1.0 BRIEF INTRODUCTION OF DENDRIMER

A dendrimer is generally described as a macromolecule, which is characterized by its

highly branched 3D structure that provides a high degree of surface functionality and

versatility. Dendrimers are a unique class of polymers which play an important role in

emerging nanotechnology. Targeted drug delivery is one of the most attractive

potential applications of Dendrimers. The present review briefly describes about

dendrimer synthesis strategies, dendrimer functionlisation properties which having

crucial importance and their potential applications such as carrie molecule,

multivalent diagnostics for MRI, Sensors, multivalent bioconjugate, Light harvesting,

catalysis, artificial enzymes, coatings, inks and other application. Due to their

multivalent and monodisperse character, dendrimers have stimulated wide interest in

the field of chemistry and biology, especially in applications like drug delivery, gene

therapy and chemotherapy.

1.1 MOLECULAR ARCHITECHTURE[[

Dendrimers are an interesting class of macromolecules that have a highly

symmetrical, hyper-branched and spherical structure. Dendrimers possess an

architecture consisting of (1) a core; (2) an interior of shells (generation); and (3) an

exterior (outermost layer), which often has terminal functional groups [Buhleier, et al,

1978] . The core determines the size and shape of the dendrimer, the interior

determines the amount of void space that can be enclosed by the dendrimer, and the

exterior allows growth of the dendrimer or other chemical modification. This unique

architecture makes dendrimers monodispersed macromolecules compared to classical

linear polymers[Richardson, et al, 2000] . In dendritic structures, the number of

terminal groups increases exponentially with a linear increase in the generation of the

dendrimer. This relationship limits the ultimate size of the dendrimer due to steric

crowding of the terminal groups. Several new branching points are available at each

repeating unit in their structure for hyperbranched growth [Tomalia, et al,

1985] .Polyamidoamine (PAMAM) dendrimers are a newer class of polymer that

possess a number of interesting and useful pharmaceutical applications. PAMAM

dendrimers have capability of enhancing the solubility of low solubility drugs. They

also have potential application for the delivery of DNA and oligonucleotide and as

platforms for the development of cancer therapeutics. Dendrimers have been shown to

1

cross cell barriers at sufficient rates to act as potential carrier/delivery systems.

Surface engineering has been found to reduce cytotoxicity and enhance transport

studies on the mechanism of dendrimer transport across epithelial cells [Newkome, et

al, 1985]. The core typically consists of a polyfunctional molecule, where the

functionality governs the number of branches that extend from it. The tetrahedral core

gives a spherical polymer whereas linear polymeric cores give rod-shaped

dendrimers[Johansson, et al, 1992].

Figure: 1.1.1 structure of dendrimer

.1.2 TYPES OF DENDRIMER

Types of dendrimers

1. Liquid crystalline dendrimers

They consist of mesogenic (liq. crystalline)monomerse.g. mesogen

functionalized carbosilane dendrimers. Functionalization of end group of carbosilane

dendrimers with 36 mesogenic units, attached through a C-5 spacer, leads to liquid

crystalline dendrimers that form broad smetic A phase in the temperature range of 17–

2

130 ◦C[Liu, et al, 2007] . claims that they have synthesized first photosensitive liquid

crystalline dendrimer with terminal cinnamoyl groups[Tomalia, et al, 1990]. They

have confirmed the structure and purity of this LC dendrimer by 1H NMR and GPC

methods. It was shown that such a dendrimer, under UV irradiation, can undergo E-Z

isomerization of the cinnamoyl groups and [2 + 2] photocycloaddition leading to the

formation of a three-dimensional network[Emrick, et al, 2000].

2. Tecto dendrimers

Tecto-dendrimers are composed of a core dendrimer, which may or may not

contain the therapeutic agent, surrounded by dendrimers. The surrounding dendrimers

are of several types, each type designed to perform a function necessary to a smart

therapeutic nanodevice the PAMAM core–shell tecto dendrimer[Boas, et al, 2004].

The Michigan Nanotechnology Institute for Medicine and Biological Sciences (M-

NIMBS) are developing a tecto dendrimers which perform the following functions:

diseased cell recognition, diagnosis of disease state, drug delivery, reporting location

and reporting outcome of therapy[Sunder,et al, 2000,] They have already made and

tested functional dendrimers which perform each of these functions. Even after a

many questions on the way they are planning to produce a smart therapeutic

nanodevice for the diseased cell like a cancer cell or a cell infected with a virus[Gong,

et al, 1999].

Fig:Schematic representation of PAMAM core–shell tecto dendrimer.

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3. Chiral dendrimers

The chirality in these dendrimers is based upon the construction of

constitutionally different but chemically similar branches to chiral core[Agarwal, et

al, 2008]. Chiral, nonracemic dendrimer with well-defined stereochemistry is

particularly interesting subclass, with potential applications in asymmetric catalysis

and chiral molecular recognition[Peterson, et al, 2003,].

4. PAMAMOS dendrimersRadially layered poly(amidoamine-organosilicon) dendrimers (PAMAMOS)

are inverted unimolecular micelles that consist of hydrophilic, nucleophilic

polyamidoamine (PAMAM) interiors and hydrophobic organosilicon (OS) exteriors.

These dendrimers areexceptionally useful precursors for the preparation of

honeycomblike networks with nanoscopic PAMAM and OS domains[Agarwal, et al,

2008].

5. Hybrid dendrimers

Hybrid dendrimers are combination of dendritic and linear polymers in hybrid

block or graft copolymer forms. The small den- drimer segment coupled to multiple

reactive chain ends provides an opportunity to use them as surface active agents,

compatibilizers or adhesives, e.g. hybrid dendritic linear polymers .

6. Peptide dendrimers

Denrimers having peptides on the surface of the traditional dendrimer

framework and dendrimers incorporating amino acids as branching or core units are

both defined as ‘peptide dendrimers’[Esfand, at al, 2000]. Also peptide dendrimers

can be defined as macromolecules that contain peptide bonds in their structure.

Because of biological and therapeutical relevance of peptide molecules, peptide

dendrimers play an important role in diverse areas including cancer, antimicrobials,

antiviral, central nervous system, analgesia, asthma, allergy and Ca+2 metabolism. On

the basis of their ability to be taken up by cells, making peptides very useful for drug

delivery[Hawker,et al, 1991]. One more interesting application of peptide dendrimers

is that can be used as contrast agents for magnetic resonance imaging (MRI),

magnetic resonance angiography (MRA), fluorogenic imaging and serodiagnosis .

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7. Glycodendrimers

The term ‘glycodendrimers’ is used to describe dendrimers that incorporate

carbohydrates into their structures . Glycodendrimers can be classified as: (i)

carbohydrate-coated; (ii) carbohydrate centered; and (iii) fully carbohydrate-based.

Glycodendrimers have been used for a variety of biologically relevant applications

such as, glycodendrimers with surface carbohydrate units have been used to study the

protein–carbohydrate interactions that are in many intercellular recognition

events[Grubbs, et al, 1997]. The accessibility of the sugars is an important

consideration for glycodendrimers used effectively to evaluate protein–carbohydrate

interactions. Like this, study of protein–carbohydrate interactions, incorporation into

analytical devices, formulation of gels, targeting of MRI contrast agents, drugs and

gene delivery systems are some of the areas where glycodendrimers are likely to be

beneficial[Guan,et al, 1999].

8. PAMAM dendrimerPerhaps the family of dendrimers most investigated for drug delivery is the

polyamidoamine (PAMAM) dendrimer[Cheng, et al, 2008] . Manysurface

modifiedPAMAMdendrimers are non-immunogenic, water-soluble and possess

terminal-modifiable amine functional groups for binding various targeting or guest

molecules[Yin, et al, 1998]. PAMAM dendrimers generally display concentration-

dependent toxicity and haemolysis.PAMAMdendrimers are hydrolytically degradable

only under harsh conditions because of their amide backbones, and hydrolysis

proceeds slowly at physiological temperatures . The internal cavities of PAMAM

dendrimers can host metals or guest molecules because of the unique

functionalarchitecture, which contains tertiary amines and amide linkages. PAMAM

dendrimers are generally prepared by divergent method and product up to generation

10 (G10) have been obtained.PAMAM dendrimers are the most extensively reported

moiety for almost all existing applications of dendrimers [Pushkar, et al, 2006]. .

1.3 SYNTHETIC APPROACH OF DENDRIMER

Dendritic polymers are synthesized using a stepwise repetitive reaction

sequence that leads to monodispersed, with a nearly perfect hyper branched topology

radiating from a central core and grown generation by generation. The synthetic

procedures developed for dendrimer preparation by controlling the critical molecular

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design parameters, such as size, shape, surface/interior chemistry, flexibility, and

topology[Sakthivel , et al, 2003]. .

1.3.1 Divergent Synthesis

In the divergent approach, dendrimers are prepared from the core as the starting point

and built up generation by generation. In the divergent way, problems occur from an

incomplete reaction of the end groups, since these structural defects accumulate with

the buildup of further generation. As the side products possess similar physical

properties, chromatographic separation is not always possible[Balogh,et al, 1998].

The higher generations of divergently constructed dendrimers always contain certain

structural defects. To prevent side reactions and to force reactions to completion large

excess of reagents is required. This may cause some difficulties in the purification of

the final product[Yiyun, et al, 2008].

Divergent synthesis of dendrimer

1.3.2.Convergent synthesis

The convergent approach starts from the surface and ends at the core, where

the dendrimer segments (dendron’s) are coupled. In this way, only a small number of

reactive sites are functionalized in each step, giving a small number of possible side-

reactions per step[Rajca, et al, 1993].In divergent method, purification of the high-

generation dendrimer becomes more difficult because of increasing similarity

between reactants and formed product. In convergent method, with proper purification

after each step, gives dendrimers without defects. On the other side, it does not allow

the formation of high generations because steric problems occur in the reactions of the

dendron’s and the core molecule [Hawker,et al, 1993].

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Convergent synthesis of dendrimer

1.3.3 Double exponential and mixed growth [

In this approach two products (monomers for both convergent and divergent

growth) are reacted together to give an orthogonally protected trimer ,which may be

used to repeat the growth process again[Liu, et al,1999] .strength of double

exponential growth is more stuble than the ability to build large dendrimers in

relatively few steps.

1.3.4 Hypercores and branched monomers growth

This method involved the pre-assembly of oligomeric species which can be

linked to give dendrimers in fewer steps or higher yields [Frechet and Tomalia,

2001].

1.4 PROPERTIES OF DENDRIMER

Nanoscale sizes that have similar dimensions to important bio-building

blocks, e.g., proteins, DNA.

Numbers of terminal surface groups (Z) suitable for bio-conjugation of drugs,

signaling groups, targeting moieties or biocompatibility groups.

Surfaces that may be designed with functional groups to augment or resist

trans-cellular, epithelial or vascular biopermeability[Rajca, et al, 1998].

7

An interior void space may be used to encapsulate small molecule drugs,

metals, or imaging moieties. Encapsulating in that void space reduces the

drug toxicity and facilitates controlled release. [[

Positive biocompatibility patterns that are associated with lower generation

anionic or neutral polar terminal surface groups as compared to higher

generation neutral apolar and cationic surface groups[Svenson, et al, 2005].

Non- or low-immunogenicity associated with most dendrime surfaces

modified with small functional groups or polyethylene glycol (PEG)[ Miller,

et al, 1997].

Surface groups that can be modified to optimize biodis-tribution; receptor

mediated targeting, therapy dosage or controlled release of drug from the

interior space.

Ability to arrange excretion mode from body, as a function of nanoscale

diameter.

Dendrimers are monodisperse macromolecules, unlike linear polymers. The

classical polymerization process which results in linear polymers is usually

random in nature and produces molecules of different size, whereas size and

molecular mass of dendrimers can be specifically controlled during

synthesis[Liu, et al, 2000].

Because of their molecular architecture, dendrimers show some significantly

improved physical and chemical properties when compared to traditional

linear polymers.

In solution, linear chains exist as flexible coils; in contrast, dendrimers form

a tightly packed ball. This has a great impact on their rheological properties.

Dendrimer solution has significantly lower viscosity than linear

polymers[Svenson, et al, 2009] . When the molecular mass of dendrimers

increases, their intrinsic viscosity goes through a maximum at the fourth

generation and then begins to decline. Such behavior is unlike that of linear

polymers. For classical polymers the intrinsic viscosity increases continuously

with molecular mass[Hong, et al, 2007].

The presence of many chain-ends is responsible for high solubility and

miscibility and for high reactivity. Dendrimers solubility is strongly

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influenced by the nature of surface groups. Dendrimers terminated in

hydrophilic groups are soluble in polar solvents, while dendrimers having

hydrophobic end groups are soluble in nonpolar solvents[Zhuo, et al, 1999]. In a solubility test with tetrahydrofuran as the solvent, the solubility of

dendritic polyester was found remarkably higher than that of analogous linear

polyester. A marked difference was also observed in chemical reactivity.

Dendritic polyesters was debenzylated by catalytic hydrogenolusis whereas

linear polyester was unreactive[Esfand, et al, 2001].

Dendrimers have some unique properties because of their globular shape and

the presence of internal cavities. The most important one is the possibility to

encapsulate guest molecules in the macromolecule interior[ Sonke and

Tomalia, 2005].

1.4.1 Encapsulation of drugs within the dendritic architecture .

Dendritic artitecture (open nature ) has led several groups to investigated

the possibility of encapsulation drug molecules within the branches of a

dendrimer .This offers the potential of dendrimers to interact with labile or poorly

soluble drugs ,enhances drug stability ,bioavailability and controlling its release ,the

nature of drug encapsulation within a dendrimer may be simple physical

entrapment ,or can involve non- bonding interactions with a dendrimer may be simple

physical entrapment ,or can involve non-bonding interactions with specific structure

within the dendrimer[Christine, et al, 2005].

1.4.2 Surface interactions between drugs and dendrimers

The external surface of dendrimers have been investigated as potential sites of

interaction with drugs .although the number of guest molecules incorporated into a

dendrimer may be dependent to a limited extent on the architecture of a

dendrimer ,the loading capacity may be dramatically increased by the formation of a

complex with the large number of groups on the dendrimer surface. The dendrimer

with each increasing generation of dendrimer[Freeman and Frechet, et al, 2005].

1.4.3 Electrostatic interaction between drug and dendrimer

The presences of large number of ionisable groups on the surface of

dendrimers provides an interesting opportunity for electrostatic attachment of

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numerous ionizable drugs ,providing the resultant complex retains sufficient water

solubility. for example electrostatic interaction can occur between the carboxyl group

of the dendrimers .it has been estimated that approximately 40 ibuprofen molecule

interact with G4 PAMAM dendrimers at pH 10.5 causing a considerable enhancement

of drug solubility[Hawker, et al, 1990].

1.4.4 Conjugation of drug to dendrimer

The covalent attachment of drugs to the surface groups of dendrimers through

hydrolysable or biodegradable linkages offers the opportunity for a greater control

over drug released by ester hydrolysis of the PEG-PAMAM (G3) conjugate was

approximately the same (within 3% ) as that of non modified penicillin [Barbara and

Maria,et al, 2001].

1.5 DENDRIMERS IN DRUG DELIVERY

The development of an efficient drug delivery system is very important to

improve the pharmacological activity of drug molecules. Dendrimers have emerged as

new alternatives and efficient tools for delivery of drug molecule[Hong, et al,. 2004].

As compare to linear polymeric carriers, the multivalent functionalities of dendrimers

can be linked to drug molecules or ligands in a well-defined manner and can be used

to increase the binding efficiency and affinity of therapeutic molecules to receptors

via synergistic interaction[Patel, et al, 2007].

1.5.1 Oral drug delivery

Oral drug delivery systems are very important to the field of medicine, since

most of the common illnesses are treated via oral rout of medication. Dendrimers

those are able to hold medication with good durability but also biodegradable within a

biological system[. By combining the ideas of drug carriers and degradability,

research has recently focused on controlled degradation of dendrimers and release of

compounds via oral route. Encapsulation and conjugation of drug with dendrimers

have shown immense employment for delivery of hydrophobic and labile

drugs[Tomalia, et al, 1985] . Transport of dendrimers throughout epithelial part of

gastrointestinal tract depends upon its characteristics. Packaging a drug in a dendrimer

host not only makes it soluble but also allows it to bypass the transporter protein that

would normally stop it from being absorbed in the intestines after it has been taken

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orally. Anticancer and antihypertensive drugs are most promising candidate for oral

rout using dendrimer as carrier[Soulie, et al, 1999]

1.5.2 Ocular drug delivery

The topical application of active drugs to the eye is the most prescribed route

of administration for the treatment of various ocular disorders. It is generally agreed

that the intraocular bioavailability of topically applied drugs is extremely poor. These

results mainly due to drainage of the excess fluid via nasolacrimal duct and

elimination of the solution by tear turnover. Several research advances have been

made in ocular drug delivery systems by using specialized delivery systems such as

polymers, liposomes, or dendrimers to overcome some of these disadvantages.

1.5.3 Transdermal drug delivery

In recent era, dendrimers have found applications in transdermal drug delivery

systems. Generally, bioactive drugs have hydrophobic moieties in their structure,

resulting in low water-solubility that inhibits efficient delivery into cells[Morgan, et

al, 2003]. Dendrimers designed to be highly water-soluble and biocompatible have

been shown to be able to improve drug properties such as solubility and plasma

circulation time via transdermal formulations and to deliver drugs efficiently. The

viscosity imparting property of a dendrimers solution allows for ease of handling of

highly concentrated dendrimer formulations for these applications. Dendrimers have

been shown to be useful as transdermal drug delivery systems for nonsteroidal anti-

inflammatory drugs (NSAIDs), antiviral, antimicrobial, anticancer, or

antihypertensive drugs[Caminati, et al, 1990].

1.5.4 Targeted gene delivery

Dendrimers can act as carriers, called vectors, in gene therapy. Vectors

transfer genes through the cell membrane into the nucleus. Currently liposomes and

genetically engineered viruses have been mainly used for this[Thomas, et al, 2004].

PAMAM dendrimers have also been tested as genetic material carriers. Cationic

dendrimers (Polypropylenimine (PPI) dendrimer) deliver genetic materials into cells

by forming complexes with negatively charged genetic materials through electrostatic

interaction. Cationic dendrimers lend themselves as non-viral vectors for gene

delivery because of their ability to form compact complexes with DNA[Newkome, et

11

al, 2001]. Another potential application could be the use of dendrimers coated with

sialic acid for the influenza virus to attach to the cell surface. In addition, dendrimers

are non-immunogenic and are thus uniquely suited as carrier structures for drugs or

bioactive molecules without degradation in immune system[Adronov, et al, 2000].

1.5.5 Dendrimers in pulmonary drug deliveryDendrimers have been reported for pulmonary drug delivery also. During one

study, efficacy of PAMAM dendrimers in enhancing pulmonary absorption of

Enoxaparin was studied by measuring plasma anti-factor Xa activity, and by

observing prevention efficacy of deep vein thrombosis in a rodent model[Jain, et al,

2001] . G2 and G3 generation positively charged PAMAM dendrimers increased the

relative bioavailability of Enoxaparin by 40%, while G2.5 PAMAM, a half generation

dendrimers, containing negatively charged carboxylic groups had no effect.

Formulations did not adversely affect mucociliary transport rate or produce extensive

damage to the lungs. respectively, as compared to free drugs[Boris and Rubinstein, et

al, 1996].

1.6 LIPOSOME DRUG DELIVERY SYSTEM

Liposomes are the leading drug delivery systems for the systemic

(intravenous) administration of drugs. There are now liposomal formulations of

conventional drugs that have received clinical approval and many others in clinical

trials that bring benefits of reduced toxicity and enhanced efficacy for the treatment of

cancer and other life-threatening diseases[Louvet, et al, 2000]. The mechanisms

giving rise to the therapeutic advantages of liposomes, such as the ability of long-

circulating liposomes to preferentially accumulate at disease sites such as tumours,

sites of infection and sites of inflammation are increasingly well understood. Further,

liposome-based formulations of genetic drugs such as antisense oligonucleotides and

plasmids for gene therapy that have clear potential for systemic utility are increasingly

available[Thomas, et al, 2005] . This work reviews the liposomal drug delivery field,

summarises the success of liposomes for the delivery of small molecules and indicates

how this success is being built on to design effective carriers for genetic drugs[Cottu,

et al, 2000

1.6.1 liposome with dendrimer

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This is the first study demonstrating the successful encapsulation of a 'guest-

host' oxaliplatin-dendrimer complex (i.e. oxaliplatin [guest] contained in a

poly(amidoamine) (PAMAM) dendrimer (G=4) [host]), which is then in turn

encapsulated within a liposome to produce a 'modulatory liposomal controlled release

system' (MLCRS)[ Malik, et al 2000]. These new constructs have exhibited unique,

modulatory drug release profiles compared to traditional drug-liposome (guest-host

complexes). Furthermore, they have manifested enhanced oxaliplatin activity against

lung cancer cell lines DMS114 and NCI-H460 with a (10x) one order of magnitude

higher growth inhibition activity against the NCI-H460 cancer cell line[Tomalia,

2005].

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1.7 DRUG PROFILE

1.7.1 Oxaliplatin

Oxaliplatin is a platinum containing antineoplastic agent[Jansen, et al, 1995] . It is thought to exert its cytotoxic action in a similar manner to alkylating agents by

causing inter- and intrastrand cross links in DNA, inhibiting DNA synthesis and

inducing apoptotic cell death. l Oxaliplatin is licensed for the first line treatment of

metastatic colorectal cancer in combination with 5-fluorouracil and folinic

acid[Jevprasesphant, et al, 2003] . l The addition of oxaliplatin to 5-fluorouracil and

folinic acid significantly improves the overall response rate in patients with

previously untreated colorectal cancer[Wiseman, et al, 1999].

1.7.2 Identification

Molecular weight 395.296 gm/mol

Chemical formula C8H12N2O4Pt

Structure

Synonyms Oxaliplatin [Usan:Inn:Ban] Oxaliplatino [Spanish] Oxaliplatinum [Latin] Oxaloplatine [French] Oxaloplatino [Spanish][ Bleiber, et al,

1996]IUPAC name (3aR,7aR)-octahydro-2',5'-

dioxaspiro[cyclohexa[d]1,3-diaza-platinacyclopentane-2,1'-cyclopentane]-3',4'-dione

Specific optical rotation + 74.5 to + 78.0 (dried substance

Solubility Water: slightly

Methanol: very slightly

14

Ethenol: practically soluble

Acetone: practically soluble

Melting point 198ºc and 292ºc[Giaccheti, et al, 2000]

PH 3 to 9[De Gramont, et al, 2000]

Common trade name Eloxatin

Classification Alkylating agent

Additional Names  [(1R,2R)-1,2-cyclohexanediamine-N,N¢][oxalato (2-)-O,O¢]platinum; oxalato(1R,2Rcyclohexanediamine)platinum(II); Pt(oxalato)(trans-l-dach); oxalato (trans-l-1,2-diaminocyclohexane)platinum (II); oxalatoplatin; oxalatoplatinum; l-OH[13,14,15]

1.7.3 Pharmacology

1.7.3.1 Mechanism of action

Oxaliplatin belongs to a new class of platinum agent. It contains a platinum

atom complexed with oxalate and diaminocyclohexane (DACH). The bulky DACH is

thought to contribute greater cytotoxicity than cisplatin and carboplatin[Misset, et al,

2000]. The exact mechanism of action of oxaliplatin is not known. Oxaliplatin forms

reactive platinum complexes which are believed to inhibit DNA synthesis by forming

interstrand and intrastrand cross-linking of DNA molecules. Oxaliplatin is not

generally cross-resistant to cisplatin or carboplatin, possibly due to the DACH group

and resistance to DNA mismatch repair[Freyer,et al, 2000]. Preclinical studies have

shown oxaliplatin to be synergistic with fluorouracil and SN-38, the active metabolite

of irinotecan[Carraro,at al 2000] Oxaliplatin is a radiation-sensitizing agent[Culy, at

al, 2000].It is cell-cycle-phase nonspecific[Graham,et al, 200

1.7.4 Pharmacokinetics

Distribution Minimal in plasma accumulation ;in

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erythrocytes does not diffuse into plasma

or act as drug reservoir

Volume of distribution Ultrafilterable platinum;581+261 L6

Plasma protein binding 70-95%[Souglakos, et al, 2002]

Metabolism Rapid nonengymatic biotransformation to

reactive platinum complexes

Excretion Platinum is mainly by renal excretion and

tissue distribution while platinum

metabolites are mainly by renal excretion

[Scheithauer, et al,2003].

Terminal half life Platinum elimination from

erythrocytes :48 days

1.7.5 Pharmacodynamics

Oxaliplatin exhibits a wide spectrum of both in vitro cytotoxicity and in vivo

antitumour activity in a variety of tumour model systems, including human colorectal

cancer models. Oxaliplatin also demonstrates in vitro and in vivo activity in various

cisplatin resistant models. A synergistic cytotoxic action has been observed in

combination with fluorouracil both in vitro and in vivo[Andre,at al, 1999].

1.7.6 Uses

Primary uses : colorectal cancer

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Other uses : breast cancer ,gastric canser ,germ cell cancer head and neck

cancer,lung cancer ,non small cell,ovarian cancer ,pancreatic cancer,prostate cancer

[Giacchetti, et al, 2000]

1.7.7 Side effects

Organ site Side effects

Allergy/immunology

blod /bone marrow

Febrile neutropenia

Anaphylaxis(0.5-2%)

Anemia(64-83%,severe 4-5%)

Immune hemolytic anemia(rare)

Neutropenia:single agent(15%,severe 3%)with fluorouracil

and leucovorin(76%,sever 4%)[Goldberg, et al, 2004]

Constitutional

symptoms

demetology /skin

Fever 36%

Extravasation hazard:irritant

Alopecia 2%

Gastrointestinal Emetogenic potential:heigh moderate

Diarrhea:single agent(41%,severe 5%),with fluorouracil and

leucovorin(58%,severe 10%)

Nausea,vomiting

Hepatic Liver function abnormalities

Infection Infection 23%

Neurology Central neuro toxicity(rare)

1.8 LITERATURE REVIEW

17

M P Toraskar et al (2011) has reported that the dendrimers, a nanoparticles based

drug-delivery system have numerous applications in pharmaceuticals such as

enhancing the solubility of poorly soluble drugs, enhancing the delivery of DNA and

oligonucleotides, targeting drug at specific receptor site and ability to act as carriers

for the development of drug delivery systems.

V. Rajesh Babu et al (2010) has reported that the dendrimers are a new class of

synthetic polymers which have the structure like a tree or star shape, with a central

core, interior branches and terminal groups which decorate the surface.

Barbara Klajnert et al (2001) has reported that the dendrimers are a new class of

polymeric materials. They are highly branched, monodisperse macromolecules. The

structure of these materials has a great impact on their physical and chemical

properties. As a result of their unique behaviour dendrimers are suitable for a wide

range of biomedical and industrial applications. The paper gives a concise review of

dendrimers’ physico-chemical properties and their possible use in various areas of

research, technology and treatment.

G.srinubabu et al (2005) has reported that the a simple and reproducible

spectrophotometric method has been developed for the estimation of Oxaliplatin in

pure and dosage forms. The method is based on the reaction of the drug with 1, 10

Phenanthroline in presence of ferric chloride, which forms a blood red colored

complex exhibiting maximum absorption at 520 nm.

Claudia Burzlet et al (2000) has reported that the to evaluate the therapeutic

efficacy of oxaliplatin and to analyze the pharmacokinetics of both ultrafiltrable (free)

and protein–bound platinum in patients with metastatic colon cancer.

Norbert Maurer et al (2001) has reported that the liposomes are the leading drug

delivery systems for the systemic (intravenous) administration of drugs. There are

now liposomal formulations of conventional drugs that have received clinical

approval and many others in clinical trials that bring benefits of reduced toxicity and

enhanced efficacy for the treatment of cancer and other life-threatening diseases.

18

Nahid latif et al (1984) has reported that the liposomes, the artificial phospholipid

vesicles, have the capacity of entrapping water soluble substances in their aqueous

compartments. Of the many possible potentials of liposomes their application in

immunology is most significant. Recent studies have shown an adjuvant and a carrier

effect of liposomes to a number of antigens.

AndrewE et al (2006) has reported that the Pegylated liposomal oaliplatin is a

formulation of oxaliplatin in which the molecule itself is packaged in a liposome

made of various lipids with an outer coating of polyethylene glycol. Liposomal

technology is being used in increasing amounts in the therapy of a variety of cancers,

including ovarian cancers. This article reviews the mechanistic actions of this

formulation, the Phase II and Phase III data that helped define the role of pegylated

liposomal oxaliplatin in recurrent ovarian cancer, as well as a discussion of some of

the side-effects and their managemen.

Buleier, E et al, (2009) has reported that the dendritic polymers are belonging to a

special class of macromolecules. They are called "Dendrimers." Similar to linear

polymers, they composed of a large number of monomer units that were chemically

linked together. Due to their unique physical and chemical properties, dendrimers

have wide ranges of potential applications. These include adhesives and coatings,

chemical sensors, medical diagnostics, drug-delivery systems, high-performance

polymers, catalysts, building blocks of supermolecules, separation agents and many

more(116).

Aristarchos Papagiannaros et al ( 2007), has reported that the liposomes

incorporating oxaliplatin-PAMAM complex exhibited release properties which were

advantageous compared to the conventional type of liposomal oxaliplatin in terms of

oxaliplatin toxicity and its availability to the tumor site. This liposomal formulation

may show improved therapeutic properties in vivo.

Andreas Wagne et al, (2010) has reported that the liposomes, spherical vesicles

consisting of one or more phospholipid bilayers, were first described by Bangham

and coworkers. Today, numerous lab scale but only a few large-scale techniques are

available. However, a lot of these methods have serious limitations in terms of

19

entrapment of sensitive molecules due to their exposure to mechanical and/or

chemical stress.

Rok Podlipec et al, (2010) has reported that the Liposome-cell interaction which is

exchange of lipids, absorbtion of liposomes on cell membranes and specially fusion or

endocytosis, can be described with proper equations of mass action.

Buleier, E et al, (2009) has reported that the dendritic polymers are belonging to a

special class of macromolecules. They are called "Dendrimers." Similar to linear

polymers, they composed of a large number of monomer units that were chemically

linked together. Due to their unique physical and chemical properties, dendrimers

have wide ranges of potential applications. These include adhesives and coatings,

chemical sensors, medical diagnostics, drug-delivery systems, high-performance

polymers, catalysts, building blocks of supermolecules, separation agents and many

more.

Gardikis K, et al, (2010) has reported that the liposomal locked-in dendrimers

(LLDs), the anticancer drug oxaliplatin was loaded into pure liposomes or LLDs and

the final products were subjected to lyophilization. The loading of oxaliplatin as well

as its in vitro release rate from all systems was determined and the interaction of

liposomes with dendrimers was assessed by thermal analysis and fluorescence

spectroscopy. The results were very promising in terms of drug encapsulation and

release rate, factors that can alter the therapeutic profile of a drug with low therapeutic

index such as oxaliplatin. Physicochemical methods revealed a strong, generation

dependent, interaction between liposomes and dendrimers that probably is the basis

for the higher loading and slower drug release from the LLDs comp to pure

liposomes.

20

1.9 AIM OF OBJECT

Cancer is a large heterogeneous class of disease in which a group of cells

display uncontrolled growth ,invasion that intrudes upon and destroys adjacent tissues

and often metasizes ,wherein the tumour cells spread to other location in the body via

the lymphatic system or through the blood stream[Augustin, et al, 1998] .these three

malignated properties of cancer differentiate malignant tumors from benign

tumors ,which do not grow uncntollably ,directly invade locally ,or metastazie to

regional lymph nodes or distant body sites like brain ,bone ,liver or orther organs

[Sorbye, et al, 2004].

Platinum-based complexes are important drugs for the treatment of cancer

diseases. Compared to the commonly used Pt(II) compounds oxaliplatin, the recently

reported complexes containing Pt(IV) seem to have several advantages; they are safer,

can be used orally, have a higher scope of anticancer effect, and do not show cross-

resistance to cisplatin[Bennett, et al, 1999]

Liposomes incorporating the oxaliplatin-PAMAM complex exhibited release

properties which were advantageous compared to the conventional type of liposomal

oxaliplatin in terms of oxaliplatin toxicity and its availability to the tumor site. This

liposomal formulation may show improved therapeutic properties in vivo[ Bangham,

et al, 1964].

In the present investigation include to prepare liposomal formulation of

oxaliplatin-pamam complex and their characterization .

The present work is based on following report :

1.Stathopoulos GP et al (2006) has reported that the lipoxal is a liposomal

oxaliplatin, which reduces the cytotoxic agent's adverse reactions without reducing

effectiveness. Our objectives were to determine the adverse reactions, dose-limiting

toxicity (DLT) and the maximum tolerated dose (MTD) of lipoxal.

2.Moraes ML et al (2008) has reported that the cationic pamam dendrimers

were incorporated in the aqueous interior of liposomes order to increase the

encapsulation efficiency for the anticancers drugs.

21

3.Moraes ML et al (2008) has reported that the loading of drug indeed

increased proportionally to dendrimer generation while the system decreased.

4.Papagiannaros A et al (2005)  has reported that the first study

demonstrating the successful encapsulation of a 'guest-host' oxaliplatin-dendrimer

complex (i.e. oxaliplatin [guest] contained in a poly(amidoamine) (PAMAM)

dendrimer (G=4) [host]), which is then in turn encapsulated within a liposome to

produce a 'modulatory liposomal controlled release system' (MLCRS).

22

1.10 PLAN OF WORK

1.Identification of oxaliplatin

a. Melting point

b. U.v spectroscopy

c. I .R spectroscopy

d. Solubility profile

2. Analytical method development

a. Preparation of calibration curve of oxaliplatin in phosphate buffer solution using

uv method (PH-7.4,λmax 207).

b.Preparation of calibration curve of oxaliplatin in water using uv method at λmax

207nm

c.Preparation of calibration curve of oxaliplatin in water using hplc method

d.Preparation of calibration curve of oxaliplatin in plasma using hplc method

3.Preparation of formulation

Preparation of liposome of oxaliplatin with and without dendrimer

Characterization of liposome

1.Particle size determination

a.Optical microscopic method

b.Particle size and size distribution

2.Estimation of dendrimer in external phase

3. Effect of Tris dendrimer and NH2 surface on encapsulation efficiency

4. Invitro release study

5. Stability studies

a. Effect of aging

23

b. Effect of temperature

4. Determination of pharmacokinetics parameters

a. AUC

b. tmax

c. Cmax

24

This chapeter presnts preparation and characterization of dendrimer

phospholipid composition. An attempet was made to understand the formulation

behavior during hydration by various microscopic technique and to estabilish the

presence of dendrimer in the liposomal structure. we have also proved that the

presence of dendrimer modulates the encapsulation, release and pharmacodynamics

properties of the encapsulated drug. The details of experimental methods are as

follows .

2.0 MATERIALS

1. Instruments: Magneticstir with hot plate (simco microscope), Ultrasonic bath

sonicater (relex), Systronics double beam spectrophotometer, Shimadzu hplc,

Electronic balance (Vibra &Essae),whatman filter paper, 2.4nm semipermeable

membrane .

2. Drugs and chemicals: PAMAM dendrimer (Nanosynthons,USA), oxaliplatin(sun

pharma) cholesterol(sigma chemical), lecithine (himedia), HPLC water(rankem),

potassium dihydrogen phosphate(merkindia limited), HPLC grade

methanol(qualigens), choloroform(qualigens).

3. Animal and housing: In the present study male albino rats (150-200g) were used.

All the animals were procured from the disease-free animal house of Central Drug

Research Institute, Lucknow, India. The animals had free access to food and drinking

water as per CPCSEA dietary norms. They were subjected to natural light-dark cycle

(12 hours each). The animals were acclimatized for at least 5 days to the laboratory

conditions prior to experimentation. The experimental protocol was approved by the

Institutional Animal Ethics Committee dated 13-04-2012. The care of the animals

was taken as per the guidelines of CPCSEA, Ministry of Forests & Environment, and

Government of India(Approval no- IAEC/ceu/10).

2.1 METHODS

2.1.1 Preformulation study: Preformulation may be described as a phase of the

research and development process where we characterizes the physical, chemical and

mechanical properties of a new drug substance, in order to develop stable, safe and

25

effective dosage form.Thus we performed this study for authentication and

identification of the drug.

2.1.2 Identification of Oxaliplatin

Drug was identified as per B.P (2010)

2.1.3 Appearance

Physical appearance of the drug was noted by visual observation

2.1.4 Chemical test

Chemical test of the drug sample were performed according to B.P (2010)

Method: Dissolve 0.01 gm in water and dilute to 50 ml with the same solvent.

2.1.5 Melting point

Melting point of drug sample was determined using melting point apparatus.

2.1.6 Solubility profile

The solubility of drug at room temperature was determine in different

solvents.

2.1.7 Scanning of drug

Accurately weight quantity of drug (10mg) was taken in10 ml volumetric

flask, dissolved in 1 ml of water and volume was made upto 100 ml with HPLC grade

water.

Drug solution (0.001%) in water and scanned in range 200-400 nm by

Systronics double beam spectrophotometer and absorption maxima were noted.

2.1.8 Infrared spectroscopy

The IR spectrum of drug sample was obtained by KBr disc method by shimadzu IR

Spectrophotometer CDRI Lucknow (sample code-oxs).

26

2.2 ANALYTICAL METHODS DEVELOPMENT

A calibration curve we were used to figure out the concentration of drug in a sample,

by comparing the sample to a set of predetermined data.

2.2.1 Preparation of calibration curve of oxaliplatin in phosphate buffer solution

using U.V method (PH-7.4, λmax 207)

The wavelength of maximum absorbtion (λmax) was determined by scanning

the drug. The media used for oxaliplatin was phosphate buffer (PH-7.4). The buffer

was prepared according to I.P (2007).

Preparation of stock solution and standard curve: Accurately weight 10 mg of

oxaliplatin was dissolved in sufficient quantity of water and volume was made up to

100 ml with hplc grade water .This gave a stock solution of 0.1 mg/ml. Aliquots were

prepared by transferring 0.2,0.4,0.6,0.8 and 1.0 in 10 ml of volumetric flask then

volume was made upto 10 ml with phosphate buffer solution this gave a

concentration of 2,4,6,8 and 10 µg/ml .The absorbance was measured at 207 nm

against phosphate buffer solution as a blank .

2.2.2 Preparation of calibration curve of oxaliplatin in water using U.V method

at λmax 207nm

The oxaliplatin was estimated spectrophotometrically in water by systronics

double beam spectrophotometer. The calibration curve was prepared as follows .

Preparation of stock solution and standard curve: Accurately weighed 10

mg of oxaliplatin was dissolved in sufficient amount of water and volume was made

upto 10 ml with hplc grade water to produce stock solution of concentration 1 mg/ml.

Aliquots were prepared by transferring 0.2,0.4,0.6,0.8 and 1.0 ml in 10 ml of

volumetric flask, then volume was made upto 10 ml with HPLC water this gave a

concentration of 2,4,6,8 and 10 µg/ml . The absorbance was measured at 207 nm

against water as a blank.

27

2.2.3 Preparation of calibration curve of oxaliplatin in water using HPL method

Equipment: The HPLC was performed with a modular system consisting of a

variable wavelength UV visible detector and auto sampler.

Mobile phase: Mobile phase consisted of a mixture of methanol: water (25:75).

Preparation of stock solution and standard curve: Accurately weight quantity of

drug 10 mg was taken in 10 ml volumetric flask ,dissolved in sufficient quantity of

water and volume was made upto 100 ml with HPLC grade water. This gave a stock

solution of .1 mg/ml.

Aliquots were prepared by transferring 0.1,0.2,0.3 and 0.4 ml in 10 ml

volumetric flasks and volume was made up to 10 ml using mobile phase (methanol

and water in the ratio of 25:75), then all aliquots were filtered by whattman filter

paper this gave a concentration of 1,2,3 and 4 µg/ml. The solution was then injected

in the 200 µl loop attached to the pump, the mobile phase was run at the rate of 1

ml/min. Detection were done at 207 nm sample concentration were calculated by

measuring covered area and plotting against standard concentration.

2.2.4 Preparation of calibration curve of oxaliplatin in plasma using HPLC

method.

The HPLC method reported by Srinubabuin et al. (2006) was followed for

estimation of oxaliplatin in biological sample.

Equipment: The hplc was performed with a modular system consisting of a variabe

wavelength U.V. visible detector and auto sampler.

Mobile phase: Mobile phase consisted of a mixure of methanol: water (25:75).

Preparation of stock solution and standard curve: Accurately weight quantity of

drug 10 mg was taken in 100 ml volumetric flask ,dissolved in sufficient quantity of

water and volume was made upto 10 ml with HPLC grade water. This gave a stock

solution of .1mg/ml.

28

Aliquots were prepared by transferring 0.1,0.2,0.3, 0.4 and 0.5 ml in 10 ml

volumetric flasks this gave a concentration of 1,2,3,4 and 5 µg/ml mixed with 0.2

ml of rat plasma homogenated and the volume was made upto 10 ml then all aliquots

were filtered by whatman filter paper. The solution was then injected in the 200 µl

loop attached to the pump. The mobile phase was run at the rate of 1ml/min.

Detection were done at 207 nm sample concentration were calculated by measuring

peak height and plotting against standard concentration.

2.3 PREPARATION OF LIPOSOMAL FORMULATION WITH AND

WITHOUT PAMAM DENDRIMER

Liposomal preparation having different lipid composition, aqueous phase

composition or quantity of oxaliplatin in feed were prepared by film hydration method

encoded and summarized in (Table 4.2.1) The lipid (2.59×10ˉ5m)consisting of soya

lecithine ,cholesterol in 1.5:1:1 molar ratio, with and without PAMAM

dendrimer(1.4x10ˉ7m ) form a film that was hydrated with 10 ml aqueous phase. The

liposomes were sonicated at 60 w for 2min with a bath sonicator,. Oxaliplatin

solution (1mg/ml) was then added to the dispersion and allowed to stand in 60ᵒ bath

for 6 hrs with mild stirring with PH adjustment to 7.4.

2.3.1 Characterization

2.3.2 Particle size determination

2.3.3 Optical microscopic method

Particle size of denrimeric liposomal formulations was determined by optical

microscopy in which the eye piece micrometer were calibrated by stage micrometer.

A 50µl of dispersion was placed on a microscopy glass slide below the objective of

the microscope and was spread evenly over the slide and a cover slip was placed over

the sample .They were left to equilibrate until the particles slow down their visible

movements. Then particle counts of 100 numbers for its size were determined for

each formulation under 10 X magnification. Values are expressed as average particle

size( Robinson, et al, 1994).

29

2.3.3 particle size and size distribution

It was determined by Malvern Particle Size analyzer from CDRI Lucknow (sample code were: TD101, SD104, ND104).

2.3.4 Estimation of dendrimer in external phase

Presence of dendrimer in external phase in blank experiment was observed by

shift in absorbtion maximum of copper sulfate. 1% copper sulfate solution was added

to the 1ml of formulation and uv absorbtion spectra was recorded against the

formulation blank. The shift in absorbtion maximum was considered as a qualitative

measured for the presence of the dendrimer(Diallo et al.,1999).

2.3.5 Effect of Tris dendrimer and NH2 suface on encapsulation efficiency

It was dertermined by taking .1ml of formulation in 10 ml of volumetric flask

and volume was made upto 10ml with hplc water this gave a concentration of

0.01mg/ml then the solution was analysed by Spectrophotometrically at 207nm.

2.3.6 In -vitro release study

The release of oxaliplatin was studied by placing the formulation equivalent

to 1mg of drug in pretreated dialysis tube with dialysis membrane. The tube was

immersed into vessel containing 100ml of phosphate buffer on hot plate .The

release studies were carried out at temperature 37ᵒc under mechanical stirrer at 50

rpm. At fixed interval of 1hr ,2hr ,3hr upto 24hr samples were withdrawn from the

solution and oxaliplatin content were determined by Spectrophotometrically.

2.3.7 Stability studies

All the selected formulations were subjected to a stability testing for 0,1,2 and

3 days at a room temperature 37ºc .Then all the selected formulations were visually

analyzed for the change in appearance ,pH or drug content (Table :4.1.10).

2.3.8 In-vivo studies

30

Determination of Pharmacokinetic oxaliplatin

The albino rats (average wt.250 gm) were divided into three groups each

containing six rats. First group was administered drug solution equivalent to 15

mg/kg of oxaliplatin through i.v route. Second group was administered NH2D(G4)L2

liposomal formulation and third group was administered OHD(G4)L3 liposomal

formulation. The blood sample was collected at 5,10,15,20,25 hr time intervals from

the tail vein in a heparinized syringe. The sample were centrifuged and plasma were

collected. From each sample 200 µl of plasma were taken in different volumetric

31

3.0 PREFORMULATION

3.1 Identification of Oxaliplatin

The identification test were perform according to B.P 2010. All identification

test were positive. Chemical test was positive that is found similar to reported as per

B.P (2010). The melting point was found 198-292ºC which is similar to reported by

[Overington, et al, 2006], all the data are shown in. (Table 3.1).

Table 3.1 Physicochemical parameter of oxaliplatin

Parameter Observation Appearance

Melting point

White crystalline powder

198ºc and 292ºc

3.2 Scanning of drug

The drug sample was scan in spectrophotometer in the range λmax 200-400nm

.The absorption maxima for oxaliplatin were found at 216,273,254 and 264nm, which

is fully complied with pharmacopoeia specification. The drug sample was almost 99%

pure as analyze by official method (Figure 3.2).

Fig 3.2 U.V spectra of oxaliplatin (Reported )

32

33

3.2 Fig U.V spectra of oxaliplatin (sample)

34

3.3 Infrared spectroscopy

The infrared spectra of the drug was performed and compared with the

standard spectra of drug, which confirm the identity of drug is shown in (Figure 3.3).

3.3 Fig IR spectra of oxaliplatin (Reported )

 

3.3 Fig I R spectra of oxaliplatin (sample)

35

 

 

The powder drug was studied for solubility in various solvents. It was slightly

soluble in water , very slightly in methanol and practically soluble in ethanol and

acetone (Table 3.4).

Table 3.4 Solubility of oxaliplatin in different solvent

Solvent Solubility

Water Slightly

Methanol Very slightly

Ethanol Practically soluble

Acetone Practically soluble

3.5 Analytical method development

After identification on assessment of purity of sample, calibration curves were

prepared in phosphate buffer (PH-7.4) solution, HPLC grade water and in biological

36

sample for oxaliplatin using spectrophotometric method, for in vitro performance

evaluation during formulation development (Table 3.5,3.6,3.7,3.8 and figure 3.5, 3.6,

3.7, 3.8). The spectrophotometric method was selected to its sensitivity and

simplicity. The absorption maxima (λmax) selected was 207 nm for oxaliplatin shows

excellent linearity and obeys bears-lamberts law in the concentration used (20-100

µg/ml) for oxaliplatin . The correlation coefficient values were greater than 0.99 for

the drug.

Table 3.5.1 Standard curve data of oxaliplaatin in Phosphate buffer at λmax

. 207nm

S.no. Conc. (µg/ml) Absorbance (nm)

1.

2.

3.

4.

5.

2

4

6

8

10

0.540

0.772

0.831

0.842

0.972

Fig 3.5.1 Standard curve of oxaliplatin in phosphate buffer at λmax

37

207nm

1 2 3 4 5 6 7 8 9 10 110

0.2

0.4

0.6

0.8

1

1.2

f(x) = 0.1016 xR² = 0.999939764871648

Conc(ug/ml)

Abso

rban

ce(n

m)

Table 3.5.2 Standard curve data of oxaliplatin in Water by UV method at

λmax 207nm

S.NO Conc.(µg/ml) Absorbance(nm)

1.

2.

3.

4.

5.

2

4

6

8

10

0.13

0.15

0.23

0.37

0.63

38

Fig 3.5.2 Standard curve of oxaliplatin in Water by UV Method at λmax

207nm

 

1 2 3 4 5 6 7 8 9 10 110

0.1

0.2

0.3

0.4

0.5

0.6

0.7

f(x) = 0.0610508816062502 xR² = 0.999760628769013

Conc(ug/ml)

Abso

rban

ce n

m

Table 3.5.3 Standard curve data of oxaliplatin in water by HPLC method at

λmax 207nm

S.NO Conc(µg/ml) Area Height Area% Height%

1. 1 82162 218799 46.595 23.737

2. 2 156419 16258 21.219 9.811

3. 3 295231 17327 29.896 18.629

4. 4 337860 13573 23.023 18.711

Fig 3.5.3 Standard curve of oxaliplatin in water by HPLC method at

39

λmax 207nm

0.5 1 1.5 2 2.5 3 3.5 4 4.50

500000

1000000

1500000

2000000

2500000

3000000

f(x) = 609666.666666667 xR² = 0.999695480924973

Conc (ug/ml)

Area

Table 3.5.4 Standard curve data of oxaliplatin in Biological sample by

HPLC method at λmax 210 nm

S.NO. Conc.

(µg/ml)

Area Height Area% Height%

1 1 294374 13573 23.023 18.711

2 2 534940 17327 29.896 18.629

3 3 954425 23204 24.516 14.557

4 4 959599 16258 21.219 9.811

5 5 21205005 218799 46.595 23.757

40

Figure 3.5.4 Standard curve of oxaliplatin in biological Sample By HPLC

method at λmax 210n

0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.50

5000000

10000000

15000000

20000000

25000000

f(x) = 4096363.63636364 xR² = 0.999870781310171

Conc(ug/ml)

Area

3.6 LIPOSOMAL FORMULATION WITH AND WITHOUT DENDRIMER

Liposomal formulation ,differing in lipid composition aqueous phase

composition or quantity of oxaliplatin in feed were prepared by film hydration method

encoded in (Table: 4.1). To summarize three type of liposomal formulations were

prepared neutral and negatively charged soyalecithine and cholesterol basic liposomal

formulations. PAMAM dendrimer G4 generation were included in these formulation

in which NH2 surface PAMAM dendrimer was used in lipid film formation and Tris

dendrimer was used in hydration step.

3.6 Table Coding of liposomal formulation with or without PAMAM

dendrimer .

Formulation

code

LIPID FILM COMPOSITION Aqueous phase

(m)Lecithine (m) Cholestrol (m) Dendrimer (m)

41

SI L1 2.59×10-5 2.21×10-5 dr(2.5×10-5).wa

NH2 D(G4) L2 9.4×10-5 5.4×10-5 1.4×10-7 dr(2.5×10-5).wa

OH D(G4) L3 9.7×10-4 2.1×10-4 dr(2.5×105).OH

(5.5×10-7)

SI L1:- Simple liposome 1

NH2 D(G4) L2:- NH2 dendrimer (G4) liposome 2

OH D(G4) L3:- OH dendrimer liposome 3

Molecular weight of NH2 dendrimer : 14215

Stock solution : 2 mg/ml

Molecular weight of OH dendrimer : 18128

Stock solution : .1 mg/ml

Molecular weight of oxaliplatin : 397.29gm/mol

Stock solution of drug : 1mg/m

3.6.1 Particle size determination

Liposomal formulation containing dendrimer were sonicated for reduction in

size and . The size of freshly filtered liposome were given in( Table 4.2.2,4.2.3) . The

particle size SI L1 of liposomal formulation without dendrimer could be more than

NH2 D(G4) L2 and OH D(G4) L3.The particle sizes reported have were determined

by instrumentally using optical microscope and malveren particle size analyzer

3.6.1 Table Average particle size by Optical Microscope

FORMULATION CODE AVERAGE PARTICLE

SIZE(µm)

SI L1 0.76 ± 0.034

NH2 D(G4) L2 0.16 ± 0.02

42

OH D(G4) L3 0.74 ± 0.02

3.6.1 Fig Average particle size by Optical Microscope

SL1 NH2(4G)L2 OHDL30

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

formulation code

size(

µm)

[

3.6.1 Table Average particle size by Malvern Particle Sizer

FORMULATION CODE AVERAGE PARTICLE SIZE (µm)

SI L1 0.78 ±0.012

NH2 D(G4) L2 0.18±0.014

OH D(G4) L3 0.67±0.011

3.6.1 Fig Average particle size by Malveren Particle Sizer [

43

SI LI NH2 D(G4) L2 OH D(G4)L30

0.10.20.30.40.50.60.70.80.9

formulation code

size(

µm)

3.6.2 Estimation of dendrimer in external phase

The copper sulfate solution have absorbtion maximum at 209nm on addition

of dendrimer the maximum is shifted to 216nm indicating red shift. This can visually

be observed by change in color of copper sulfate from dark blue to light blue. This

reaction is very tentative and could detect small amount of dendrimer in solution by

shift in absorbtion maxima of copper sulfate solution .

44

3.6.2 Fig UV spectra of copper sulfate

45

3.6.2 Fig Estimation of dendrimer in external phase

46

3.6.3 Invitro release study

The release was lowered in dendrimeric liposomal formulation than simple

liposome (Table:). The charge of liposomal lipid was not found to affect the release of

drug with different type of PAMAM dendrimer. The study shows that it is possible to

modulate the release of drug from the dendritic liposomal formulations by changing

the composition of lipid and dendrimer.

3.6.3 Table Drug release of oxaliplatin from dendritic liposomal formulation

TIME (hr) % DRUG RELEASE SI L1 OH D(G4) L3 NH2 D(G4) L2

1 19.6 15.7 9.2 2 37.7 25.9 19.6 3 49.4 38.6 28.3 4 69.3 47.9 36.8 5 79.7 57.34 47.8 6 79.15 63.60 58.1 7 79 .39 63.19 58.11 8 79.67 63.47 58.27 9 63.57 58.47 10 63.67 58.67 24 63.87 58.79

3.6.3 Fig Drug release of oxaliplatin from dendritic liposomal formulation

0 1 2 3 4 5 6 7 8 9 10 240

10

20

30

40

50

60

70

80

90

NH2 D(G4)L2OHD(G4)L3SI L1 

Time(hr)

% D

rug

rele

ase

47

3.6.4 Effect of Tris dendrimer and NH2 surface on drug entrapment

The encapsulation of drug was prapotional to the Tris and NH2 surface

dendrimer (Table:3.4.4) .The liposome containing G4 NH2 surface dendrimer were

able to encapsulate drug approximately two or four times greater than OH D(G4) L3

and SI L1 another possibility may be that the proportion of the dendrimers attached

with the surface phosphate group was unable to encapsulate drug due to their open

structure.

3.6.4 Table Effect of Tris dendrimer and NH2 suface on drug entrapment

FORMULATION CODE ENCAPSULATION EFFICIENCYSI L1 68%NH2 D(G4)L2 92.9%OH D(G4)L3 87.9%

3.6.5 Stability studies

The stability of 4th generation dendrimer containing formulation (NH2 G4D

L2) was slightly greater than( OH G4D L3) and (SI L1) formulation. Perhaps because

of the protective effect of higher generation dendrimer on the encapsulation of drug.

The formulation kept at 37ºc were clear. There was drop in pH values from adjusted

7.2 to 5.2 suggesting that there was hydrolysis of phospholipids and pH drop was

produced despite that use of fully hydrogenated phospholipid .Overall the stability of

the formulation was sufficiently promising for its successfully applicability.

3.6.5 Table Stability Studies

Serial no Formulation Days Appearance pH Drug content(%)

1. SI L1 0 Clear 6.7 681 Clear 6.7 67.12 Clear 6.3 66.43 Clear 6.3 65.8

2. NH2 D(G4)L2 0 Clear 7.2 92.91 Clear 7.2 91.52 Clear 7.1 91.33 Clear 7.1 89.2

3. OH D(G4)L3 0 Clear 5.4 87.91 Clear 5.4 86.12 Clear 5.3 84.63 Clear 5.2 83.1

48

3.6.6 In –vivo studies

From the plasma clearance data of oxaliplatin from formulation ,the pharmacokinetics

parameters were calculated by open one compartment model indicated that AUC of

formulation or oxaliplatin increased by 3.74, 3.96, 4.30 for OH D(G4)L3, NH2

D(G4)L2 respectively than free drug .The AUC also increased with different

concentration and types of PAMAM dendrimer .This may be due to open to globular

structure of dendrimer .

3.6.6 Table Determination of pharmacokinetics parameters

Time Drug FormulationOH D(G4)

Formulation NH2D(G4)

0 11 ± 0.1 10 ± 0.21 8 ± 0.145 15.1 ± 0.14 17.2 ± 0.36 20.1 ± 0.4610 12.5 ± 0.22 20.9 ± 0.14 23.8 ± 0.2815 10.9 ± 0.24 20.11 ± 0.16 23.15 ± 0.2520 20.8 ± 2.7 23.14 ± 1.0525 20.5 ± 2.5 23.11 ± 1.05

3.6.6 Fig Determination of pharmacokinetics parameter

0 5 10 15 20 25 300

5

10

15

20

25

Drug OHD(G4)L3NH2D(G4)L2

Time(hr)

Plas

ma

drug

conc

entr

ation

49

3.6.6 Table Pharmacokinetic parameter

Pharmacokinetics parameters

Drug (oxaliplatin) Formulation OH D(G4)l2

Formulation NH2 D(G4)l3

Cmax( µg/ml) 18.7 19.8 21.5

tmax (hr) 5 5 5

Auc (µg h/ml) 3.74 3.96 4.30

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4.0 SUMMARY AND CONCLUSION

Dendrimers are a new class of synthetic polymers which have the structure

like a tree or star shape, with a central core, interior branches and terminal groups

which decorate the surface. Cavities inside the core and the interior branches can be

modified to carry hydrophobic and hydrophilic drugs. Recently, dendrimers have

successfully proved themselves as promising nanocarriers for drug delivery because

they can render drug molecules a greater water-solubility, bioavailability, and

biocompatibility. The terminal groups on the surface can also be adapted to carry

drugs. Thanks to the unique molecular architecture it is possible to design the

hyperbranched polyester in numerous ways to reach desired properties in different

application.

Liposomal drugdelivery systems have come of age in recent years, with

several liposomal drugs currently in advanced clinical trials or already on the market.

It is clear from numerous pre-clinical and clinical studies that drugs, such as antitumor

drugs, packaged in liposomes exhibit reduced toxicities, while retaining, or gaining

enhanced, efficacyons. Liposomal drugs exhibit reduced toxicities and retain, or gain

enhanced, efficacy compared with their free counterparts. Liposomes that allow

enhanced drug delivery to disease sites, by virtue of long circulation residence times,

are now achieving clinical.

In the present investigation include to prepare liposomal formulation of

oxaliplatin-pamam complex with different concentration of PAMAM dendrimer and

their characterization.

Two types such as NH2 surface and Tris dendrimer of different concentration

of pamam dendrimer was used for encapsulation of oxaliplatin into liposome through

film hydration method . Liposomal preparation having different lipid composition,

aqueous phase composition or quantity of oxaliplatin, The lipid consisting of soya

lecithine ,cholesterol in 1.5:1:1 molar ratio, with and without PAMAM dendrimer

form a film that was hydrated with 10 ml aqueous phase with. The liposomes were

sonicated at 60 w for 2min with a bath sonicator and Oxaliplatin solution (1mg/ml)

was then added to the dispersion and allowed to stand in 60ᵒ bath for 6 hrs with mild

stirring with PH adjustment to 7.4. Then chraeteriation had done after preparation .

51

Particle size of denrimeric liposomal formulations was determined by optical

microscopy in which the eye piece micrometer were calibrated by stage micrometer.

Then dispersion was placed on a microscopy glass slide below the objective of the

microscope and was spread evenly over the slide and a cover slip was placed over the

sample. Then particle counts of 100 numbers for its size were determined for each

formulation. Particle size and size distribution was determined by Malvern Particle

Size analyzer from CDRI Lucknow.

Presence of dendrimer was observed by shift in absorbtion maximum of

copper sulfate. 1% copper sulfate solution was added to the 1ml of formulation and uv

absorbtion spectra was recorded against the formulation blank..

Effect of Tris dendrimer and NH2 suface on encapsulation efficiency was

dertermined by taking .1ml of formulation in 10 ml of volumetric flask and volume

was made upto 10ml with then the solution was analysed by Spectrophotometrically

at 207nm.

Invitro release study of oxaliplatin was studied by placing the formulation

drug in pretreated dialysis tube with dialysis membrane. The tube was immersed into

vessel containing phosphate buffer on hot plate .The release studies were carried out

at temperature 37ᵒc at 50 rpm. At fixed interval upto 24hr samples were withdrawn

from the solution and oxaliplatin content were determined by Spectrophotometrically.

Stability studies was found by placing the formulation under room temperature

for 3 days and where the formulation were relatively stable .

Invivo studies were performed in male albino rats (150-200g) . All the

animals were procured from the disease-free animal house of Central Drug Research

Institute, Lucknow .Then pharmacokinetics parameters such as AUC Cmax and

tmax was found for drug and different formulation of PAMAM dendrimeric

liposomal system and bioavilability were determined from the plasma profile of

oxaliplatin .,

Conclusion : Thus liposomal drug delivery system incorporating a complex of

oxaliplatin-PAMAM G4 dendrimers was prepared and compared to simple liposomal

formulation encapsulating oxaliplatin with the same lipid composition regarding

release properties, encapsulation efficiency, stability studies, particle size and

52

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