =

Vd = i.v bolus dose/Co

8.) Total Clearance151 : units of clearance rate clt → volume/unit time

Total clearance is the sum of metabolic clearance, renal clearance, etc. It

is constant for a given drug.

Rate of elimination of a drug from the body is directly proportional

to the plasma concentration.

dx/dt α Cp

dx/dt = clt . Cp

9.) Elimination Rate Constant : It is the sum of all individual rate

constants used to calculate the rate at which drugs are removed from the

system.

It is found by two methods : i) Excretion Rate Method

ii) Sigma Minus Method

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4.Experimental Investigation

4.1.1 MATERIALS USED

1) Ciprofloxacin – procured from the Dr.Reddy‟s Labs, Hyderabad.

2) Amoxicillin – gift sample from Tiny Pharma, Tirupati

3) Ofloxacin-gift sample from Pharmafabrikon,Maduai

4) Ampicillin- gift sample from Pharmafabrikon,Maduai

5) Chitosan – gift sample from the Fisheries College and Research

Institute, Thoothukudi, TamilNadu, India.

6) Sepia officinalis (cuttlefish) ink – procured from fish vendors in

Thoothukudi and authenticated by Dr.R.Santhanam, Professor,

Fisheries College and Research Institute, Thoothukudi, India

7) Hydrochloric acid – Qaligens Fine Chemicals (GLAXOSMITHKLINE

PHARMACEUTICALS, MUMBAI).

8) Sodium acetate - Qaligens Fine Chemicals (GLAXOSMITHKLINE PHARMACEUTICALS MUMBAI).

9) Calcium chloride (Sd Fine Chem Ltd., Mumbai).

10) Peptone – Merck Ltd., Mumbai.

11) Yeast extract - Himedia Laboratories, Mumbai.

12) Beef extract - Himedia Laboratories, Mumbai.

13) Sodium chloride – Sd – Fine Chem Ltd.,Mumbai.

14) Agar-agar Himedia Laboratories, Mumbai

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4.1.2 INSTRUMENTS USED:-

1) UV- double beam spectrophotometer – Analytical Instruments

Pvt.Ltd.

2) Laminar Air Flow (Model No Car 1200 Clean air, Chennai)

3) Centrifuge - (R-24 Remi)

4) Sonicator

5) Dissolution Test apparatus – (TDT – 08L – Electro lab)

6) Scanning Electronic microscope

7) Zetasizer

8) FTIR (Analytical Instrument Pvt .Ltd.

9) Differential Scanning Calorimetry

Experimental animals used:-

1) Healthy Rabbits

Group I – 4Nos.

Group II – 4Nos.

4.2 General Physical Characterization of Sepia officinalis

4.2.1 Organoleptic properties :

Taste, colour and odour are observed using physical appearance..

Many of the properties are observed.

4.2.2 Particle Size analysis :

Eyepiece micrometer was first calibrated by using stage

micrometer which consists of 100 divisions. Each division of stage

105

micrometer = 10µm. A drop of given polymer solution was diluted with

water. The drop spreads over a glass slide and viewed through

microscope. The diameters of particles were measured and average

diameter average particle size was determined.

4.2.3 Bulk Density :

The sample was introduced into a graduated cylinder. Cylinder was

fixed on a table of bulk density apparatus and allowed to tap. The volume

of cylinder was noted and tapped for 500 times. If the subsequent

volume is reduced to some volume, then it is noted as the Bulk Volume.

The Bulk Density is given by mass per bulk volume.

4.2.4 True Density :

First an empty specific gravity bottle was weighed (w1). Then add

some amount of sample was added and it was weighed again(w2). Then w

the weight of specific gravity bottle + drug + water (w3) was observed.

Then the weight of the specific gravity bottle and water (w4) was noted.

True density of the polymer was determined.

4.2.5 Porosity :

25 ml of measuring cylinder was filled with a given powder and the

displacement liquid was added upto 12.5ml mark. All the liquid volume

of the powder was displaced. The flask was then emptied and the same

was filled with water upto 12.5 ml mark and its weight was determined.

Using these readings, bulk density and true density of given powdered

sample were calculated.

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4.2.6 Angle of Repose :

The powder was poured through funnel and its height and radius

were measured.

4.2.7 Viscosity :

Viscometer was washed thoroughly with water and rinsed with

acetone and dried. The liquid was taken with the help of pipette and

interfused into viscometer from the broad end of viscometer. Then it was

sucked from other end. Then the liquid was allowed to flow by removing

the rubber till liquid reaches the mark. The time was noted with the help

of stop clock. After reaching lower mark of bulb.The Stop clock was

stopped. The density of liquid under test was determined.

4.2.8 Surface Tension :

Stalgamometer was cleaned well with water and rinsed with

acetone. A rubber tube was attached with a screw clip at top of

stalganometer to regulate the flow of liquid. The flattened end was dipped

in beaker containing liquid and sucked through rubber tube till the level

reached mark „A‟, when the screw clip was closed. Then the liquid was

allowed to fall from mark „A‟ to mark „B‟. This was repeated for three

times and was filled with test liquid and the no. of drops was counted

from mark „A‟ to mark „B‟.

4.2.9 pH determination :

The pH was determined by using a pH meter

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4.2.10 Melting point :

The melting point of polymer was determined by using melting

point apparatus.

4.2.11 Solubility :

The solubility tests were performed by using different solvents.

4.3 ANALYTICAL METHODS

4.3.1 Estimation of Ciprofloxacin:

A spectroscopic method based on the measurement at extension at

530nm was used for the estimation of ciprofloxacin.

4.3.2 STD – Graph of Ciprofloxacin HCl in 0.1N HCl:

100mg of Ciprofloxacin HCl was first dissolved in little amount of

0.1N HCl and later made up to 100ml by using 0.1N HCl and further

dilutions were made by using 2ml of ferric chloride and 0.1N HCl to

obtain concentration ranging from 10 to 20 mg/ml.

The absorbance of solution was measured at 540nm using ECICO – UV –

Visible Spectrophotometer. The readings obtained is tabulated in Table

5.1. The standard curve of Ciprofloxacin HCl in 0.1N HCl is shown in

Graph 5.1

4.3.3 ESTIMATION OF AMOXICILLIN.

100 mg of Amoxicillin was taken and dissolved in 100ml of sodium

acetate buffer butter so that it contains 1000μg/ml. The latter was

diluted to have the concentrations of 10μg/ml, 20μg/ml, 40μg/ml 80

108

μg/ml, and 100μg/ml. The absorbance was observed under U.V. Visible

Spectrophotometer at 285nm.

4.3.4 ESTIMATION OF AMPICILLIN

The stock solution was prepared by taking 100mg of pure drug of

ofloxacin and dissolved in 100ml of 0.1N Hydrochloric acid to get

1000μg/ml.From the solution of 1000μg/ml,0.1ml.0.2ml,0.4ml,0.8ml

and 10ml samples were taken and diluted upto 10ml in volumetric flask

to get the concentration of 10,20,40,80,100 μg/ml respectively.The

absorbance was measured for the serial dilutios at 268 nm using UV-

Visible spectrophotometer

4.3.5 ESTIMATION OF OFLOXACIN

The stock solution was prepared by taking 100mg of pure drug of

ofloxacin and dissolved in 100ml of 0.1N Hydrochloric acid to get

1000μg/ml.From the solution of

1000μg/ml,0.1ml.0.12ml,0.14ml,0.16ml.0.18,0.20 and 10ml samples

were taken and diluted upto 10ml in a volumetric flask to get the

concentration of 10,12,14,16,18,20 μg/ml respectively.The absorbance

was measured for the serial dilutios at 293 nm using UV-Visible

spectrophotometer