17

1.1 INTRODUCTION, OBJECTIVES AND SCOPE OF THE

INVESTIGATION

A nanoparticle is a particle having one or more dimensions of the

order of 100nm or less. Novel particles that differentiate nanoparticles

form the bulk material typically develop at a critical length scale of under

100nm1.

In nanotechnology, a particle is defined as a small object that

behaves as a whole unit in terms of its transport and properties.

In terms of diameter, fine particles cover a range between 100 and

2500nm, while fine particles on the otherhand are sized between 1 and

1000nm. Nanoparticles may or may not exhibit size related properties

that differ significantly from those observed in fine particles or bulk

materials. (Wikipedia, the free Encyclopedia). Protein nanoparticles are of

gelatin, albumin, gliadin and legumin3.

Nanoparticle research is currently an area of intense scientific

research due to a wide variety of potential applications in biomedical,

optical and electronic fields.

Nanoparticles form an effective bridge between bulk materials and

atomic or molecular structures2. The properties of materials change as

their size approaches the nano sale and as the percentage of atoms of the

surface of a material becomes significant.

Suspensions of nanoparticle are possible because the interaction

of the particle surface with the solvent is strong enough to overcome

18

differences in density which usually result in a material either sinking or

floating in a liquid. Nanoparticles often have unexpected visible

properties because they are small enough to confine their electrons and

produce quantum effects4.

Nanoparticles have a very high surface area to volume ratio. This

provides a tremendous driving force for diffusion especially at elevated

temperatures. The large surface area to volume ratio also reduces the

incipient melting temperatures of nanoparticles.

The unique size dependent properties of nanomaterials make them

very attractive for pharmaceutical applications. Cytotoxic effects of

certain engineered nanomaterials towards malignant cells form the basis

for nanomedicine. It is inferred that size, three dimensional shape,

hydrophobicity and electronic configuration make nanoparticles an

appealing subject in medicinal chemistry. The unique structure of

nanoparticles coupled with immense shape for derivatization forms a

base for exciting developments in therapeutics. Solid Lipid Nanoparticles

(SLN) forms an alternative colloidal carrier system for controlled drug

delivery. Because of their versatility and wide range of properties,

biodegradable polymeric nanoparticles are being used as novel drug

delivery systems. Further, this class of carrier holds tremendous

promise in the areas of cancer therapy and controlled delivery of vaccines

19

1.1.2 Classification:

Nanoparticles are often referred to cluster, nanospheres, nano –

rods, nano – fibers and nano – caps. Nanoparticles are made of semi

conducting materials may also be labeled quantum dots if they are small

enough (typically about 10nm) their quantization electronic energy level

occurs. Such nanoparticles are used in biomedical applications as drug

carriers or imaging agents

A prototype – nanoparticle of semi – solid nature is the liposome.

Various types of liposome nanoparticles are currently used clinically as

delivery systems for anti – cancer drugs and vaccines.

1.1.3 Characterisation:

It is done by Transmission Electron Microscopy/Scanning Electron

Microscopy, (TEM/SEM), Atomic Force Microscopy (AFM), Dynamic Light

Scattering (DLS), X-ray Photoelectron Spectroscopy (XPS), Powder X-ray

Diffractometry (XRD), Fourier Transform Infrared spectroscopy (FTIR),

Matrix Assisted Laser. Desorption Time of Flight mass spectrometry

(MALD –TOF) and Ultra – Violet Visible spectroscopy

1.2 CONTROLLED DRUG DELIVERY SYSTEMS6

1.2.1 INTRODUCTION:

The controlled drug delivery systems are gaining greater attention

in recent years owing to their importance and manifold advantages.

These systems are designed to release one or more drugs continuously in

a predetermined pattern for a fixed period of time either systematically or

20

to a specified target organ. Drug release from these systems should be at

a designed predictable reproduceable rate. By employing this system, the

safety, improved efficiency of drugs and patients compliance could be

assumed. Through, better control of plasma drug level and less frequent

dosing, the objectives of controlled drug delivery system can be fully

achieved. Though, these systems have been designed for oral, parenteral,

implantations and transdermal routes, oral routes are considered to be

the most convenient and common modes of administration Oral route

includes systems in the form of coated pellets, matrix tablets, poorly

soluble drug complexes and ion exchange resin complexes. Osmotic

preparations are known to release drug over an extended period of time

either in a continuous manner (sustained release) or as a series of pulses

(timed release). Among the various approaches, microencapsulation and

microcapsules have been accepted as reliable methods to achieve

controlled release.

1.2.2 MICROENCAPSUALTION

It is a process in which small, discrete solid particles or liquid

droplets are surrounded and enclosed by an intact shell and the

resulting materials are microcapsules. The capsule shells can be

designed to release their contents at specific rates under specific set of

conditions. Though, a variety of wall materials are used for the above,

polymeric substances having film forming properties are most suited for

microencapsulation.

21

1.3 AMOXICILLIN 7, 8

Heptane – 2 carboxylic acid. 6 [(Amino (4-hydroxyphenyl) acetyl)]

Amino – 3.3- dimethyl -7oxo-trihydrate.

D (-) - Amino –p-hydroxy benzyl Penicillin C16 H19 N3 O5 S. 3H2O

Preparation: - By Acetylation of 6 – Amino penicillanic acid with D (-) – 2

– (p–hydroxylphenyl glycine)

Properties: The solubility is at 1g in 370 ml water and 2000 ml alcohol.

It is a fine white to off-white crystalline powder with bitter taste. High

humidity and temperatures over 37ºC adversely affects its stability.

By the oral route, 75-90% is absorbed. An oral dose of 250mg will

provide a peak plasma concentration of about 4µg/ml. 50-72% is

eliminated by renal tubular secretion. The half life is about 1hr when

renal function is normal and 8-16hr in renal failure.

Uses: It is chemically p.hydroxyampicllin and has an antibacterial

spectrum similar to that of ampicillin drug except that it is less active

against Clostridium, Salmonella, Streptococcus and Shigella. Like

ampicillin, it is destroyed by β – lactamases and hence, it cannot be used

to treat infections caused by resistantstrain in bacteria of the β –

22

lactamase producing typhi. It cannot be given parent orally. It is the drug

of choice for infections by penicillinase producing Staphylococcus.

1.4 CIPROFLOXACIN HYDROCHLORIDE9, 10

3-Quinoline carboxylic acid

1-cyclopropyl – 6 fluro-1, 4 dihydro-4-oxo-7–(1 – piperazinyl) monohydro

chloride, monohydrate

C17H18FN3O3·HCl·H2O 385.82

Preparation: It is a pale yellow amphoteric crystal prepared from 3-

chloro-4-fluroamiline by condensation with ethyl ethoxy methylene

malonate to form the imine which is thermocycized to ethyl 7-chloro-6-

fluro-4 hydroxyguinoline-3-carboxyl-N-alkylation with cyclopropyl iodide

followed by nucleophilic displacement of the 7 chlorogroup by N-methyl

piperazine and hydrolysis of the ester affords the product.

Properties: It is soluble at 1g in 25ml water. The oral bioavailability is

about 70-80%. A dose of 0.5g yields plasma concentration 12hrs after

administration of about 0.2μg.Urinary excretion accounts for the

elimination of 40-50% of the dose. 20-35% is eliminated in feces. There

is hepatic biotransformation of four known metabolites which account for

15%. The halflife is about 4hrs.

23

Uses: It is used in the treatment of bone and joint infections caused by

certain microbes. Further, it is an unlabelled but authoritatively

alternate drug for the treatment of gonorrhea and salmonella infections.

The molecular weight value of ciprofloxacin-loaded PEBCA

nanoparticles was shown to be reduced as compared with unloaded

nanoparticles. Drug release from the colloidal carrier in medium

containing esterase was found to be very slow (a maximum of 51.5% after

48hrs) suggesting that this release resulted from bioerosion of the

polymer matrix. F-NMR analysis demonstrated that ciprofloxacin

entrapped into nanoparticles was only in its neutral form. Ciprofloxacin

HCl-loaded nanoparticles of chitosan, lipid, (SLNs) albumin and gelatin

showed sustained drug release avoiding burst effect of the free drugs.

Further, ciprofloxacin nanoparticles and SLNs can act as promising

carriers for sustained ciprofloxacin release.

AMPICILLIN

C16H14N3O4S (6R)-6(alpha phenyl-D-glycyl amino) pencillanic acid

It is an antibacterial agent,effective against various types of

bacteria.The daily dose is 2-6 g in frequent intervals.It is a crystalline

white substance sparingly soluable in water but insoluable in

24

ethanol,chloroform and fixed oils; soluable in acids and alkali

hydroxides.It should be stored in well closed containers in a cool and dry

place

OFLOXACIN

Ofloxacin inhibits an enzyme called DNA gyrase that is an essential

component of the mechanism that passes genetic information onto

daughter cells when a cell divides.

9-fluoro-2,3-dihydro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo-

7H-pyrido[1,2,3-de]-1,4-benzoxazine-6-carboxylic acid hemihydrate

1.5 Sepia officinalis – A SOURCE OF NATURAL POLYMER OF DRUG

DELIVERY106, 107, 108

Sepia melanins are negatively charged pigments that are

hydrophobic, containing phenolic or indolic compounds.

These melanins are of the following types

Eumelanins – black or brown in colour

Pheomelanin – yellow or reddish in colour

Pyomelanin – brownish in colour

25

Sepia melanins are dark in colour and they are used in the

preparation of UV-absorbing optical lenses and in cosmetic

creams.

They are conductive to electricity.

Melanins are mainly used in pharmaceutical formulations and

drug delivery systems in nanotechnology.

In human physiology, melanins play main role in imparting

pigmentation to hair, skin and eyes, as a free radical scavenger

and increases the speed of nerve and brain messages.

Melanins are synthesized by free-living microbes, even facultative

microbes like “Cryptococcus neoformans”8 in soils. Melanin

production in these offers an advantage of survival from

environmental predators which produce hydrolytic enzymes. It is

due to sequestration of enzymes on melanin or stearic hindrance.

Melanins offer protection from UV-light and prevent photoinduced

damage.

1.5.1 Evidence for melanins bind to drugs in-vitro

(i) Isotherm analysis of adsorption of drugs by melanin:

Binding of Gentamicin, Methotrexate and Chlorpromazine to melanins

is explained by Isotherm binding equations to characterize the

adsorption of drugs to synthetic and sepia melanins.

Best fit Freundlich equation for Gentamicin6.

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[q = qo (KC) 1/n] dm3.g-1

where, q = amount absorbed (m.mol.g-1)

qo = adsorption capacity

K = energy of absorption

C = equilibrium solution concentration of solute and

heterogeneity index 1/n (between 0 and 1)

(ii) Scatchard plot analysis of drug binding by melanin:

This method involves usage of radio-labelled compounds to

demonstrate the presence of heterologous binding sites.

Aminoglycoside antibiotics like Gentamicin and Kanamycin7 have

„2‟ binding sites on synthetic DOPA melanin.

For Kanamycin, association constants for strong and weak binding

sites were 3 X 10-5 and 4 X 10-3 m-1 respectively.

0.64µm Kanamycin is required to saturate binding sites in 1 mg

melanin.

Scatchard plot type analysis with melanins reveals that high

and low affinity binding sites for cocaine, amphetamines and

anti-arrythmics quinidine, disopyramide and metoprolol.

1.5.2 Absorption studies with Anti-fungals:

Amphotecin-B and caspofungin bind to melanin which uses 2

methods. They are

27

The melanin produced by C.neoplasms and synthetic melanin to bind

to these anti-fungal drugs was interfered from experiments,

incubating melanins with various compounds and anti-fungal activity

of solution was determined.

Testing of anti-fungal solutions in MIC and time-kill studies were

performed by removing melanin particles by centrifugation and

testing.

1.5.3 Binding of compounds by melanin in human’s in-vivo

Binding of drugs to host melanin damages certain tissues and causes

pathogenecity. For example in Parkinson‟s disease, there is a loss of

pigment in melanonic dopaminergic neurons in substantia nigra of

the brain.

In Parkinson‟s disease, 1-methyl 4-phenyl 1, 2, 3, 6 - tetrahydro

pyridine (MPTP)9 caused damage of substantia nigra neurons which

are concentrated with melanin.

Phenothiazines caused parkinsonian symptoms and secondary which

are reversible. The specific retention of other drugs which concentrate

at the pigmented tissues causes the damage of cells like skin, eye and

inner eye.

The complex interactions depend on diverse factors like cysteine

content, pH and ionic Interactions7.

Chloroquine accumulates in dermal melanocytes and hair follicles

where it causes irreversible hearing loss, tinnitus and dizziness.

28

Hearing loss is due to effect on 8th cranial nerve. Quinine accumulates

in melanin in the Stria nascularis of cochlea and causes cellular

degeneration.

Aminoglycosides8 become positively charged at the physiological

pH. Because of its high molecular weight, its penetration in the

tissues is reduced. Further, administration of this drug can cause

permanent vestibular and auditory ototoxicity.

Aminoglycosides when administered as intravitreal injection,

caused ocular pigmentation can partially protect retina turn damage.

Thioureylenes when incorporated into melanin like propylinic

uracil cause a loss or depigmentation of hair.

Ravuconazole, which is similar to voriconazole is effective against

Aspergillus fumigates and Aspergillus flavus.

1.5.4 Cuttlefish ink (sepia)

Cuttlefishes are the ink producing marine invertebrates and they

belong to the Phylum Mollusca and Class Cephalopoda which include

similar ink producing animals such as octopus and squid. The

cuttlefishes are soft bodied swimming animals provided with a large head

ringed by tentacles and an internal cuttle bone made of chiefly calcium

carbonate. These animals possess an ink pouch (sac) in which, a

brownish black fluid called „sepia‟ secreted by them is stored.

Cuttlefishes are known to display natural camouflage. In order to escape

from predators at the times of emergency, cuttlefishes darken the

29

environment by ejecting a gelatinous and mildly narcotic dark brown ink

to stun attackers and this defensive response gives them time to escape.

Further, the melanin particles of sepia are easily miscible in sea

water and remain dispersed in solution for more than 14 days.

Factors controlling ink production: The ink production and ejection in

cuttlefishes are affected and modulated by N-methyl-d-aspartate (NMDA)

- nitric oxide (NO)-cyclic GMP (cGMP) signaling pathway, Glutamate

NDMA receptor and NO synthase, the enzyme which is responsible for

the synthesis of NO has been detected in immature ink gland cells.

1.5.5 Extraction of sepia

The crude ink obtained from the ink sac is boiled with caustic

soda, filtering the extract and then adding HCl for precipitating the

colouring matter. The liquid ink may also be dried by combining with

lactose and then ground.

Characteristics of sepia: The liquid of cuttlefish ink has a grainy texture

and is alkaloid. Hence, it is not preferred by predators especially fish.

The ink is not poisonous and acts solely as a decoy device. The main

constituents of the ink are melanin and mucus. Melanin is a natural

melanoprotein containing 10 – 15% protein. The melanin binding protein

through aminoacid containing sulphur which is sistein.

The ink gland contains a variety of melanogenic enzymes including

tyrosinase, a peculiar dopachrome rearranging enzyme (which catalyses

the rearrangement of dopachrome to 5, 6 – dihydroxyindole) and a

30

peroxidase presumably involved in later stages of melanin biosynthesis.

The ink is also believed to contain dopamine and L-DoPA and small

amounts of aminoacids, including taurine, aspartic acid, glutamic acid,

alamine and lysine.

Human use: While the flesh of cuttlefish is used as a food source, its ink

finds applications in food colouring and in the preparation of pastries

and sauces. As an important dye, cuttlefish ink has been used for

centuries by humans for writing, drawing and in photographic works.

Sepia ink is available in Italy as hard dark chips. These are smelly

and hard to grind small enough to form an ink. They do not dissolve

readily in water. Mixing sepia powder with gum Arabic water to make

little cakes letting them dry and rubbing them up water when an ink is

needed.

1.5.6 Protective and therapeutic uses:

Sepia is a long standing homeopathic remedy for females because

it is effective for all menstrual and menopausal complaints. It also helps

combat persistent sadness and depression. That is sepia can lift the

mood of melancholy people urging them to take a more positive approach

to their lives. Vaginal discharge and even severe pain from endometriosis,

the growth of uterine cells in the abdominal cavity may be greatly

relieved by sepia. Migraines, liver weakness constipation, hair loss,

exhaustion and poor circulation with its resulting chillness can also be

treated with sepia remedy.

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Sepia is known to soothe disturbances of the metabolism and ANS.

It also helps restore hormonal balance in women positively affecting

uterus and ovaries. Further, sepia improves blood circulation in the

organs especially those in abdominal cavity.

1.5.7 Bioactive properties:

The bioactive properties of ink gland of cuttlefish have been

studied for antibacterial, antiviral and anticancer agents. Purified cuttle

fish ink with a mixture containing mainly of a conjugated glucide (in

which agar, protein and lipid units are combined) ink may be effective in

fighting cancer. It was tested on 15 mice which were implanted with

tumours. The compound present in ink works by activating

macrophages, a type of WBC near the site of tumour. This would

increase the body‟s immune response to the tumour cells rather than

fighting the cancer cells directly.

Cytotoxicity: An uronic acid with rich peptidolglycon isolated from the

ink of cuttlefish Sepia pharaonis showed cytotoxicity against human

cervical cancer.

1.5.8 Radio-protective effect:

Irradiation leads to immunosuppression, haemopoiesis injury as

well as subhealth of human being. The protective and therapeutic effects

of cuttlefish ink on haemopoiesis in 60 Co gamma radiated model mice

were investigated. The results showed that the cuttlefish ink showed

significant effect on granulopoiesis. It is suggested that the increases of

32

antioxidant level in mice, the improvement of bone marrow

haematopoietic micro environment and the inducement of cellular

factors promoted the proliferation and differentiation of CFU–S (colony

forming unit in spleen) and CFU–GM (colony forming unit of granulocyte

and monocyte) and thus enhance the defensive system of organism.

1.6 CHITOSAN11, 12, 13

It occurs naturally in fungi, yeasts, marine invertebrates and arthropods.

Chitosan is the principal component of exoskeletons of marine

crustaceans from which supplements are often derived.

SYNONYMS: Chitosan hydrochloride or 2-Amino–2-deoxy-(1, 4) – β – D-

gluco pyranosoamine or β-1, 4 – poly-D-glucosamine or poly – (1, 4 – β –

D – gluco pyranosoamine).

Chemical name: Poly-β-(1, 4) – 2 – Amino – 2 – deoxy – D – Glucose.

Empirical Formula

Partial deacetylation of chitin results in the production of chitin

which is a polysaccharide comprising copolymers of glucosamine and N-

acetylglucosamine. The degree of deacetylation necessary to obtain a

soluble product must be greater than 80-85%. Chitosan is available with

different molecular weights (10000 to 1000000).

33

Structural Formula:

Uses: Chitosan is widely used as an excipient in oral and other

pharmaceutical formulations. It is used as a coating agent, disintegrant,

film-forming agent, mucoadhesive, tablet binder and viscosity increasing

agent.

1.6.1 Application in Pharmaceutical Formulations:

The suitability and performance of chitosan for drug delivery

applications have been investigated. It is used in controlled drug delivery

application as a component mucoadhesive dosage forms and rapid

release dosage forms in improved peptide delivery and for gene delivery.

Chitosan has been processed into several pharmaceutical forms

including gels, films, microspheres tablets and coatings for liposome.

Furthermore, chitosan may be processed into drug delivery systems

using several techniques including spray drying, coacervation, direct

compression and conventional granulation processes.

Although, the carriers are of the same size (200nm), drug loading

capacity of chitosan is 20 times higher for nanoparticle than for

liposome. Polysaccharide based nanoparticles of chitosan are prepared

34

by covalent cross linking, ionic cross linking polyelectrolyte complex and

the self assembly hydrophobically modified polysaccharides. Chitosan is

non-toxic, biocompatible and biodegradable and these properties make

chitosan a good candidate for conventional and novel drug delivery

systems. Chitosan forms colloidal particles and entraps bioactive

molecules through a number of mechanisms including chemical cross

linking, ionic cross linking and ionic complexation. Because of high

affinity of chitosan for cell membrane, it has been used as a coating

agent for liposome formulations. Chitosan is only soluble in acidic

solution with <pH6 and loses its change in >pH6. Therefore, it will be

insoluble in aqueous media. Synthesis of quaternary derivatives of

chitosan to improve solubility in wide pH range for increasing its

potential as an enhancer has been investigated. A number of factors

such as degree of polymerization, level of deacetylation, types of

quarternisation, installation of various hydrophobic substances, metal

complexation and combination with other agents influence the structure

characteristics of chitosan. Biodegradable, non-toxic and non-allergic

nature of chitosan encourages its potential use as a carrier for drug

delivery systems in all targets.

1.6.2 Properties:

Chitosan occurs as odourless white or creamy powder or flakes.

Fibre formation is quite common during precipitation and the chitosan

35

may look „cotton like‟. Chitosan is a cationic polyamine with a high

change density at pH <6.5. It is a linear polyelectrolyte with reactive

hydroxy and aminogroups. The presence of a number of aminogroups

allows chitosan to react chemically with anionic systems which results in

alteration of physico – chemical characteristics of such combinations.

The nitrogen in chitosan is mostly in the form of primary aliphatic

aminogroups. Chitosan therefore, undergoes reactions typical of amines.

All functional properties of chitosan depend on the chain length, chain

density and charge distribution. Further, salt form, molecular weight,

degree of deacetylation and pH are known to influence chitosan in

pharmaceutical applications. Particle size distribution is < 30μm.

Chitosan is sparingly soluble in water and is practically insoluble in

ethanol (95%), other organic solvents and neutral or alkali solution at pH

> 6.5. Chitosan dissolves readily in dilute and concentrated organic

acids and to some extent in inorganic acids (except phosphoric and

sulphuric acids). Upon dissolution, aminogroups of the polymer become

protonated resulting in a positively charged polysaccharides and

chitosan salts (chloride, glutamate, etc.) that are soluble in water.

Solubility of chitosan is affected by the degree of deacetylation. Solubility

is also greatly influenced by the addition of salt to the solution.

36

1.6.3 Stability and storage:

Chitosan powder is a stable material at room temperature

although, it is hygroscopic after drying. Hence, it should the stored at a

temperature of 2 - 8ºC.

1.6.4 Preparation:

Chitosan is prepared by chemically treating the shells of

crustaceans such as shrimps and crabs. The basic preparatory process

involves the removal of protein by treatment with alkali and of minerals

such as calcium carbonate and calcium phosphate by treatment with

acid. Before these treatments, the shells are ground to make them more

accessible. The shells are initially deproteinized by treatment with an

aqueous sodium hydroxide 3.5% solution. The resulting product is

neutralized and calcium is removed by aqueous HCl 3.5% solution at

room temperature to precipitate chitin. The chitin is dried so that, it can

be stored as a stable intermediate for deacetylation to chitosan at a latter

stage. N-deacetylation of chitin is achieved by treatment with an

aqueous sodium hydroxide (40-45%) solution at elevated temperature

(110ºC) and the precipitate is washed with water.

The crude sample is dissolved in 2% acetic and the insoluble

material is removed. The resulting clear supernatant solution is

neutralized with an aqueous sodium hydroxide solution to give a purified

white precipitate of chitosan. The product can then be further purified

and ground to a fine powder or granules.

37

Chitosan, the deacetylated polymer of N-acetyl-D-glucosamine (chitin)

is water soluble and chemically similar to cellulose.

1.6.5 Pharmaceutical Uses:

Chitosan is believed to affect cholesterol levels and weight

because it has positively charged aminogroups at the same pH as the

gastrointestinal tract. These aminogroups are believed to bind to

negatively charged molecules such as lipids and bile preventing their

absorption and storage by the body. The action of chitosan in cholesterol

management may be explained by the theory that ingested chitosan salts

react with fatty acids and binds lipids because of hydrophobic

interactions; these bound lipids are extracted rather than absorbed.

Animal studies in rats, mice and chickens indicate that chitosan

decreases very low density lipoprotein-cholesterol levels while increasing

high density-lipoprotein (HDL)-cholesterol levels. In vitro studies have

also shown that O-carboxy methyl chitosan beads absorb low-density

lipoprotein (LDL) cholesterol.

Chitosan acts as a „Fat Blocker‟. Chitosan is the only edible fibrin

with positive charge in nature. The resulting molecule called chitosan -

fat polymer is too large to be absorbed through the intestinal wall and

therefore excreted via feces without digestion.

38

1.7 Biopharmaceutics:

It deals with the inter-relationships of physicochemical properties of

the drug in dosage form in which the drug is given and the route of

administration on the rate and extent of systemic drug absorption. The

factors which influence biopharmaceutics include:

(i) protection of the activity of the drug within the drug product;

(ii) the release of the drug from a drug product;

(iii) the rate of dissolution of the drug at the absorption site and

(iv) the systemic absorption of drug.

The dynamic relationships existing in biopharmaceutics are shown

hereunder.

Studies in biopharmaceutics use both in-vitro and in-vivo methods. In-

vitro methods are procedures employing test approaches and equipments

without involving laboratory animals and humans. In-vivo methods on

the otherhand involve human subjects and laboratory animals14

39

1.7.1 Pharmacokinetics:

It involves the kinetics of drug absorption, distribution and

elimination (i.e. excretion and metabolism). The drug distribution and

elimination are together often termed as „drug disposition‟. The study of

pharmacokinetics involves both experimental and theoretical

approaches. The experimental aspects of pharmacokinetics involve the

development of biological sampling techniques, analytical methods for

the measurement of drugs and metabolites, the procedures that facilitate

data collection and manipulation. The theoretical aspect of

pharmacokinetics involves the development of pharmacokinetic models

that predict drug disposition after drug administration.

1.7.2 Bioavailability:

It refers to the measurement of the rate and extent of active drug that

reaches the systemic circulation and is available at the site of action.

Physicochemical characteristics of the drug:

The physicochemical properties of the solid drug particles not only

affect dissolution kinetics, but are important considerations in designing

the dosage form.

Solubility, pH and drug absorption:

The solubility – pH profile is a plot of the solubility of drug at

different pH values. While a basic drug is more soluble in acidic medium

forming a soluble salt, an acid drug is more soluble in the intestine

forming a soluble salt at more alkaline pH. The solubility pH profile gives

40

a rough estimation of the completeness of dissolution for a dose of drug

in the stomach or in intestine. Solubility may be improved with the

addition of an acidic / basic excipient15.

Stability, pH and drug absorption:

The pH – stability profile is a plot of the reaction rate constant for

drug degradation versus pH. If drug decomposition occurs by an acid or

base catalysis, some precision of the degradation of the drug in the

gastrointestinal tract may be made.

Particle size and drug absorption:

The effective surface area of the drug is measured enormously by a

reduction in the particle size. Because dissolution takes place at the

surface of solute (drug), the greater surface area the more rapid the rate

of drug dissolution. The geometric shape of the particle also affects the

surface area and during dissolution, the surface is constantly changing.

Particle size and particle size distribution studies are important for

drugs that have low water solubility. Many hydrophilic drugs are very

active intravenously but are not very effective when given orally due to

poor absorption. Smaller particle size results in an increase in the total

surface area of the particles thus enhancing water penetration into the

particles and increases the dissolution rates.

Polymorphic crystals, solvates and drug absorption: 16

Polymorphism refers to the arrangement of a drug in various

crystal forms or polymorphs which have the same chemical structure,

41

but different physical properties such as solubility, density, hardness,

and compression characteristics. Some polymorphic crystals have much

lower aqueous solubility than the amorphous forms causing a product to

incompletely absorb. A drug that exists as an amorphous form generally

dissolves more rapidly than the same drug in a more structurally rigid

crystalline form. Some polymorphs are metastable and may get converted

into more stable forms overtime.

Polymeric drugs:

Polymers have been used to prolong drug release in controlled

release dosage forms. The basic components of site-specific polymer

carriers are:

(i) The polymeric backbone,

(ii) A site specific component for recognizing the target (horning

device),

(iii) The drug covalently attached to the polymer chain and

(iv) Functional chains to enhance the physical characteristics of the

carrier system.

The molecular weight of the polymer carrier is an important

consideration in designing the dosage forms. Generally large molecular

weight polymers have longer residence time and diffuse more slowly.

Insoluble polymers are used either as regular carriers or formulated into

microparticles and nanoparticles.

42

Polymeric backbone

1.7.3 Bioavailability17:

These studies are performed for both approved active drug

ingredients and therapeutic moieties not yet approved for marketing by

FDA. Further, these studies are used to define the effect of changes in

the physicochemical properties of drug substance and the effect of the

drug product (dosage form) on the pharmacokinetics of the drug.

Relative and Absolute availability: The area under the drug

concentration-time curve (AUC) is used as a measure of the total amount

of drug that reaches the systemic circulation. The AUC is dependent on

the total quantity of available drug FDo divided by elimination rate

constant „K‟ and the apparent volume of distribution VD. F is the fraction

of the dose absorbed. After IV administration, F is equal to unity because

the entire dose is placed into systemic circulation. Therefore, the drug is

considered to be completely available after IV administration. After oral

administration of the drug, F may vary from 0(no drug absorption) to

1(complete drug absorption).

43

Relative Availability (Apparent availability):

It is the availability of a drug from its product as compared to a

recognized standard. The availability of drug in the formulation is

compared to the availability of the drug in a standard dosage

formulation, usually a solution of the pure drug evaluated in a crossover

study. The relative availability of two drug products gives at the same

dosage level and by the same route of administration can be obtained

with the following equation.

Relative availability = (AUC)A_

(AUC)B

where drug product B is the recognized reference standard. This fraction

may be multiplied by 100 to give percent relative availability. When

different doses are administrated, a correction for the size of dose is

made as in the following equation.

Relative availability = (AUC) A/dose A

(AUC)B/dose B

Urinary drug excretion data may also be used to measure relative

availability, as long as the total amount of the intact drug, excreted in

the urine is collected. The percent relative availability using urinary

excretion data can be determined as follows:

Percent relative availability = (Du) (Ax) X 100

(Du) (Bx)

Here (Du) is the total amount of drug excreted in the urine

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Absolute availability:

The absolute availability of the drug is the systemic availability of a

drug after extravascular administration (eg. oral, rectal, transdermal and

subcutaneous). The absolute availability of a drug is generally measured

by comparing the respective AUCs after extravascular and IV

administration. This measurement may be performed as long as VD and

K are independent of the route of administration. Absolute availability

after oral drug administration using plasma data can be determined as

follows:

Absolute availability = (AUC) po /dose po = F

(AUC) IV/ dose IV Z

Absolute availability using urinary drug excretion data can be

determined by the following:

Absolute availability = (Du)x po/dosepo

(Du)x po/doseIV

1.7.4 EVALUATION OF IN-VIVO BIOAVAILABILITY DATA:

A properly designed bioavailability study is performed in-vivo. The

data are then evaluated using both pharmacokinetic and statistical

analysis methods. The evaluation may include a pharmacokinetic profile,

steady – state plasma drug concentrations, rate of drug absorption

occupancy time and statistical evaluation of the pharmacokinetic

parameters.

45

Pharmacokinetic Profile: Plasma drug concentrations versus time

curve define the bioavailability of the drug from the dosage form. The

bioavailability data should include a profile of the fraction of a drug

absorbed and it should rule out dose dumping or lack of a significant

food effect. The bioavailability data should also demonstrate the

controlled -release characteristics of the dosage form compared to the

reference or immediate release drug products.

Steady -state plasma drug concentration:

The fluctuation between the C∞max (peak) and C∞min (trough)

concentration may be calculated as follows.

Fluctuation = C∞max - C∞min

C∞av

where C∞av is equal to (AUC)/T

An ideal extended release dosage form should have minimum

fluctuations between Cmax and Cmin. A true zero-order release will have no

fluctuations. In practice, the fluctuation in plasma drug levels after the

extended release dosage form should be less than the fluctuation after

the same drug given more immediate release dosage

Rate of drug absorption:

The rate of drug absorption from the conventional or immediate

release dosage form is generally of first order, whereas, the drug

absorption after the extended release dosage form may be zero order,

first order or an intermediate order. For many controlled release dosage

46

forms, the rate of drug absorption is of first order with an absorption rate

constant Ka which is smaller than the elimination rate constant k the

pharmacokinetic models when ka<k is termed flip–flop

pharmacokinetics.

Occupancy Time: For drugs for which the therapeutic window is known,

the plasma drug concentrations should be maintained above the

minimum effective drug concentration (MEC) and below the minimum

toxic drug concentration (MTC). The time required for the maintenance of

the plasma drug levels within the therapeutic window is known as

occupancy time

1.7.5 Bioequivalence Studies: 18

Bioequivalent drug products that have the same systemic drug

bioavailability will have the same predictable drug response. However,

variable clinical responses among individuals that are unrelated to

bioavailability may be due to differences in the pharmacodynamics of the

drug. Differences in pharmacodynamics i.e. the relationship between

drug and receptor site may be due to difference in receptor sensitivity to

the drug. Bioequivalence is established if the in-vivo bioavailability of a

test drug product does not differ significantly in the product‟s rate and

extent of drug absorption. A drug product that differs from the reference

material in its rate of absorption, but not in it‟s extent of absorption may

be considered bioavailable if the difference in the rate absorption is

intentional and appropriately reflected in the labeling and the rate of

47

absorption is not detrimental to the safety and effectiveness of the drug

product.

1.7.6 Statistical Evaluation:

Variables subjected to statistical analysis generally include plasma

drug concentrations at each collection time, AUC (from zero to last

sampling time), AUC (from zero to infinity), Cmax, tmax and elimination half

life t1/2. Statistical testing may include an analysis of variance (ANOVA)

computation of 90% and 95% confidence intervals on the difference in

formulation means and the power of ANOVA to detect a 20% difference

from the reference mean

1.7.7 Pharmacokinetics of oral absorption: 19

The systemic absorption of a drug from the G.I. tract or any other

extravascular site is dependent on the physicochemical properties of the

drug, the dosage form, and the anatomy and physiology of absorption

site. Further, surface area of gut, stomach emptying rate, G.I mobility

and blood flow to the absorption site may affect the rate and extent of

drug absorption. The overall rate of drug absorption may be described

mathematically as a first order or zero order input process. Most

pharmacokinetic models assume first order absorption unless an

assumption of zero order absorption improves the model significantly

and it has been verified experimentally.

The rate of change in the amount of drug in the body dDB/dt is

dependent on the rates of drug absorption and elimination.

48

The rate of drug accumulation in the body at any time is equal to

the rate of drug absorption less the rate of drug elimination.

dDB = dDGI - dDe

dt dt dt

During the absorption phase of a plasma level time curve, the rate of

drug absorption is greater than the rate of drug elimination.

dDGI > dDe

dt dt

At the time of peak drug concentration in the plasma which corresponds

to the time of peak absorption, the rate of drug absorption just equals

the rate drug elimination and there is no change in the amount of drug

in the body.

dDGI = dDe dt dt

1.7.8 Model of drug absorption and elimination:

Immediately after the time of peak drug absorption, some drug

may still be at the absorption site (i.e., in the GI tract). However, the rate

of drug elimination at this time would be faster than the rate of

absorption

49

dDaI < dDe

dt dt

When the drug at the absorption site becomes depleted, the rate of

drug absorption approaches zero or of DGI/dt =0. The elimination phase

of the curve then represents only the elimination of drug from the body

usually a first order process. Therefore, during the elimination phase, the

rate of change in the amount of drug in the body is described as a first

order process.

dDB = -kDB

dt where, k is the first order elimination rate constant

Zero – order absorption Model:

In this model drug in the GI tract DGI is absorbed systemically at a

constant rate ko. Drug is eliminated from the body by a first order rate

process with a first order rate constant k.

The rate of elimination at any time by first order process is equal to

DBk. The rate of input is ko. Therefore, the change per unit time in the

body can be expressed as

dDB = ko - kDB

dt

DGI

ko

DBVD

ko

50

One compartment model for zero – order drug absorption and first order

drug elimination

Integration of this equation with substitution of VD Cp for DB produces.

Cp = ko (1- e–kt)

VDk

The rate of drug absorption is constant and it continues until the

entire amount of drug in gut DGI is depleted. The time at which drug

absorption is continuous is equal to DGI/ko. After this time, the drug is

no longer available for absorption from the gut. The drug concentration

in the plasma will decline in accordance with first order elimination rate

process.

First order absorption model:

This model assumes a first order impact across the gut wall and

first order elimination from the body. This model applies mostly to the

oral absorption of drugs in solution or rapidly dissolving dosage

(immediate release) forms such as tablets, capsules and suppositories. In

addition, drugs given by intramuscular aqueous injections may also be

described using a first order process.

After administration, the drug is absorbed from the absorption site

by a first order process. In the case of a drug given orally, the drug

dissolves in the fluids of GI tract and is absorbed into the body according

to a first order process. The rate of disappearance of drug from the GI

tract is described by the following

51

dDGI = ka DGI F

dt

where, ka is the first order absorption constant from GI tract, F is

the fraction absorbed and DGI is the amount of drug in solution in GI

tract at anytime.

Integration of the above differential equation gives

DGI = Doe-kat

where, Do is the dose of drug. The rate of drug elimination is described

by a first order rate process for most drugs and is equal to -kDB. The rate

of drug change in the body dDB/dt is therefore the rate of drug in, minus

the rate of drug out as given by the following differential equation.

dDB = Rate in – Rate out dt

dDB/dt=Fka

where, F is the fraction of drug systemically absorbed

1.7.9 One compartment model for first order absorption and first

order elimination:20

F may vary from 1 for a fully absorbed drug to zero for a drug

completely unabsorbed. The maximum concentration is cmax and the

time needed to reach maximum concentration is tmax. The time needed to

reach maximum concentration is independent of dose and is dependent

on the rate constants for absorption (ka) and elimination k.

52

tmax = Inka – Ink = In (Ka/k)

ska – k ka – k

The time for maximum drug concentration tmax is dependent only

on the rate constants ka and k. The rate of drug excretion after a single

oral dose of drug is given with the following formula

dDu = Fke kaDo

dt

where, dDu/dt = rate of urinary drug excretion

K = fraction of dose absorbed

F = first order renal excretion constant

1.8 Biopharmaceutic considerations:

The prime considerations in the design of a drug product are safety

and efficiency. The drug product must effectively relieve the active drug

at an appropriate rate and amount to the targeted site, so that, the

intended therapeutic effect is achieved. The finished dosage form should

not produce any additional side effects or discomfort due to the drug

and/or the excipient. Ideally, all the excipients in the drug producer

should be inactive ingredients above or in combination in the final

dosage form.

The finished drug product is a compromise of various factors

including therapeutic objectives, pharmacokinetics, physical and

chemical properties, manufacturing cost, and patient acceptance. Most

importantly the drug product should meet the therapeutic objective by

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delivering the drug with maximum bioavailability and minimum or nil

adverse effects.

Biopharmaceutical considerations in drug product design

Pharmacodynamic considerations21

Therapeutic objectives

Toxic effects

Adverse reactions

Drug considerations

Physical and chemical properties of drugs.

Drug product considerations

Pharmaceutics of drug

Bioavailability of drug

Route of drug administration

Designed drug dosage form

Designed dose of drug

Patient considerations

Compliance and acceptability of drug product cost

Manufacturing considerations

Cost

Availability of raw materials

Stability

Quality control.