Targeted Drug Delivery Systems_brain

INDEX CHAPTER NO. 1.

CONTENTS

INTRODUCTION 1.1

CONCEPT OF TARGETING

1.1.1

RATIONALE OF DRUG TARGETING

1.1.2

CLASSIFICATION OF DRUG TARGETING

1.1.2.1

PASSIVE TARGETTING

1.1.2.2

INVERSE TARGETTING

1.1.2.3

ACTIVE TARGETTING

1.1.2.4

DUAL TARGETTING

1.1.2.5

DOUBLE TARGETTING

1.1.2.6

COMBINATION TARGETTING

1.2

DRUG TARGETTING FOR RECEPTOR

1.2.1

ENDOCYTOSIS

1.2.2

LIGAND MEDIATED TARNSCYTOSIS

1.3

CHEMICAL DRUG TARGETTING

1.3.1

CDS FOR KIDENY

1.3.2

LUNG AS A TARGET ORGAN

1.3.3

CDS FOR LIVER TARGETTING

1.3.4

LYMPHATIC TARGETTING

PAGE NO.

2.

REVIEW OF LITERATURE 2.1 2.1.1 2.1.2 2.1.3 2.2 2.2.1 2.2.2 2.2.2.1 2.2.2.2 2.2.3 2.3 2.3.1 2.3.2 2.3.3 2.3.4 2.4 2.4.1 2.4.2 2.5

LIPOSOMES LIPOSOME FORMATION METHOD OF LIPOSOME PREPERATION CHEMICAL CHARACTERIZATION NANOPARTICLES PREPEARTION TECHNIQUES CHARACTERIZATION OF NANOPARTICLES SIZE AND MORPHOLOGY SPECIFIC SERFACE APPLICATIONS OF NANOPARTICLES RELEASED ERYTHROCYTES DRUG TARGETTING DRUG TARGETTING TO RES ORGANS RES IN OXYGEN DEFICIENCY THERAPY NOVEL SYSTEM(NANOERTHROSOMES) MAGNETIC DRUG TARGETTING SYSTEM PRINCIPLES OF MAGNETIC TARGETTING APPLICATIONS MICROSPHERES

3.

DRUG DELIVERY TO BRAIN

4. 5.

APPLICATION TO TARGETED DRUG DELIVERY TO BRAIN CONCLUSION

6.

REFERENCES

12

CHAPTER -1

INTRODUCTION: Because the brain is tightly segregated from the circulating blood by a unique membranous barrier, the blood-brain barrier (BBB), many pharmaceuticals cannot be efficiently delivered to, or sustained within the brain; hence, they are ineffective in treating cerebral diseases. Therefore, drug delivery methods that can provide brain delivery, or eventually preferential brain delivery (i.e. brain targeting), are of particular interest. To achieve successful delivery, an understanding of the major structural, enzymatic, and active transport aspects related to the BBB, and of the issues related to lipophilicity and its role in CNS entry, is critical. During the last years, considerable effort was focused in the field of brain-targeted drug delivery. Various more or less sophisticated approaches, such as intracerebral delivery, intracerebroventricular delivery, intranasal delivery, BBB disruption, nanoparticles, receptor mediated transport (vector-mediated transport or ‘chimeric’ peptides), cell-penetrating peptides, prodrugs, and chemical delivery systems, have been attempted. These approaches may offer many intriguing possibilities for brain delivery and targeting, but only some have reached the phase where they can provide safe and effective human applications. Site-target indexing and the use of targeting enhancement factors can be used to quantitatively assess the site-targeting effectiveness from a pharmacokinetic perspective of chemical delivery systems. Drug targeting is the delivery of drugs to receptors or organs or any other specific part of the body to which one wishes to deliver the drug exclusively. The drug therapeutic index(TI) as measured by its pharmacological response and safety. Drugs minimizing its interaction with non target tissue. The desired differential distribution of drug by its targeted delivery would spare the rest of the body and thus significantly reduce the over all toxicity while ma intaining it's therapeutic benefits. The targeted or site specific delivery of drugs is indeed a very attractive goal because this provides one of the most potential ways to improve the the rapeutic index of the drug. The need for targeted delivery of drugs is best illustrated with peptide drugs where failure in the clinic may not be due to a poor intrinsic activity, but rather due to transport factors including widespread disposition,rapid catabolism and excre tion, variable or in efficient extravasations, and the subsequent high dosing levels required

13

to obtain a therapeutic effect (Tomlinson etcal.1986). Earlier work done between late 1960s and the mid 1980s'stressed the need for drug;-carrier systems primarily to alter the pharmacokinetics of the already proven drugs whose efficacy might be improved by altering the rates of metabolism in liver or clearance by the kidneys (Pozanski&Juliano, 1984). These approaches generally were not focused to achieve site-specific or targeted delivery such as getting a cytotoxic drug to cancerous tissue while sparing other normal, though equally sensitive tissue (Papahadjopoulos, 1978)1.

1.1 CONCEPT OF TARGETING: 1.1.1 RATIONALE OF DRUG TARGETING (CARRIERS): Carrier is one of the most important entities essentially required for successful transportation of the loaded drug (s) Delivery systems developed and exploited in the last decade for ligand directed receptor mediated targeting are mainly focuses on liposome's and microparticulates, bioconjugates (drug-antibody conjugate,

drug-

polymer conjugates, drug-immunotoxin conjugates), fusogenic proteins and peptides and certain polymeric and macromolecular delivery systems.

An ideal drug carrier

engineered as a targetable device should have the following features. •

It must be able to

cross anatomical barriers and

in

case of tumour

chemotherapy tumour vasculature. •

It must be recognized specifically and selectively by the target cells and must maintain the avidity and specificity of the surface ligands.

•

The linkage of the drug and the directing unit (ligand) should be stable in plasma, interstitial and other biofluids.

•

Carrier should be non-toxic, non-immunogenic and biodegradable particulate or macromolecule and after recognition, and internalization, the carrier system should release the drug moiety inside the target organs, tissues or cells.

•

The bimolecular used for carrier navigation and site recognition should not be ubiquitous otherwise it may cross over the sites, defeating the concept of targeting2.

1.1.2 CLASSIFICATION OF DRUG TARGETING: The various approaches of vectoring the drug to the target site can be broadly classified as 1. Passive targeting 2. Inverse targeting 14

3. Active targeting (Ligand mediated targeting and Physical targeting) 4. Dual targeting 5. Double targeting 6. Combination targeting

1.1.2.1 PASSIVE TARGETING: Systems that target the systematic circulation are generally characterized as" passive" targeting delivery systems (i.e. targeting occurs because of the body's natural response to the physicochemical characteristics of the drug or drug -carrier system. It is a sort of passive process that utilizes the natural course of attributed to inherent characteristics) biodistribution of the carrier system, through which, it eventually accumulate in the organ compartment (s) of body. The ability of some colloids to be taken up by the RES especially in liver and spleen has made them as ideal vectors for passive hepatic targeting of drugs to these compartments.

1.1.2.2 INVERSE TARGETING: One strategy applied to achieve inverse targeting is to suppress the function of RES by a pre-injection of a large amount of blank colloidal carriers or macromolecule like dextran sulphate. This approach leads to RES block-ade and as a consequence impairment of host defense system Alternative strategies include modification of the size, surface charge, composition, surface rigidity and hydrophilicity of carriers for desirable biofate.

1.1.2.3 ACTIVE TARGETING: The natural distribution pattern of the drug carrier composites is enhanced using chemical, biological and physical means, so that it approaches and identified by particular biosites. The facilitation of the binding of the drug-carrier to target cells through the use of ligands or engineered homing devices to increase receptor mediated (or in some cases receptor independent but epitope based ) localization of the drug and target specific delivery of drug (s) is referred to as active targeting.. The targeting approach can further be classified it into three different levels of targeting.

15

(1)First order targeting (organ compartmentalization). (2)Second order targeting (cellular targeting). (3)Third order targeting (intracellular targeting). (1) FIRST ORDER TARGETING: It refers to restricted distribution of the drug-carrier system to the capillary bed of a predetermined target site, organ or tissue. Compartmental targeting in lymphatic, peritoneal cavity, plural cavity, cerebral ventricles, lungs, joints, eyes, etc., represents first order targeting it could also be categorized as a level of passive targeting. Large liposome (10u or above ) are rapidly removed via mechanical filtration of lungs and from this size range down upto 150nm are removed by tissue macrophages originated in the liver and spleen, which are the natural target for these vesicles.

(2) SECOND ORDER TARGETING: The selective delivery of drugs to a specific cell type such as tumour cells and not to the normal cells is referred as second order drug targeting.

(3) THIRD ORDER TARGETING: The third order targeting is defined as drug delivery specifically to the intracellular site of target cells. An example of third order targeting is the receptor based ligand-mediated entry of a drug complex into a cell by endocytosis, lysosomal duration of carrier followed by release of drug intracellular or gene delivery to nucleolus.

(4) LIGAND MEDIATED TARGETING: Targeting components, which have been studied and exploited are pilot molecules themselves (bioconjugates) or anchored as ligands on some delivery vehicle (drug -carrier system). All the carrier systems, explored so far, in general, are colloidal in nature. They can be specifically functionalized using various biologically relevant molecular ligands including antibodies, polypeptides, oligosaccharides (carbohydrates), viral proteins and fusogenic residues. Ligand mediated activity targeting could be achieved asing specific uptake mechanisms such as receptor dependent uptake of natural low density lipoproteins (LDL) particles and synthetic lipid microemulsions of partially reconstituted LDL particles coated with the apoproteins .

16

(5) PHYSICAL TARGETING (TRIGGERED RELEASE): The selective drug delivery programmed and monitored at the external level (ex vivo) with the help of physical means is referred to as physical targeting; In this mode of targeting, some characteristics of the bioenvironmental are used either to direct the carrier to a particular location or to cause selective release of its contents . The first such approach reported is the temperature sensitive liposomes, which were developed and applied to tumour by (Weinstein and co-workers, 1979).

Table-1.1 : Passive Hepatic Targeting for Macrophage Associate Diseases Macrophage associated

Drugs proposed for encapsulation

Leishmaniasis; brucellosis; candidiasis

Antimalarial and antiinfective

Intracellular fungal infections Histoplasmosis: i Antifungal (Amphotericin B) systemic Mycoses Histiocytes medullar Reticulosis; monocyte & Cytotoxic drugs hairy Cell leukemia; Hodgkin's disease Viral infected diseases Hepatitis

Anti-viral drugs

Enzyme storage diseases Gaucher's disease, Glucocerebroside and Other enzymes mucoliposes Type II & III!

1.1.2.4 DUAL TARGETING: This classical approach of the drug targeting employs carrier molecules, which have their own intrinsic antiviral effect thus synergies the antiviral effect of the loaded active drug. Based on this approach, drug conjugates can be prepared with fortified activity Profile against the viral replication.

1.1.2.5 DOUBLE TARGETING: For a new future trend, idrug targeting may be combined with another methodology, other than passive and active targeting for drug delivery systems. The combination is made between spatial control and temporal control of drug delivery.

1.1.2.6 COMBINATION TARGETING:

17

Combination targeting for the site- specific delivery of proteins and peptides these targeting systems are equipped with carriers, polymers and homing devices of molecular specificity that could provide a direct approach to target site. Modification of proteins and peptides with natural polymers, such as polysaccharides, or synthetic polymers, such as poly (ethylene glycol), may alter their physical characteristics and favour targeting the specific compartments, organs or their tissues within the vasculature 3.

1.2 DRUG TARGETING FOR RECEPTOR: LIGAND DRIVEN RECEPTOR MEDIATED DRUG DELIVERY Design and development of potential carriers for cell specific delivery of therapeutics are immensely dependent on the selectivity of the carrier to the cellular receptors distributed variable at intracellular sites and on the surface of cellular systems. Other crucial factors include the anatomical and pathological barriers that have to be circumvented, enroute before recognition site(s) are arrived. Intracellular mesogenic constraints as well as physiologic constraints are also encountered following receptor recognition, similarly, cellular internalization is equivocally critical for intracellular rounding4.

1.2.1 ENDOCYTOSIS: Endocytosis (phagocytosis and pinocytosis) has been defined as the internalization of

plasma membrane with concomitant engulfment of plasma membrane with concomitant engulfment of extracellular cargo/fluM. The process serves to selectively retrieve and assimilate various macromolecules from extracellular fluid for a variety of cellular

18

functions. It is the main cellular activity involved in the internalization of the extracellular cargo and their vesicular coat proteins, which are subsequently processed via different pathways to appropriate intracellular targets. Phagocytosis is the engulfment of the endogenous and exogenous particulate materials, such as bacteria, erythrocytes, latex beads, colloidal particles and immunoglobulin molecules. It is performed by the phagocytic cells of the hepatic sinusoids, the tissue fixed macrophages (histocytes) and the blood macrophages or monocytes that the fluid phase and receptor mediated pinocytosis are not separate cellular events, but they are different facets of the same event. Non specific adsorption piocytosis is responsible for the uptake of many non-glycosylated proteins particularly following cellular damage or protein denaturation.

1.2.2 LIGAND MEDIATED TRANSCYTOSIS: Transcytosis is the process by which intracellular ligand or extracellular cargo internalized at one plasma membrane domain of a polarized cell which is transported via vesicular intermediates to the contra lateral plasma membranes. Much of the characterization of transcytosis in the basolateral to apical direction. Accordingly, the polymeric immune globulin receptor (plgR), a protein specialized for basal to apical transcytosis that recycles at the apical membrance as part of its transcytotic sojourn, has been used apparently as an apical endocytic carrier to introduce vector DMA into cells that express the PlgR. The apical to basal transcytosis has been however identified at molecular levels in Cacao 2 cells. This protein as well as additional ones should provide powerful tools for future characterization of transcytosis pathway. Ideally some of these proteins may represent better putative carriers for the apical to basal transcytosis of therapeutic agents. Transcytosis pathway has been well established for the protein sorting an trafficking of transferrin, polymeric Ig, and viral pathogens. With respect to the delivery and transport of pharmaceuticals, characterization of this pathway should lead to the advances in the development of transcellular drug delivery.

1.3 CHEMICAL DRUG TARGETING: 1.3.1 CDS FOR KIDNEY: Kidney possesses high concentrations of L-glut amyl transpeptidase and L-amino acid decarboxylase. Selective delivery of dopamine in kidney has been obtained after administration

of

L-Y

glutamyl

dopa

produces

almost

5

times

higher

concentrations of dopamine in kidney compared with equivalent dose of L-dopa.

19

1.3.2 LUNG AS A TARGET ORGAN: The lung possesses the next highest levels of nearly all the metabolic enzymes found in liver an in some cases even higher specific activities in certain cell types. Additionally, in contrast to all other tissues, the lung receives total venous return first, so it in an ideal position to regulate the concentration of substrates in the blood before they reach the arterial circulation hence avoiding problems that may be associated with a hepatic first pass and permitting a more efficacious sequestration of a drug entity.

1.3.3 CDS FOR LIVER TARGETING: Liver is an important organ and is considered to be focal point of metabolic ativities in body. Targeted drug delivery to liver is achieved using bile acid transport system associated with the sinusoidal membrane of the hepatocytes developed bile acid prodrug of chlorambucil for liver targeting. et. receptor

al.

1992.

Hepatic

asialoglycoprotein

mediated endocytosis has been exploited for antiviral drug targeting to liver

parenchymal cells.

1.3.4 LYMPHATIC TARGETING: The lymphatic system is regarded as an integral and necessary part of the vascular system. Its main function is to collect the excessive tissue fluid and return it back to the blood. Lymphatics are numerous in number and distributed throughout the body. Their major physiological function is to maintain the body's water balance, thus acting as body's drainage system. The intestinal lymphatic system consists of a network of vessels distributed throughout the small and large intestine. They play a major role in the absorption of variety of nutrients, lipids including long chain fatty acids, triglycerides, cholesterol esters fluids, lipid soluble vitamins some xenobiotics (e.g. DOT). The potential advantage of transporting drug through the intestinal lymphatic system includes. Avoidance of hepatic first-pass metabolism.Selective treatment of diseases and infections of the mesenteric lymphatic system.Directing the delivery of appropriate agent to various sites of intestinal and thoracic lymphatic system.Enhancement of the absorption of large macromolecules

such

a

peptides

an

particulates.Inhibition

of

cancer

cell

metastasis.Lymphocytic targeting and receptor mediated targeting via the low density lipoproteins receptor to regions of the lymphatic that are directly supplied by lymph from mesenteric

lymphatic’s.

These regions are often poorly perfused by

the

systemic 20

circulation making it difficult to attain adequate drug concentration at the target organ/cell after the compound has absorbed via the portal blood

Reduction in local gastrointestinal

irritation and toxicity .An overall modulation in the rate of drug input thus providing a sustained delivery5.

21

CHAPTER-2

REVIEW OF LITERATURE: 2.1 LIPOSOMES: Liposomes have attracted a considerable amount of intevest for potential use as a drug delivery system owing to their suitable characteristics. They consist of one or more concentric phospholipid bilayers enclosing an aqueous space. They are biocompatible, biodegradable, and normally nonimmunogenic. More importantly, they are capable of loading both hydrophilic and hydrophobic drugs in the aquesous and bilayer phase respectively. Drugs encapsulated in liposomes are protected from enzymatic degradation and other inactivation processes. There are basically two different modes in liposome targeting passive and active targeting. The former takes advantage of the fact that systemically injected liposomes are rapidly and efficiently taken up by phagocytic cells of the reticuloendothlial system (RES) located Mainly the live and the spleen Some of the advantage of liposome are as follows: • Provides selective passive targeting to tumour tissue. • Increased efficacy and therapeutic index. • Increased stability via encapsulation • Reduction in toxicity of the encapsulated agent. • Site avoidance effect. •

Improved pharmacokinetic effects (reduced elimination, increased circulation life times)

•

Flexibility to couple with site-specific lignads to achieve active targeting

2.1.1 MECHANISM OF LIPOSOME FORMATION: In order to understand why liposomes are formed when phospholipids are hydrated it requires a basic understanding physicochemical features of phospholipids. Phospholipids are amphipathic (having affinity for both aqueous and polar moieties) molecules as they have a hydrophobic tail and a hydrophilic or polar head. The hydrophobic tail is composed of two fatty acid chains containing 10-24 carbon atoms and 0-6 double bonds in each chain. The polar end of the molecule is mainly phosphoric acid bound to a water soluble molecule. The hydrophilic and hydrophobic domains/segments within the molecular geometry of amphiphilic lipids orient and self orgainzie in ordered supramolecular structure when confronted with solvents.In aqueous 22

medium the molecules in self assembled structures are oriented in such a way that the polar portion of the molecule remains in contact with the polar environment and at the same time shields the non-polar part. Among the amphiphiles used in the drug delivery viz. soaps, detergents, polar lipids, the latter (polar lipids) are often employed to form concentric bilayered structures. However, in aqueous mixture these molecules are able to form various phases, some of them are stable an others remain in the metastabel state. At high concentrations of these polar lipids, liquid-crystalline phases are formed that upon dilution with an excess of water can be dispersed into relatively stable colloidal particles. The macroscopic structures most often formed include lamellar, hexagonal or cubic phases dispersed as colloidal Nanoconstructs (artificial membranes) referred to a liposomes, hexasomes or cubosomes , respectively.The most common natural polar phospholipids are phosphatidylcholine (PC). These are amphipathic molecules in which a glycerol bridge links to a pair of hydrophobic acyl hydrocarbon chains with a hydrophilic polar head group, phosphocholine explains that the fatty chains are embedded in the hydrophobic inner region of the membrane, oriented at an angle to the plane of the membrane surface, the hydrophilic head group, including the phosphate portion. Points out towards the hydrophilic aqueous environment. Molecules of PC are not soluble (rather dispersible) in aqueous media in the physical chemistry sense, as in aqueous media they align themselves closely in planer bilaryer sheets to minimize the unfavorable interactions between the bulk aqueous phase and long hydrocarbon fatty acyl chain. Such interactions are completely eliminated when the sheets fold over themselves to form closed, sealed and concentric vesicles. The large free energy change between an aqueous and hydrophoble environment explains the most favored orientation of lipids to assemble as concentric bilayer structures that exclude confrontation between aqueous and hydrophobic domains.Thus the amphipathic (amphiphilic) nature of Phospholipids and their analogues render them the ability to form closed concentric bilayers in the presence of water. Liposomes (lipid vesicles) are formed when thin lipid films or lipid cakes (of amphiphilic nature) are hydrated and stacks of liquid crystalline bilayers become fluid and swell.

2.1.2 METHODS OF LIPOSOME PREPARATION: Liposomes are manufactured in majority using various procedures in which the water soluble (hydrophilic) materials are entrapped by using aqueous solution of these materials as hydrating fluid or by the addition of drug/drug solution at some stage during the manufacturing of liposomes (Ostro, 1987, 1989; Talsma and Cromunelin, 1992). The lipid soluble (lipophilic) materials are solubilized in the organic solution of the constitutive lipid (s) and then evaporated to a dry drug containing lipid film followed by its hydration. These methods involve the loading of the entrapped agents before or during the manufacturing procedure (passive loading ) However, certain types of compounds with ionizable groups,

Fig-2.1:liposome drug delivery then membrane phospholipids are disrupted, they can reassemble themselves into tiny spheres, smaller than a normal cell, either as bilayers or monolayers. These are liposomes. The lipids in the plasma membrane are chiefly phospholipids like phosphatidyl ethanolamine and cholesterol. Phospholipids are amphiphilic with the hydrocarbon tail of the molecule being hydrophobic; its polar head hydrophilic. As the plasma membrane faces watery solutions on both sides, its phospholipids accommodate this by forming a phospholipid bilayer with the hydrophobic tails facing each other.

Liposomes can be composed of naturally-derived phospholipids with mixed lipid chains (like egg phosphatidylethanolamine), or of pure surfactant components like DOPE (dioleoylphosphatidylethanolamine). Liposomes, usually but not by definition, contain a core of aqueous solution; lipid spheres that contain no aqueous material are called micelles, however, reverse micelles can be made to encompass an aqueous environment.

2.1.3 CHEMICAL CHARACTERIZATION OF LIPOSOMES: Various chemical analysis methods used for quantitative and qualitative tests of liposomal components prior to and after the preparation are critical characteristics of liposomes (Barenholz and Cromellin 1994). These methods become more essential to characterize liposomes, which require lipid stability cropping up from oxidatin, lipid peroxidation, hydrolysis and degradation ni various environment used in their manufacturing6.

TABLE-2.1: Liposome Characterization with their Quality Control Assays –

Fracture correlation

electron

microscopy,

spectroscopy.

Laser

photon light

scattering, gel permeation and gel exclusion. Surface charge Phase behaviour Drug release 3. Bioligical Chractcrization Sterility Pyrogenicity

Free- flow electrophoresis. Freeze-fracture electron microscopy. Diffusion cell/ dialysis

Animal

Monitoring survival rates. Histology and

Aerobic or anaerobic cultures Rabbit fever responses test pathology.

Characterization parameters

Analytical method/ Instrumentation

1. Chemical Characterization Phospholipid concentration

Lipid phosphorus content using Barlett assay Stewart assay, HPLC

Cholesterol concentration

Cholesterol oxidase assay and HPLC

Phospholipid peroxidation

UV absorbance. TBA ( for endoperoxidase), iodometric (for hydroperoxidase) and GLC.

Phospholipid hydrolysis

HPLC and TLC and fatty acid concentration

Cholesterol auto-oxidation

HPLC and TLC

Anti- oxidatant degradation

HPLC and TLC

pH

pH meterr

Osmolarity

Osmometer

2. Physical Characterization Vesicle shape and surface morphology Transmission electron microscopy , freeze-fricture electron microscopy Vesicle size and size distribution Dynamic light scattering, transmission electron Submicron range

microscopy,

Micron range

microscopy,

zetasizer

Transmission

electron

2.2 NANOPARTICLES: The colloidal earners based on biodegradable and biocompatible polymeric systems have largely influenced the controlled and targeted drug delivery concepts. It was realized that the nanoparticles loaded bioactives could not only deliver drug (s) to specific organs within the body but delivery rate in addition could be controlled as being by standers, burst, controlled, , pulsatile or modulated. The possibilities and potentials further prompted the work and as a result a great deal of related information covering preparation methodologies, characterization. Engineering, bio-fate and toxicology has been gathered. The understanding that relates to the biodistribution in particular has propelled and motivated the development of functionally designed nonoparticulates6.

2.2.1 PREPARATION TECHNIQUES OF NANOPARTICLES: The selection of the appropriate method for the preparation'of nanoparticles depends on the physicoehemical characteristics of the polymer. Various Proteins and Polysaccharides used for the prepartion of Nano particles: Proteins Gelatin Albumin Lectins Legunin Vicilin

Polysaccharides Alginate Dextran Chitosan Agarose Pullulan

The drug to loaded. on the contrary, the preparation techniques largely determine the inner structure, in vitro release profile and the biological fate of these polymeric delivery systems .Two types of systems with different inner structure are apparently possible matrix type system

consisting

of

entanglement

of

oligomeror

polymer

units

(nanoparticles/nanoshpheres). A reservoir type of system comprised of an oily core surrounded by an embryonic polymeric shell (Nanocapsules). The Polymers are strictly structured to a nanometric size range particle (s) using appropriate methodologies.

FIG-2.2: Flowchart of preparation of nanoparticles

These methodologies are conveniently classified as follows: 1.

Amphiphilic macromolecule cross-linking.

a. Heat cross-linking b. Chemical cross-linking

2.

Polymerization based methods a. Polymerization of monomers in situ b. Emulsion(micellar) polymerization c.Dispersion polymerization

d.Interfacial condensation polymerization e.Interfacial complexation

3.

Polymer precipitation methods a. Solvent extraction/evaporation b. Solvent displacement (nanoprecipitation) c. Salting out

FIG-2.3:NANOPARTICLE

2.2.2 CHARACTERIZATION OF NANOPARTICLES: The nanoparticles are generally characterized for size, density, electrophoresis mobility, angle of contact and specific surface area.

2.2.2.1 SIZE AND MORPHOLOGY: The particle size is one of the most important parameters of nanoparticles. Particles size and sizing of sub-optical particulates is a different procedure, as it involves not only procedural variability, but some of the surface associated properties may even change during sizing procedure. Two main techniques are being used to determine the particle size distribution of nanopartitcles and include photon correlation spectroscopy (PCS) and electron microscopy. The latter (TEM) and freeze -fracture techniques. The size evaluation of nanoparticale dispersion demonstrates better results with freeze-fracturing microscopy and photon correlation spectroscopy as quantitative methods. The freeze-fracturing with poly (methyl methacrylate) is confronted with and interrupted by in process particles aggregation which only yields a few discrete particles for size measurement of analysis. The electron microscopy however, could be adopted as an alternative option, which measures individual particles for size and its distribution. It is relatively less time consuming. Additionally, freeze fracturing of particles allows for

morphological

determination of freeze fracture procedures, TEM permits differentiation among nanocapsules, nanoparticles and emulsion droplets. Similarly, scanning electron microscopy is much less time consuming. However, since particles are based on organic and non-conductive material, they require from 30-50 nm. Thus determined size should be denoted as gold- coated particle size rather than as particle size.

Table2.2: Different Parameters and Characterization Methods for Nanoparticles: Parameter.

Characterization Method (S)

Particle size and size distribution

Photon correlation

Spectroscopy

(PCS)

laser defractometnnry transmission electron microscopy scannming Electron Microscopy. Charge determination

Laser doppIcr Anemomclry Zeta potentiometer

Surface hydrophobicity

Water contact angle measurements rose bengal binding hydrophobic interaction chromatography X- ray photoelectron spectroscopy

Chemical analysis of surface

Static secondary ion mass spectrometry Sorptometer .

Carrier- drug interaction

Differential scanning calorimetry.

Nanoparticle dispersion stability

Critical flocculation temperature (CFT)

Release profile

In vitro release characteristic under physiolgic and sink conditions

Drug stability

Bioassay of drug extracted from nanoparticles Chemical analysis of drug.

2.2.2.2 SPECIFIC SURFACE: The specffic surface area of freeze-dried nanoparticles is generally determined with the help of sorptometer (Kreuter, 1983). The equation can be used in the calculation of specific surface area. Inmost of the cases, the measured and calculated specific surface areas fairly compare while in some cases the residual surfactant could affect deviation in measured values. The surfactant coating apparently reduces the specific surface area.

2.2.3 APPLICATION OF NANOPARTICLES : Nanoparticles with different composition and characteristics have been formulated and investigated for various therapeutic applications sevral different types of biodegradable polymers including biopolymers (e.g. gelation, albumin, casein, polysaccharide, lectin etc.) and synthetic polymers (polycaprolactone, polyesters, polyanhydrides, polycyanoacrylates) with various drug release characteristics ranging from several hours to several months have been used to formulate sustained release nanoparticles. It is the submicron size of this delivery system, which makes it more efficient in certain drug therapy applications, such as in intracellular localization of therapeutic agents. These systems, in addition to sustained drug delivery have been investigated for various therapeutic applications7.

2.3 RESEALED ERYTHROCYTES: These are prepared by placing RBC's in hypotonic media which leads to rupturing of cell membrane & formation of pores (dia 200-500A) through which intracellular & erxtracellular exchange takes place. In this way a drug compound in extra cellular media entrees the RBC's are loaded into RBC's.. Resealed erythrocytes are biodegradable, non-immunogenic. Resealed erythrocytes can be used to target drug to liver & spleen. Desirable properties of released erythrocytes: •

Biodegrability Circulate throughout the circulatory system.

•

Large quantities of material can be encapsulated within small volume of cells.

•

Can be utilized for orgn targeting within RES.

A wide variety of bioactive gents can be encapsulated within them. Erythrocytes biocompatible provided that compatible cells are used in patients there is no possibility of triggered immunological response8.

2.3.1 DRUG TARGETING: A drug delivery should ideally be site-specific & target oriented in order to exhibit maximal therapeutic index & minimum side & toxic effects. It has been observed that osmotically loaded erythrocytes can act as drug carriers in systematic circulation, whereas chemically surfaces modified erythrocytes are targeted to organs of the mononuclear phagocytic system/ reticuloendithelial system (MPS/RES) because of change incorporated in the membranes that are recognized by macrophage cells.

FIG -2.4: RESEALED ERYTHROCYTES

2.3.2 DRUG TARGETING TO RES ORGANS: The damaged erythrocytes are quickly removed from circulation by phagocytic kupffer cells located in liver & spleen. Though, released erythrocytes have been proposed for passive autovectorization to MPS/RES system where modified surfaces characteristics lend them selectivity & specificity towards target cells (mainly liver & spleen).

2.3.3 RES IN OXYGEN DEFICIENCY THERAPY: Released erythrocytes are also used in cases of oxygen deficiency where an improved oxygen supply is required as in the following cases: 1. High altitude conditions (Where partial pressure of oxygen is low). 2. Small number of alveoli (Where lung exchange surface is low). 3. Increased resistance to oxygen diffusion in the lungs.

2.3.4 NOVEL SYSTEM (NANOERYTHROSOMES): An erythrocyte based new drug carrier, named nanoerythrosome has been developed which is prepared by extrusion of erythrocyte ghosts to produce small vesicles having an average diameter of 100 nm. Daunorubicin was covalently conjugated to the nEryt using glutraledhyde as homobifunctional liking arm. This given has a higher antoneoplastic index than free drug 9.

2.4 MICROSPHERES : The term microsphere is defined as a spherical particle with size varying from 50 nm to 2 nm, containing a core substance. Microspheres are,in strict sense, a spherical empty particles. However, the term microcapsules and microsphere are often used synonymously. The microsphere are characteristically free flowing powders consisting of proteins or synthetic polymers, which are biodegradable in nature and ideally having particle size less than 200 micrometer.

2.4.1 TECHNOLOGY AND APPLICATIONS: 2.4.1.1 POWDERS AND GRANULATES : Free-flowing powders and granulates are needed for a variety of industrial processes. These, however, do not always meet the exacting standards which modern manufacturing demands of them, due to their varying grain size distribution and odd shapes. These properties are detrimental to efficient processing and lead to agglomeration, inexact dosage, abrading with loss of material, or low reproducibility of castings. Pharmaceutical applications require highly reproducible dosage and the controlled release of active agents, which can not be achieved with conventional powders and granulates. The use of small and perfectly round Microspheres with exactly the same size circumvents all of the disadvantages that are encountered while using powders and granulates. These Microspheres are free-flowing and roll with practically no friction, that means there is no

abrasion, guaranteeing a dust-free environment.

Fig- 2.5: Flow chart of microsphere synthesis

2.4.2 PROCESSING CHARACTERISTICS: Microsphere production units have a minimal space requirement (15 to 40 ft2), the energy consumption is very low and they are noiseless during operation. These units operate at atmospheric pressure or slightly above and can be designed to be explosion-proof and/or according to the GLP/ GMP guidelines. Microsphere production units from BRACE need practically no maintenance, therefore only a minimal staff is required. Metal oxide spheres as molded (yellow), dried (yellow transparent), calcined (black) and sintered (black, smallest). The shrinkage in diameter corresponds to their solid content during sintering. Units are delivered with automated controls and can be delivered as remote controlled and enhanced10.

2.4.3 TYPES OF MICROSPHERES: There are very few restrictions on the types of Microspheres than can be produced. With the right combination of liquid precursor, solidification process, and subsequent treatments, a wide range of Microspheres can be produced. •

Dry metal oxide Microspheres produced on the basis of a sol (Al2O3, ZrO2, HfO2, TiO2, CeO2, SiO2, and mixed oxides) can be used as highly sinteractive press-feed for the production of high-tech ceramics.

•

Through calcining, the pore size and surface area of the Microspheres can be tailored to exacting specifications. These Microspheres make excellent catalystcarriers, homogeneous catalysts, or filtering materials. Unusually effective and abrasion resistant Microspheres for grindingther materials are made from sintered Al, Zr, and Hf-oxides.

Fig -2.6:Comparison of size of microsphere •

Monodispersive alumina Microspheres.

•

Ultra Spherical Microspheres .

•

Microspheres with a monodisperse grain sizedistribution and the smallest divergence are manufactured by BRACE.

•

perfectly spherical Microspheres .

•

monodisperse grain size, narrow sizedistribution with diameters between 50µm and 5000µm nonabrading, therefore dust-free, free flowing, porous, large surface area, soft or rigid for embedding pharmaceuticals, biomass (e.g., yeast or enzymes) or other heterogeneous catalysts with or without coating.

2.4.4 USES OF MICROSPHERE: Microspheres produced from molten materials (inorganic, organic, alloys, and polymers) can be used for dosing, proportioning, compounding, coloring, and light stabilization. Microspheres with dissolved or embedded active agents, with or without coating, are used for numerous pharmaceutical and cosmetic products. Soluble chemical compounds can be incorporated into Microspheres by precipitation

for use in the agricultural, food, pharmaceutical, and cosmetics industries. Suspensions are used to produce Microspheres with embedded enzymes or bacteria. With our special double nozzle systems, Microspheres with encapsulated materials can be obtained. Especially for the encapsulation of water, aqueous solutions or cells, a microsphere with a liquid core and a solidified shell can be produced. The shell and the core material can be chosen as appropriate: alginate, PVA, PEI, PEG, wax, metal oxides, gelatin, hydroxylcelluloses, etc11.

CHAPTER-3

DRUG DELIVERY SYSTEM IN BRAIN Drug delivery to specific locations in the brain is a challenging task in the treatment of diseases related to Central Nervous System (CNS) such as brain tumors, epilepsy, Parkinson's, Alzheimer's and Huntington's diseases owing to the blood-brain barrier (BBB). There is an interest from medical community in delivering Glial-Derived Neurotrophic Factor (GDNF) and Brain-Derived Neurotrophic Factor (BDNF) drugs at specific locations to CNS [1]. It is also known that these drugs consisting of big molecules cannot overcome the BBB. A promising solution to this problem is to deliver the required drug into the targeted location by invasive techniques as the convection enhanced delivery (CED).

In this work, computational fluid dynamics (CFD) techniques are utilized to study invasive drug delivery in multi-dimensional brain geometries with the consideration of the chemical interactions that the drug undergoes while it diffuses into the brain tissue. A challenge is to accurately reconstruct the three-dimensional structure of the human brain. We are able to resolve very accurately the brain geometry and render physiologically consistent the distribution of the complex brain inner organization. We distinguish between gray and white matter and assign transport properties of relevance according to the data obtained by MR images or histological data. We will quantify with numerical simulations the diffusive and convective transport phenomena in the porous brain tissues and the effectiveness of the drug release to a desired region. This approach will help to evaluate precisely the penetration depth of the drug and the concentration profiles need to surpass set thresholds in order to ensure proper efficacy of the drug. We rigorously examine the variables that influence CED and pose constraints to the treatment. These include effect of infusate (bulk) flow rate, concentration of the infusate, drug diffusivity, effect of molecular weight of the drug, and effect of white matter anisotropy, infusate leak-back by considering metabolic uptake by the parenchyma cells and re-absorption of the bulk fluid [2]. Understanding the parameters that could possibly influence the convective delivery of drugs in the CNS is very important because, it will improve the current medical approaches. The proposed methodology will provide a systematic approach to optimally choose catheter dimensions, infusion rates, drug concentrations etc. The information obtained from these accurate simulations could be used to model inverse

kinetic problems capable of predicting the mass diffusivity of the drug and the kind of metabolism that actually takes place.

Brain targeted transcranial route of drug delivery of diazepam The term transcranial route means the brain targeted transfer of drug molecules across the cranium through the layers of the skin and skin appendages of the head, arteries and veins of the skin of the head, the cranial bones along with the diploe, the cranial bone sutures, the meninges and specifically through the emissary veins. The administration of drugs through the scalp in ayurvedic system for the diseases associated with the brain was evaluated with a view to develop a novel targeted route for central nervous system drugs. It is expected to circumvent the systemic side effects of oral route. Diazepam was dissolved in an oil medium and applied on scalp as practiced in the ayurvedic system. Thirty rats were tested on the rotating rotarod for muscle relaxant effect of diazepam. Five groups of rats tested were the control, diazepam i.v. injected (280 µg/0.1 ml) group, two groups treated with transcranial diazepam oil solution (1.5 mg/0.2 ml) and the transcranial blank vehicle treated groups. Holding time in triplicate for each rat on the rotating rotarod was measured. The holding times following each treatment was statistically compared (one-way ANOVA). The pooled average times for the control, diazepam i.v. injected, diazepam oil solution transcranial treated two groups and the blank vehicle treated groups were 35.45, 4.73, 16.5, 15.39 and 33.23 seconds respectively. The two groups subjected to the brain targeted transcranial route showed a statistically significant decrease (50% drop) in the holding time against the control group indicating the centrally acting muscle relaxant effect due to absorption of diazepam into the brain through the proposed route. Man entertained a special care in all matters relating to the head because the head housed the brain. The effects of a bath are remarkably different from that of a body wash. A head injury, however trivial, is considered a precarious situation and a pimple on the face may be fatal. The apprehensions mentioned above and the drug delivery route that is being undertaken in the present study is incidental to a special anatomical feature of the skull. The emissary veins draining blood from extracranial sites into the

intracranial sinuses pierce a series of foramina present in the cranial bones. Seven major sinuses within the skull are interconnected by a number of anastomosing veins, which finally drain intracranially into the jugular veins giving ample scope for the diffusion of the drug molecules into the nerve tissue of the brain. There are thirteen emissary veins connecting extracranial sites of the head with the intracranial sinuses[1]. The emissary veins are present in all higher animals starting with aves[2] and their presence in the horse is well established. The arteries of the scalp send small twigs to the underlying bones of the scalp. The spongy diploe within the flat skull bones is also well supplied by numerous small diploic branches from arteries both on external and internal surfaces of the skull[3]. These anatomical arrangements of the vascular system are made use of in the investigations to develop the brain targeted transcranial route (abbreviated TCR) of drug delivery. In the ayurvedic system there are five methods, namely Shirodara, Shiroabyanga, Shiropichu, Shirovasthi and Shiropralepa in which drugs are delivered by the transcranial route[4]. Most of these ayurvedic preparations are oil based. There are many household ayurvedic medicinal head oils for minor ailments such as headaches, sinusitis, vertigo and migraine. An important dosage design feature in this study is the use of the essentially non polar active diazepam drug moiety dissolved in an oil medium as against the use of polar salt forms in aqueous media that are popular with the modern formulations. Diazepam was selected as the screening agent since it is a prototypical benzodiazepine acting in the central nervous system. Diazepam has central depressant and centrally acting skeletal muscle relaxant effects[5]. The skin area of the head which drains venous blood through emissary veins into the intracranial locations is probably the region lying above the circular contour drawn through the angles of the mouth and the ears. Therefore the venous blood draining the eyes, ears and the nose are also drained by this route. The proposed transcranial route is intended to circumvent the side effects encountered during treatment by the oral route. Some of the central nervous system diseases such as epilepsy need long term therapy. There are prospects of adopting the transcranial route for several groups of drugs. They include antiepileptics, antipsychotics,

tranquilizers, analeptics, antiparkinsonian drugs, those acting on the endocrine glands located in the brain, drugs related to diseases of the labyrinth, glaucoma, anticoagulants and those employed in the treatment of drug addiction. CNS side effects of other drugs could be counteracted by administering the specific antagonists through transcranial route. There is a good prospect in adopting the transcranial route in veterinary practice as well. Development of subscalpal injections is another possibility. The proposed brain targeted transcranial route of drug delivery could be viewed as a parallel drug delivery system to that of metered dose inhalers in diseases of the respiratory system. Ethical clearance for animal experiments was granted by the Ethical Review Committee of the Medical Faculty, Colombo. Both male and female inbred SpragueDawley rats weighing between 165-230 g were used. They were divided into five groups of six rats including three from either sex. In the animals that were subjected to the treatment by transcranial delivery route the hair of the scalp was trimmed with scissors without injuring the skin. It was done within the trapezoidal area of the head bound by the pair of eyes and ears, closer to the line joining the ears than the eyes. They were kept at room temperature (around 30°) and fed with adult rat meal pellets and water without any restriction. They were appropriately numbered with picric acid. Rotating rotarod, constant rpm: The equipment was devised by having an electrically driven horizontal circular rod 18 mm in diameter, 29 cm in length rotating constantly at 15 rpm, mounted on two side panels 43 cm above the base. The rod was covered with no. 300 silicon carbide abrasive paper to provide roughness for the rats to grip the rod firmly. Reformulation of human diazepam i.v. injection for rats: Diazepam i.v. injection 5 mg/ml, 2 ml ampoules, manufactured by Lab Renaudin, France was reformulated into i.v. injection for rats. Adult human reference i.v. dose of diazepam 10 mg/60 Kg[6] is equivalent to 35 µg/210.6 g rat on a weight basis. Considering the ten fold metabolic rate in rats as against man an eight fold increase in the dose was considered, i.e., 35 µg×8=280 µg of diazepam per rat. Since the strength

of the commercial diazepam injection is 5 mg/ml or 5 µg/µl the number of microlitres required for 280 µg of diazepam from the original commercial injection is 280/5 µl = 56 µl. Volume to be injected into a rat was restricted to 100 µl. Therefore the volume of the diluting vehicle needed was 100 µl-56 µl=44 µl. The diluting vehicle was a 1:2 cosolvent of propylene glycol:50% ethyl alcohol. On the above basis the i.v. injection formula for rats was prepared aseptically using twenty times the following amounts to yield 2 ml of the injectable solution. 56 µl of diazepam i.v. injection 5 mg/ml (equivalent to 280 µg of diazepam), propylene glycol 14 µl and 50% ethyl alcohol 30 µl. Formulation of diazepam oil solution 1.5 mg / 0.2 ml for transcranial route (TCR): It was prepared by dissolving 30 mg of diazepam (250 mesh powder) in 1 ml isopropyl alcohol by shaking for 15-30 min and then adding 3 ml of sesame oil to the above solution and kept mixing until a clear solution formed. The product was left overnight for complete dissolution. The dose and the volume (1.5 mg in 0.2 ml) for TCR administration was decided based on the results of preliminary trials. The trials indicated that the doses which are a few multiples of the intravenous dose did not elicit marked central effects on the animals by the TCR. The volume 0.2 ml was the amount that could be accommodated in the restricted scalp area of the rat without undue spreading. Blank oil vehicle 0.2 ml for TCR administration was prepared by dissolving 1 ml of isopropyl alcohol in 3 ml of sesame oil. Disposable insulin syringes of 100 units/1 ml capacity with 29 gauge needle were used for rat dorsal tail vein i.v. injection as the positive control and also to deliver the oil based formulation drop wise onto the scalp of the animals. The animals were divided into the following five groups. Group 1, untreated animals (blank). Group 2, reformulated diazepam tail vein i.v. injected animals (positive control). Group 3, transcranial diazepam oil solution treated animals tested 15 minutes after drug application (test group A). Group 4, same as Group 3 but tested 45 minutes after drug application (test group B). Group 5, transcranial blank vehicle treated animals tested 15 minutes after the application (control group).

One animal at a time was placed on the rotating rod. Rats fell off the rod when the grip was lost. The holding time on the rod in seconds was observed for each animal. Each rat was subjected to three rotarod trials, five minutes apart. Group 2 was tested on the rotarod 15 minutes after the i.v. injection. Group 3 (test group A) and Group 4 (test group B) were tested as follows. Each rat was treated with 0.2 ml of the transcranial diazepam oil formulation containing 1.5 mg of diazepam using insulin syringe. The oil solution in 1/3 quantities were delivered drop wise on to the hair trimmed area of the scalp leaving a gap between the needle end and the skin in three stages as follows. First application at 00:00 time, 2nd application at 00:15 minutes and 3rd application at 00:45 minutes. The first application was followed by gentle rubbing on the scalp with a gloved finger previously smeared in the diazepam oil formulation by stroking ten times in a cranial to caudal direction. This was to facilitate dispelling any air pockets and to bring the oil solution into intimate contact with the skin of the scalp. Each animal was tested thrice 5 minutes apart on the rotating rotarod starting 15 minutes after the 3rd application in Group 3 and starting 45 minutes after the 3rd application in Group 4, respectively. Accordingly animals were tested one hour after the first application in Group 3 and one and a half hours after the first application in Group 4. In testing Group 5 the animals were treated with blank oil solution similar to Group 3 and tested 15 minutes after the 3rd application of the vehicle. Statistical data analysis was done using SPSS software package. One-way ANOVA and Dennett T 3 Post Hoc test was done to compare mean holding times between the groups.

Holding time on the rotating rotarod: Mean value of three trials were calculated for each animal in all five groups. The individual means of time in seconds on the rotating rotarod of six animals in each group were pooled to get the mean holding time on the rod for each group [Table - 1] Statistical comparison of mean holding times between groups showed significant difference between the groups (1-way ANOVA, P < 0.05). 1-way ANOVA followed by multiple comparisons with Dunnett T3 Post Hoc test showed statistically significant difference between group 1 and groups 2, 3 and 4, while there was no significant difference between groups 1 and 5. There was no statistically significant difference between the mean holding times between group 3 and 4 (test groups A and B) while both groups 3 and 4 showed a statistically significant difference from group 2 and 5.

The mean holding times on the rotarod for group 1 (untreated) and the transcranial blank vehicle treated group (Group 5) being not significantly different (35.45 and 33.23 s, respectively) suggests that there are no effects of either isopropyl alcohol or sesame oil or scalp stroking on the holding time and therefore on the muscle tone/grip. The diazepam i.v. injected group 2 as expected had a significant effect on the mean holding time, bringing it down to 4.73 seconds, further proving the known muscle relaxant effect of diazepam. The two groups subjected to transcranial route treatment with diazepam showed statistically significant reduction in mean holding times from that of the untreated and the vehicle treated groups and this reduction was almost by 50% [Figure - 1]. These results suggest that the diazepam drug molecules have been conveyed transcranially in to the nerve tissue of the rat brain under the experimental conditions described here. However the mean holding times of the transcranially treated groups were longer than in the i.v. treated group and these differences were statistically significant, suggesting that the amount of diazepam delivered by the transcranial route to the CNS is significantly lower than that delivered by the i.v. route. This difference in diazepam

delivery may be due to the novel experimental route and the properties of the oily formulation of diazepam prepared for transcranial application. The results of the experiments further indicate that after a certain point irrespective of the concentration of the drug in the oil solution, the volume applied and the time allowed before testing the response to the drug tend to even out unlike in the conventional oral or parenteral routes. This is evident by the fact that the holding times are nearly the same for two transcranially treated groups despite the substantially longer time allowed for the group 4 to effect the diffusion of the drug. There appear to be a wide therapeutic window for diazepam administered by the transcranial route. On these findings we suggest further experiments with improved formulations of diazepam and other CNS drugs to establish transcranial route as an effective and convenient route of drug administration.

CHAPTER-4

APPLICATION OF TARGETED DRUG DELIVERY SYSTEM IN BRAIN: Scientists began to study targeted drug delivery, because the traditional drug delivery system had many disadvantages, such as high toxic effect and high minimum effective dose. In traditional drug delivery system, after the patient takes some drugs, the drugs will distributed throughout his body through the systemic blood circulation. Only a small amount of drugs can reach the affected organ which it needs to act on. Since many drugs have some toxicity, they can kill some helpful bacteria or normal cells in some normal organs. The targeted drug delivery system can overcome these shortcomings and deliver the drugs right to the specific organ, without having any adverse effects on other healthy organs and tissues. Actually, the targeted drug delivery can be used to cure many diseases, such as the cardiovascular diseases and diabetes. However, the most important application of the targeted drug delivery is to treat the cancerous tumor. There are two kinds of targeted drug delivery. The first one is active targeted drug delivery, such as some antibody drugs. The second one is passive targeted drug delivery, such as the Enhanced Permeability and Retention effect (EFR-effect). Some Important applications are given below.

(1) Magnetic drug targeting: Tumor targeting: Magnetic drug targeting allows the concentration of drugs at a defined target site generally and importantly, away from the reticular endothelial system (RES) with the aid of a magnetic field. Site-directed drug targeting is one way of local or regional antitumor treatment. The drug & an appropriate Ferro fluid are formulated into a pharmaceutically stable formulation which is usually injected through the artery that supplies the target organ or tumor in the presence of an external magnetic field. Prolonged retentions of the magnetic drug carrier at the target site alleviate or delay the RES clearance & facilitates extra vascular uptake. For effective retaining of magnetic drug carrier, the magnetic forces must be high

enough to counteract liner flow rates within the organ or tumor tissue (between 10 & 0.05 cm/s depending on vessel size & branching pattern . There is increase in drug concentration in the target tissue after administration of the drug dose has been observed.The efficiency of chemotherapy treatment may be enhanced to a great extent by magnetically assisted delivery of cytotoxic agent to the specific site. There are a large number of magnetic carrier systems which demonstrates increasing drug concentration efficiency at the tumor site. Magnetism can play very important role in cancer treatment. The first clinical cancer therapy trials using magnetic microspheres were performed by Lubbe et al. in Germany for the treatment of advanced solid tumor while current preclinical research is investigating use of magnetic particles loaded with different chemotherapeutic drugs such as mitoxantrone, paclitaxel. Non invasive permanent magnetic field for one hour way found to induces lethal effects on several rodent & human cancers. Anticancer drugs reversibly bound to magnetic fluids & could be concentrated in locally advanced tumors by magnetic field that or arranged at tumor surface outside of the subject. In case of brain tumors, the therapeutic ineffectiveness of chemotherapy is mainly due to the impervious nature of the blood-brain barrier (BBB), presence of drug resistance and lack of tumor selectivity. Various novel biodegradable magnetic drug carriers are synthesized and their targeting to brain tumor is evaluated in vitro and in animal models. New cationic magnetic aminodextran micro spheres (MADM) have been synthesized. Its potentiality for drug targeting to brain tumor was studied. this particles were retained in brain tissue over a longer period of time. A magnetic fluid has been reported to which the drugs, cytokines & other molecule can be chemically bound to enable that agent to be directed within subject under the influence of high energy magnet. In one of such examples magnetic doxorubicin in liposome, significant anticancer effect in nude mice bearing colon cancer .

(2) Magnetic bioseparation: Bioseparation is an important phenomenon for the success of several biological processes. Therefore, prospective bioseparation techniques are increasingly gaining importance. Amongst the different bioseparation techniques, magnetic separation is the most promising. The development of magnetically responsive microspheres has brought an additional driving force into play. Particles that are bound to magnetic fluids can be used to remove cells and molecules by applying magnetic fields and-in vivo-to concentrate drugs at anatomical sites with restricted access. These possibilities form the basis for well-established biomedical applications in protein and cell separation. Additional modifications of the magnetic particles with monoclonal antibodies, lectins, peptides, or hormones make these applications more efficient and also highly specific. The isolation of various macro molecules such as enzymes, enzyme inhibitors, DNA, RNA, antibodies and antigens etc. from different sources including nutrient media, fermentation broth, tissues extracts and body fluids, has been done by using magnetic absorbents. In case of enzyme separation, the appropriate affinity ligands are immobilized on polymer coated magnetic carrier or magnetizable particles. Immobilized protein A or protein G on silanized magnetite and fine magnetotactic bacteria can be used for isolation and purification of IgG. Monosized super paramagnetic particles, Dynabeads, have been used in isolation of mRNA, genomic DNA and proteins.

(3) Magnetically induced Hyperthermia for treatment of cancer: Heat treatment of organs or tissues, such that the temperature is increased to 42–46 C and the viability of cancerous cells reduces, is known as hyperthermia. It is based on the fact that tumor cells are more sensitive to temperature than normal cells. In hyperthermia it is essential to establish a heat delivery system, such that the tumor cells are heated up or inactivated while the surrounding tissues (normal) are unaffected.

a)Intracellular hyperthermia:The alternative approach is to use fine particles as heat mediators instead of needles or rods such that hyperthermia becomes noninvasive. When fluids containing submicron-sized magnetic particles(typically 1–100nm) are injected, These particles are easily incorporated into the cells, since their diameters are in the nanometer range. These magnetic particles selectively heat up tissues by coupling AC magnetic field to targeted magnetic nano particles. As a result, the whole tumor can be heated up uniformly This is called intracellular hyperthermia. It has been shown that malignant cells take up nine times more magnetic nano particles than normal cells. Therefore the heat generated in malignant cells is more than in normal cells. Also, as blood supply in the cancerous tissues is not normal, the heat dissipation is much slower. Hence, the temperature rise in the region of tumor is higher than in the surrounding normal tissues. It is therefore expected that this therapy is much more concentrated and localized. b) Magnetic fluid hyperthermia (MFH):Magnetic fluids can be defined as fluids, consisting of ultramicroscopic particles. (~100Å) of magnetic oxide. Magnetic fluid hyperthermia is based on the fact that sub domain magnetic particles produce heat through various kinds of energy losses during application of an external AC magnetic field. If magnetic particles can be accumulated only in the tumor tissue, cancer specific heating is available, various biocompatible magnetic fluids. Cationic magnetoliposomes and affinity magnetoliposomes have been used for hyperthermia treatment. c) Combination therapy: There also exists the combination therapy which would induce hyperthermia treatment followed by chemotherapy or gene therapy. A combination of chemotherapy or radiation therapy with hyperthermia is found much more effective than hyperthermia itself. The approach involves use of magnetic carriers containing a drug to cause hyperthermia using the standard procedure, followed by the release of encapsulated drug that will act on the injured cells. It is anticipated that the combined treatment might be very efficient in treating solid tumor. Several reasons are given for the enhanced effect. Tumors are poorly vascularised and it can be hard for therapeutic agents to reach their target. Heat increases the perfusion of a tumor and therefore drugs are transported more effectively into the target tissues. In addition, heat makes blood vessels more

permeable to drugs. This occurs preferentially in tumors where blood vessels tend to be structurally incomplete. On the other hand, normal blood vessels are surrounded by a basement membrane and other perivascular cells and not significantly affected by heat. It has recently been reported that hyperthermia increases the rate of liposome leakage into tumors by a factor of 2–5 depending on the type of tumor. In normal tissues however, enhancement of liposome leakage is not reported.

(4) Magnetic control of pharmacokinetic parameter rand Improvement of Drug release: Langer et al.embedded magnetite or iron beads in to a drug filled polymer matrix and then showed that they could activate or increase the release of drug from the polymer by moving a magnet over it or by applying an oscillating magnetic field (Langer et al.,1980; Edelman and Langer,1993 ).The microenvironment with in the polymer seemed to have shaken the matrix or produced ‘micro cracks’ and thus made the influx of liquid, dissolution and efflux of drug possible thereby achieving magnetically controlled drug release. Macromolecules such as peptides have been known to release only at a relatively low rate from a polymer controlled drug delivery system, this low rate of release can be improved by incorporating an electromagnetism triggering vibration mechanism into the polymeric delivery devices with a hemispheric design; a zero-order drug release profile is achieved.

(5) Magnetic targeting of radioactivity: Magnetic targeting can also be used to deliver the therapeutic radioisotopes (Hafely, 2001).the advantage of these method over external beam therapy is that the dose can be increased, resulting in improved tumor cell eradication, without harm to adjacent normal tissues22.

Targeted therapy for brain tumours Although previously considered untreatable, brain tumours no longer carry the same prognosis as they did even a decade ago. Recent advances in drug delivery to the central nervous system have not only bypassed physiological constraints such as the blood–brain barrier, but have, in fact, changed the course of treatment for patients with malignant brain tumours. The creation of targeted therapies, which spare normal tissue and destroy tumour cells, is changing the field of neuro-oncology. In this article, we review recent developments in the delivery of drugs to tumours of the central nervous system, discuss current trends and directions in the development of novel drugs and delivery systems, and present new and cutting-edge strategies for overcoming the challenges ahead.

FIGURE 1 | Development and progression of astrocytic brain tumours.

Malignant brain tumours can arise in one of two ways. On the one hand, astrocytes undergo genetic changes accompanied by upregulation of certain receptors, such as the platelet-derived growth factor (PDGF), endothelial growth factor receptor (EGFR) or vascular endothelial growth factor (VEGF). These progressive changes culminate in the formation of a glioblastoma. On the other hand, most primary glioblastomas

arise de novo, without the need for gradual progression from an astrocytoma to a high-grade astrocytoma to a glioblastoma multiforme.

Brain tumors are one of the most lethal forms of cancer. They are extremely difficult to treat. Although, the rate of brain tumor incidence is relatively low, the field clearly lacks therapeutic strategies capable of overcoming barriers for effective delivery of drugs to brain tumors. Clinical failure of many potentially effective therapeutics for the treatment of brain tumors is usually not due to a lack of drug potency, but rather can be attributed to shortcomings in the methods by which a drug is delivered to the brain and into brain tumors. In response to the lack of efficacy of conventional drug delivery methods, extensive efforts have been made to develop novel strategies to overcome the obstacles for brain tumor drug delivery. The challenge is to design therapeutic strategies that deliver drugs to brain tumors in a safe and effective manner. This review provides some insight into several potential techniques that have been developed to improve drug delivery to brain tumors, and it should be helpful to clinicians and research scientists as well.

CHAPTER-5

CONCLUSION: The blood brain barrier (BBB) and the systemic toxicity of conventional chemotherapy present obstacles to the success of future blood-borne drug therapies of brain tumors. The work with polymer-encapsulated cancer drugs suggests an alternative and more focused treatment approach. Our experimental strategy integrates direct intracerebral drug delivery, sustained drug release from liposomes or polymer implants, and increased targeting of the drug either by chemically modifying the drug or by using tumor-specific carriers. This review will present some of the recent work on targeted drug delivery for brain cancer treatment. Cancer is one of the most challenging diseases today, and brain cancer is one of the most difficult malignancies to detect and treat mainly because of the difficulty in getting imaging and therapeutic agents across the blood-brain barrier and into the brain. Many investigators have found that nanoparticles hold promise for ferrying such agents into the brain [20-22]. Apolipoprotein E was suggested to mediate drug transport across the blood-brain barrier [23]. Loperamide, which does not cross the blood-brain barrier but exerts antinociceptive effects after direct injection into the brain, was loaded into human serum albumin nanoparticles and linked to apolipoprotein E. Mice treated intravenously with this complex induced antinociceptive effects in the tail-flick test. The efficacy of this drug delivery system of course depends upon the recognition of lipoprotein receptors. Kopelman and colleagues designed Probes Encapsulated by Biologically Localized Embedding (PEBBLE) to carry a variety of unique agents on their surface and to perform multiple functions [22]. One target molecule immobilized on the surface could guide the PEBBLE to a tumor. Another agent could be used to help visualize the target using magnetic resonance imaging, while a third agent attached to the PEBBLE could deliver a destructive dose of drug or toxin to nearby cancer cells. All three functions can be combined in a single tiny polymer sphere to make a potent weapon against cancer. Another anti-cancer drug, doxorubicin, bound to polysorbate-coated nanoparticles is able to cross the intact blood-brain barrier and be released at therapeutic concentrations in the brain [24]. Smart superparamagnetic iron oxide particle conjugates can be used to target and locate brain tumors earlier and more

accurately than reported methods [25]. It is known that folic acid combined with polyethylene glycol can further enhance the targeting and intracellular uptake of the nanoparticles. Therefore, nanomaterial holds tremendous potential as a carrier for drugs to target cancer cells. It is very difficult for the medicine to destruct the target organism at the point of infection because of complex cellular network of an organism. Target delivery of drugs, as the name suggests, is to assist the drug molecule to reach preferably to the desired site. The inherent advantage of his technique has been the reduction in the dose and side effects of the drug. By virtue of their size smaller than that of blood capillaries, intravenously administered particulate drug carriers get accumulated in the liver cells. Among the particulate drug carriers liposome's are a potential mode of delivery for the treatment of intracellular infections as the cells of mononuclear phagocytic system easily take these up. Microparticles may serve as future mode of delivery for the drugs of protein nature . Orally delivered micro particles (<5 urn in size ) are taken up by the peye's patches. This leads to induction of immune response against the antigen released from the microparticles. Also, the antigen is protected from the loss of activity in the Gl tract. A major limitation is the effective uptake of these particles from the Gl tract which is even less than 1% However, a combination of biological approach such as incorporation of specific ligands on the surface of surface of these particles enhances their uptake. Magnetic Vesicular systems have been realized as extremely useful carrier systems in various scientific domains. Over the years, magnetic microcarriers have been investigated for targeted drug delivery especially magnetic targeted chemotherapy due to their better tumor targeting, therapeutic efficacy, lower toxicity and flexibility to be tailored for varied desirable purposes

CHAPTER-6

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