LIPID PEROXIDATION AND ANTIOXIDANT DEFENCE STATUS I N LEPROSY PH.D. THESIS SUBMITTED TO RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA, BANGALORE BY M R .C.V.B ALASUBRAHMANYA P RASAD . M.Sc. RESEARCH GUIDE D R .M.V.K ODLIWADMATH . M.D. P ROFESSOR A ND H EAD . DEPARTMENT OF BIOCHEMISTRY, J. N. MEDICAL COLLEGE, NEHRU NAGAR, BELGAUM-590010. JUNE 2005
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LIPID PEROXIDATION AND ANTIOXIDANT
DEFENCE STATUS IN LEPROSY
PH.D. THESIS
SUBMITTED TORAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,
KARNATAKA, BANGALORE
BY
MR.C.V.BALASUBRAHMANYA PRASAD.M.Sc.
RESEARCH GUIDE
DR.M.V.KODLIWADMATH.M.D.
PROFESSOR AND HEAD.
DEPARTMENT OF BIOCHEMISTRY,J. N. MEDICAL COLLEGE,
NEHRU NAGAR, BELGAUM-590010.
JUNE 2005
PH.D.THESIS
LIPID P
EROXIDATION AND A
NTIOXIDANT
DEFENCE S
TATUS IN L
EPROSY
JUNE2 0 0 5
PH.D .THESIS
LIPID PEROXIDATION AND ANTIOXIDANT
DEFENCE STATUS IN LEPROSY
JUNE2 0 0 5
LIPID PEROXIDATION AND ANTIOXIDANT
DEFENCE STATUS IN LEPROSY
PH.D. THESISSubmitted to Rajiv Gandhi University of Health
Sciences, Karnataka, Bangalore,in partial fulfilment of the requirement for
the award of the degree of
Doctor of Philosophyin
Medical Biochemistry(Under The Faculty of Medicine)
By
MR.C.V.BALASUBRAHMANYA PRASAD.M.Sc.
Under The Guidance Of
DR.M.V.KODLIWADMATH.M.D.
PROFESSOR AND HEAD.DEPARTMENT OF BIOCHEMISTRY,
J. N. MEDICAL COLLEGE,NEHRU NAGAR, BELGAUM-590010.
JUNE 2005
JAWAHARLAL NEHRU MEDICAL COLLEGE,BELGAUM - 590 010.
PEROXIDATION AND ANTIOXIDANT DEFENCE STATUS IN LEPROSY" is abonafide record of research work carried out byMr. C. V. BALASUBRAHMANYA PRASAD under my guidancefor the degree of DOCTOR OF PHILOSOPHY IN MEDICAL BIOCHEMISTRY
of Rajiv Gandhi University of Health Sciences,Karnataka. The results presented in this thesis havenot previously formed the basis for the award of anydegree or fellowship.
I forward this thesis with great pleasure.
Dr. M.V. KODLIWADMATH. M.D.Research Guide,Professor and Head,Department of Biochemistry,J.N.Medical College,Belgaum - 590 010.Karnataka.
Place : Belgaum.Date :
JAWAHARLAL NEHRU MEDICAL COLLEGE,BELGAUM - 590 010.
BALASUBRAHMANYA PRASAD is a Ph.D. Student in theDepartment of Biochemistry at J.N. MedicalCollege, Belgaum and has completed his Thesis titled"LIPID PEROXIDATION AND ANTIOXIDANT DEFENCE STATUS IN LEPROSY"underthe guidance of Dr.M.V. KODLIWADMATH. M.D. Professorand Head, Department of Biochemistry,J.N.Medical College, Belgaum. He has undergonethe prescribed course of Research Work in accord-ance with the University regulations.
I have a great pleasure in forwarding it toRajiv Gandhi University of Health Sciences,Karnataka, Bangalore.
1997) I hereby declare that the thesis entitled "LIPID
PEROXIDATION AND ANTIOXIDANT DEFENCE STATUS IN LEPROSY" submittedby me for the degree of DOCTOR OF PHILOSOPHY IN MEDICAL
BIOCHEMISTRY of the Rajiv Gandhi University of HealthSciences, Karnataka, Bangalore, is the result of myoriginal and independent work done at J. N. MedicalCollege, Belgaum, during the year 2001-2004 underthe supervision of Dr.M.V. KODLIWADMATH. M.D. Profes-sor and Head, Department of Biochemistry,J.N.Medical College, Belgaum, and has not formedthe basis for the award of any Degree, Diploma,Associateship, Fellowship or other similar title pre-viously.
Mr. C. V. BALASUBRAHMANYA PRASADPh.D. Reg. No. RGUHS/Ph.D/M8/2001-02Dept. of Biochemistry,J. N. Medical College,Belgaum - 590 010.Karnataka.
Place : Belgaum.Date :
Acknowledgement
It is a great pleasure to utilize this unique opportunity to express my
deep sense of gratitude and offer my most sincere and humble regards to my
esteemed teacher and guide Shri. Rajendra C. Doijad, Assoc. Professor.
Dept of pharmaceutics, KLE’S college of pharmacy Belgaum, for his
unparalleled and excellent guidance, continuous encouragement and
support in completion of my course and dissertation successfully. His
discipline, principles, simplicity and fearless work environment was
cherished during the course.
I am thankful to Dr. F. V. Manvi, Principal K.L.E. Society’s College
of Pharmacy, for providing the facilities required for my dissertation work.
I sincerely thank Shri S.K. Krishnan Sr. Manager, Analytical
Research (HINDALCO), Belgaum, for providing me the facility of scanning
electron microscopy (SEM) and X- ray diffractrometry study at his
respective organization.
I heartily thank Mr.Suchit Chaudhary and Mr. Viond Nayak in
helping me to procure gift samples of Cisplatin from sun pharmaceutical
Mumbai & Cipla Ltd. Banglore, respectively.
I am also grateful for the invaluable guidance and kind co-operation
provided by Dr. A. R. Bhatt, Shri. C. R. Patil, Mr. Tippeswamy, Mr.
Banappa, Shri. M. B. Palkar, Shri. S. Bhongade, Shri. Noolvi and Ms.
Talath.
I extend my heartiest and dearest gratitude to my close friend
Jacqueline, Prajakta, mithra, Manish, Uday Bolmal sir and Sujata who
stood by me in every aspect. I shall cherish all the moments spent with them
throughout my life.
i
I take this opportunity to thank my seniors Deepak Kapoor, Prasun,
Ravindra singh, Shailesh, Ashok and Swati for their kind co-operation.
I extend my thanks to my colleagues Jayprakash, Biren, Nagesh,
Navneet and Lakshman who stood by me in every walks of life.
I extend my heartiest & dearest gratitude to my roommates Sunil,
Sampat, rupesh & all my juniors. I shall cherish the excellent moments
spent with them throughout my life, Special thanks to Jiten, Gopal, Asif,
Mehboob for their constant support and help in my thesis work.
I am also thankful to all the technical and non-teaching staff,
K.L.E.Society’s College of Pharmacy, for their co-operation in various
capacities.
I express my deep sense of love and affection to my beloved and
respected parents, my brothers and sisters and all other family members,
without whose encouragement, co-operation and good wishes this task
would have been impossible.
Last… but not the least, I wish to express my gratitude towards “God
– Almighty”, who gave me the strength and courage to fulfill my dream and
has showered upon me his choicest blessings.
Amber Vyas.
Date:
Place: Belgaum
ii
Affectionately Dedicated To My
Late Grand Father,
Father,
Mother,
And
My Brothers & Sisters
||| Om Shree Ganeshaya Namaha |||
LIST OF ABBREVIATIONS USED
DDS - Drug delivery system
RES - Reticulo endothelial system
MRM - Magnetically responsive microspheres
PSEP - Phase separation emulsion polymerization
CSE - Continuous solvent evaporation
CDDP - Cis-Diammine-dichloroplatinium (II)
BSA - Bovine serum albumin
HCC - Hepatocellular carcinoma
NER - Nucleotideexcision repair
DMSO - Dimethyl sulphoxide
DMF - Diethyl formamide
DMA - N,N-dimethylacetamide
PBS - Phosphate buffer saline
IR - Infrared
DDTC - Diethyldithiocarbamic acid
UV-Vis - Ultra violet visible
T - Tesla
i
TABLE OF CONTENTS
CHAPTER TITLE PAGE NO.
1 INTRODUCTION ……………………………...…… 1
2 OBJECTIVES ….…………………………………… 30
3. REVIEW OF LITERATURE ………………………. 33
4. METHODOLOGY ………………….……………… 46
5 RESULTS AND DISCUSSION……………………. 59
6 CONCLUSION …………………………………….. 102
7 SUMMARY…………………………………………. 105
8 BIBLIOGRAPHY……………………………………. 108
9 ANNEXURE…………………………………………. 117
iii
LIST OF FIGURES
Sl. No. Title of Figure Page
No.
1. METHODS OF MICROSPHERE TARGETING 11
2. CONCEPT OF MAGNETIC DRUG TARGETING 12
3. PRINCIPLE OF MAGNETIC DRUG TARGETING 12
4. APPARATUS FOR IN VITRO MAGNETIC RESPONSIVENESS
STUDY
54
5. STANDARD CALIBRATION CURVE OF CISPLATIN 83
6. DRUG ENTRAPMENT EFFICIENCY OF MAGNETIC
MICROSPHERES
84
7. PERCENT MAGNETITE CONTENT 85
8. IN VITRO MAGNETIC RESPONSIVENESS OF MAGNETIC
MICROSPHERES
86
9. PLOT OF CUMULATIVE % DRUG RELEASED Vs. TIME FOR
PURE CISPLATIN
87
10. PLOTS OF CUMULATIVE % DRUG RELEASED Vs. TIME FOR
DIFFERENT FORMULATIONS OF CISPLATIN MAGNETIC
MICROSPHERES (IN VITRO RELEASE PROFILE) [ZERO ORDER
PLOTS]
88
11. PLOTS OF CUMULATIVE % DRUG RETAINED Vs. TIME FOR
DIFFERENT FORMULATIONS OF CISPLATIN MAGNETIC
MICROSPHERES (IN VITRO RELEASE STUDIES) [FIRST ORDER
KINETICS]
89
12. PLOTS OF CUMULATIVE % DRUG RELEASED Vs. ROOT TIME
FOR DIFFERENT FORMULATIONS OF CISPLATIN MAGNETIC
MICROSPHERE (IN VITRO RELEASE STUDIES) [HIGUCHI
MATRIX]
90
vi
13. PLOTS OF LOG CUMULATIVE % DRUG RELEASED VS. LOG
TIME FOR DIFFERENT FORMULATIONS OF CISPLATIN
MAGNETIC MICROSPHERES (IN VITRO RELEASED STUDIES)
[PEPPAS PLOT]
91
14. PLOTS OF CUBE ROOT OF % DRUG RETAINED Vs. TIME FOR
DIFFERENT FORMULATIONS OF CISPLATIN MAGNETIC
MICROSPHERES (IN VITRO RELEASE STUDIES) [HIXSON
CROWELL]
92
15. COMPARISION BETWEEN AMOUNT OF DRUG DISTRIBUTED
FROM MAGNETIC MICROSPHERES WITH AND WITHOUT
MAGNETIC FIELD IN VARIOUS ORGANS
(IN VIVO TISSUE DISTRIBUTION STUDIES)
93
16. PERCENT DRUG CONTENT Vs. TEMPERATURE FOR
DIFFERENT FORMULATIONS OF CISPLATIN MAGNETIC
MICROSPHERES AFTER 60 DAYS STORAGE
94
17. PLOTS OF CUMULATIVE % DRUG RELEASED Vs. TIME OF F-3
FORMULATION AFTER 60 DAYS STORAGE
95
vii
A
LIST OF SPECTR
Sl. No. Title of Spectrum Page
No.
1.
2.
3.
4.
5.
6.
IR SPECTRUM OF CISPLATIN
IR SPECTRUM OF BOVINE SERUM ALBUMIN (BSA)
IR SPECTRUM OF MAGNETITE
IR SPECTRUM OF CISPLATIN + BSA + MAGNETITE
X-RAY DIFFRACTROGRAM OF MAGNETITE
X-RAY DIFFRACTROGRAM OF FORMULATION (F-3)
96
97
98
99
100
101
viii
LIST OF TABLES
Sl. No. Title of Table Page No.
1. FORMULATION PLAN OF CISPLATIN MAGNETIC
MICROSPHERES.
51
2. ABSORBANCE VALUES OF CISPLATIN STANDARD
SOLUTIONS AT 210 nm.
69
3. PERCENTAGE PRACTICAL YIELD OF BOVINE SERUM
ALBUMIN MAGNETIC MICROSPHERES OF CISPLATIN.
70
4. DRUG ENTRAPMENT EFFICIENCY OF MAGNETIC
MICROSPHERES.
71
5. PERCENT MAGNETITE CONTENT. 71
6. IN VITRO MAGNETIC RESPONSIVENESS OF MAGNETIC
MICROSPHERES.
72
7. IN VITRO RELEASE PROFILE FOR PURE CISPLATIN 73
8. IN VITRO RELEASE PROFILE OF CISPLATIN FROM MAGNETIC
MICROSPHERES FORMULATION-1.
74
9. IN VITRO RELEASE PROFILE OF CISPLATIN FROM MAGNETIC
MICROSPHERES FORMULATION-2.
75
10. IN VITRO RELEASE PROFILE OF CISPLATIN FROM MAGNETIC
MICROSPHERES FORMULATION-3.
76
11. IN VITRO RELEASE PROFILE OF CISPLATIN FROM MAGNETIC
MICROSPHERES FORMULATION-4.
77
12. KINETIC VALUES OBTAINED FROM IN VITRO RELEASE DATA
OF DIFFERENT MAGNETIC MICROSPHERE FORMULATIONS
OF CISPLATIN.
78
13. KINETIC VALUES OBTAINED FROM IN VITRO RELEASE DATA
OF DIFFERENT MAGNETIC MCROSPHERE FORMULATIONS
OF CISPLATIN.
79
14. IN VIVO TARGETING STUDIES OF MAGNETIC
MICROSPHERES OF CISPLATIN.
80
15. STABILITY STUDIES FOR PERCENT DRUG CONTENT [AFTER
STORAGE AT 4ºC, AMBIENT TEMPERATURE AND HUMIDITY
& AT 30ºC /65% RH].
81
16. STABILITY STUDIES- IN VITRO RELEASE STUDY OF A
SELECTED FORMULATION (F-3) AFTER ONE MONTH
STORAGE AT 4°C, AMBIENT TEMPERATURE AND HUMIDITY
AND 30ºC /65% RH.
82
v
ABSTRACT
The capability to deliver high effective dosages to specific sites in the human
body has become the holy grail of drug delivery research. Drugs with proven
effectiveness under in vitro investigation often reach a major roadblock under in vivo
testing due to a lack of an effective delivery strategy. In addition, many clinical scenarios
require delivery of agents that are therapeutic at the desired delivery point, but otherwise
systemically toxic.
Magnetically responsive albumin microspheres containing Cisplatin were
prepared by PSEP technique and were evaluated with respect to Particle size analysis by
SEM, entrapment efficiency, magnetite content, in vitro magnetic responsiveness in a
7000 Oe magnetic field, in vitro drug release studies, in vivo drug targeting studies and
stability studies.
Spherical particles of average 3-12 µm in diameter and incorporation efficiency
up to 56.37% were obtained. Result of X-ray diffractrometry confirms the presence of
magnetite in prepared Cisplatin magnetic microspheres. Using chemical analysis, it was
found that total percentage of Fe2O3 in the microspheres was between 42.53%-55.48%.
Cumulative percent drug release after 24 hours was 89.60%, 82.22%, 78.41%, and
76.35% for F-1-F-4 respectively. Results of in vitro magnetic responsiveness and in vivo
targeting demonstrated that the retention of microspheres in presence of magnetic field
was significantly more than those in the absence of the magnetic field. Stability studies
showed that maximum drug content and closest in vitro release to initial data was found
in the samples stored at 4°C. Overall, this study shows that the magnetic albumin
microspheres can be retained at their target site in vivo, following the application of
magnetic field, and are capable of releasing their drug content for an extended period of
time. This would make them a suitable depot for delivering chemotherapeutic agent(s)
in vivo.
ii
Chapter 1 Introduction
Benjamin Franklin: "If everyone is thinking alike, then no one is thinking."
INTRODUCTION
The capability to deliver high effective dosages to specific sites in the human
body has become the holy grail of drug delivery research. Drugs with proven
effectiveness under in vitro investigation often reach a major roadblock under in vivo
testing due to a lack of an effective delivery strategy. In addition, many clinical scenarios
require delivery of agents that are therapeutic at the desired delivery point, but otherwise
systemically toxic. This project proposes a method for targeted drug delivery by applying
high magnetic field gradients within the body to an injected super paramagnetic colloidal
fluid carrying a drug, with the aid of modest uniform magnetic field.1
The nonspecific distribution of drugs is wasteful and hampers the clinical
usefulness of most of these agents after their systemic administration in the body. It
increases the incidence of undesirable reaction (toxic reactions), thereby narrowing down
the therapeutic index of a drug.2
Another problem associated with systemic drug administration is the inability to
target a specific area of the body. So systemic drug therapy is an undesirable way to
attack a local disease, hence localization of chemotherapeutic agent to diseased area (i.e.
drug targeting to desired site) is more suitable and rational answer to this problem.
Drug Targeting- A “State-Of-The-Art Technique”3
Drug Delivery Systems (DDS) are divided into various subsystems. One of these,
targeting DDS, recognizes target cells and tissues of diseases such as cancer and sends
drugs and genes to the target site. Current research in this field is focusing on the
development of nanomaterials for passive targeting. Work is also being done on so-called
Dept. of Pharmaceutics, KLES’s College of Pharmacy, Belgaum. 1
Chapter 1 Introduction
"missile drugs" for active targeting DDS which can enhance the functionality of
targeting. Missile drugs are showing promise as the "wonder drugs" of the 21st century.
The concept of designing specified delivery system to achieve selective drug
targeting has been originated from the perception of Paul Ehrlich, who proposed drug
delivery to be as a “magic bullet”.
Rationale Of Drug Targeting4
The site-specific targeted drug delivery negotiates an exclusive delivery to
specific preidentified compartments with maximum intrinsic activity of drugs and
concomitantly reduced access of drug to irrelevant non-target cells. The controlled rate &
mode of drug delivery to pharmacological receptor and specific binding with target cells;
as well as bioenvironmental protection of the drug en route to the site of action are
specific features of targeting. Invariably, every event stated contributes to higher drug
concentration at the site of action and resultant lowers concentration at non-target tissue
where toxicity might crop-up. The high drug concentration at the target site is a result of
the relative cellular uptake of the drug vehicle, liberation of drug and efflux of free drug
from the target site.
Targeting is signified if the target compartment is distinguished from the other
compartments, where toxicity may occur and also if the active drug could be placed
predominantly in the proximity of target site. The restricted distribution of the parent
drug to the non-target site(s) with effective accessibility to the target site(s) could
maximize the benefits of targeted drug delivery.
Dept. of Pharmaceutics, KLES’s College of Pharmacy, Belgaum. 2
Chapter 1 Introduction
Principle & Rationale Of Drug Targeting:4
Levels Of Drug Targeting5,6
The various approaches of vectoring the drug to the target site can be broadly
classified as:
1. Passive targeting.
2. Active targeting (Ligand mediated targeting and Physical targeting).
3. Inverse targeting.
4. Dual targeting.
5. Double targeting
6. Combination targeting
1. Passive Targeting:
It is a sort of passive process, which utilizes the natural course of (attributed to
inherent characteristics) ‘homing’ of the carrier system, through which it finally identifies
and eventually approaches the intended cell lines. 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.
Dept. of Pharmaceutics, KLES’s College of Pharmacy, Belgaum. 3
Chapter 1 Introduction
2. Active Targeting:
This employs deliberately modified drug-drug carrier molecule capable of
recognizing and interacting with a specific cell, tissue or organ. Modification may include
a change in the molecular size, alteration of the surface properties, incorporation of
antigen-specific antibodies, or attachment of cell receptors-specific ligands. The Active
targeting have further classified it into three different levels of targeting:
a. First order targeting (delivery to a discrete organ).
b. Second order targeting (targeting to a specific cell type within a tissue or organ.
For example, tumor cell Vs normal cells).
c. Third order targeting (delivery to a specific intracellular compartment in the
cells. For example, lysosomes).
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. The ligands afford specific avidity to drug carrier. The engineered
carrier constructs selectively deliver the drug to the cell or group of cells generally
referred to as target. The cascade of events involved in ligand negotiated specific drug
delivery is termed as ligand driven receptor mediated targeting
Physical Targeting:
This refers to a delivery system that releases a drug only when exposed to a
specific microenvironment, such as a change in pH or temperature, or the use of an
external magnetic field.
Dept. of Pharmaceutics, KLES’s College of Pharmacy, Belgaum. 4
Chapter 1 Introduction
It requires formulation of the drug using a particulate delivery device, which by
virtue of its physical localization will allow differential release of the drug. The site
specificity is due to the exclusive generation of higher drug concentrations at the site of
localization of the device, while the drug concentration in the rest of the body is very
much diminished due to the simple dilution factor. The carrier systems employed are
either solid particulates such as microspheres, nanoparticles, or liquid colloids such as
liposomes. The particulate carriers may target liver (Kupffer cells and hepatocytes),
endothelial cells, sites of inflammation and lymph nodes. The size or surface of the
particles is crucial factors in targeting. Several anatomical compartments exist where
particles are retained due to either the physical properties of the environment or the
biophysical interactions of particles with the cellular components of the target tissue. The
delivery of drug in this manner yields a persistent and sustained supply of the drug at the
target site.
Physical or Mechanical Approach of Targeting Includes:-
a) Targeting to mononuclear phagocytic system
b) Targeting to the pulmonary region
c) Extravascular delivery
d) Mucosal delivery of antigens
e) Magnetic drug targeting
3. Inverse Targeting:
It is essentially based on successful attempt to circumvent and avoid passive
uptake of colloidal carriers by reticuloendothelial system (RES). This effectively implies
for reversion of bio-homing trend of the carrier, hence the process is referred to as inverse
targeting. One strategy applied to achieve inverse targeting is to suppress the function of
RES by pre-injection of a large amount of blank colloidal carriers or macromolecules like
Dept. of Pharmaceutics, KLES’s College of Pharmacy, Belgaum. 5
Chapter 1 Introduction
dextran sulphate. This approach leads to RES blockade and as a consequence impairment
of host defense system. Alternate strategies include modification of the size, surface
charge, composition, surface rigidity and hydrophilicity of carriers for desirable biofate.
4. Dual Targeting:
This classical approach of drug targeting employs carrier molecules, which have
their own intrinsic antiviral effect thus synergising 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. A major advantage is that the virus replication
process can be attacked at multiple points, excluding the possibilities of resistant viral
strain development.
5. Double Targeting:
For a new future trend, drug 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.
The temporal control of drug delivery has been developed in terms of control drug
release prior to the development of drug targeting. If spatial targeting is combined with
temporal, controlled release results in an improved therapeutic index by the following
two effects. First, if drug release or activation is occurred locally at therapeutic sites,
selectivity is increased by multiplication of the spatial selectivity with the local
release/activation. Second, the improvement in the therapeutic index by a combination of
a spatially selective delivery and a preferable release pattern for a drug, such as zero
order release for a longer time period of the drugs. When these two methodologies are
combined, it may be called “Double targeting”
Dept. of Pharmaceutics, KLES’s College of Pharmacy, Belgaum. 6
Chapter 1 Introduction
6. Combination Targeting:
Petit and Gombtz, 1998 have suggested the term 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 favor targeting the specific compartments, organs or their
tissues within the vasculature.
Carriers Used In Targeted Drug Delivery Systems 4,7
Carrier is one of the most important entities essentially required for successful
transportation of the loaded drug(s). They are drug vectors, which sequester, transport
and retain drug en route, while elute or deliver it within or in vicinity of target. Following
is the categorical presentation of these potential targetable systems.
Dept. of Pharmaceutics, KLES’s College of Pharmacy, Belgaum. 7
Chapter 1 Introduction
Dept. of Pharmaceutics, KLES’s College of Pharmacy, Belgaum. 8
Chapter 1 Introduction
Problems Associated With Targeted Drug Delivery Systems 5
Several problems have been identified which require alterations in targeting
strategies particularly, in vivo. These include:
Rapid clearance of targeted systems especially antibody targeted carriers.
Drug- antibody inactivation during conjugation.
Immune reactions against intravenous administered carrier systems.
Target tissue heterogeneity.
Problems of insufficient localizations of targeted systems into tumor cells.
Down regulation and sloughing of surface epitopes.
Diffusion and redistribution of released drug leading to non-specific
accumulation.
Nanoparticles are difficult to manufacture in large quantities.
Nanoparticles has bioacceptibility restrictions.
Poor stability, rapid and quantitative interception of liposomes and their contents
by cells of RES.
Magnetic Microspheres
Splendid achievements have been made in management of diseases through
invention of drugs over the past decade, which are fulfilling the challenge of modern drug
therapy i.e. optimization of the pharmacological action of the drugs coupled with the
reduction of their toxic side effects in vivo. Recently a lot of interest has been shown in
targeted drug delivery system, magnetic microspheres being one of them.
Targeting by magnetic microspheres i.e. incorporation of magnetic particles into
drug carriers8, 9 (polymers) and using an externally applied magnetic field is one way to
physically direct this magnetic drug carriers to a desired site8, 10,11. Widder et al. first
Dept. of Pharmaceutics, KLES’s College of Pharmacy, Belgaum. 9
Chapter 1 Introduction
reported on the use of magnetic albumin microspheres. Morimoto and widder and senyei
extensively reviewed their preparation and drug release properties12.
Magnetic microspheres are supramolecular particles that are small enough to
circulate through capillaries without producing embolic occlusion (< 4μ m) but are
sufficiently susceptible (ferromagnetic) to be captured in minor vessels and dragged into
the adjacent tissue by magnetic fields of 0.5-0.8 tesla (T) 13.
Evolution of Magnetic Microspheres
There are several techniques (like liposomes, resealed erythrocytes, platelets,
monoclonal antibody and non magnetic microspheres) by which drugs can be delivered to
targeted areas2.
Although above mentioned techniques are quite efficient but drug carrier in case of
liposomes, erythrocytes and platelets suffer major stability problem, hence shelf life of
such preparation is tremendously reduced or they need special storage conditions which
is not economically viable. While in monoclonal antibody preparation, selection and
isolation of an appropriate antigen for developing monoclonal antibody is again a very
brain-taxing problem. However nonmagnetic microspheres do not show any serious
stability problem but they show poor site specificity and are rapidly cleared off by RES
(reticuloendothelial system) under normal circumstances13.
Magnetic fields are believed to be harmless to biological systems and adaptable to
any part of the body. Up to 60% of an injected dose can be deposited and released in a
controlled manner in selected non-reticuloendothelial organs.
So magnetic targeting of microspheres was developed to overcome two major
problems encountered in the drug targeting namely RES clearance (RES readily takes up
Dept. of Pharmaceutics, KLES’s College of Pharmacy, Belgaum. 10
Chapter 1 Introduction
a variety of microparticles including liposomes, microspheres as well as other colloidal
particles) and target site specificity. Figure 1 shows methods of microsphere targeting.
Fig.1: Methods of microsphere targeting
Principle Of Magnetic Drug Targeting 4,14
Magnetic drug delivery by particulate carriers is a very efficient method of
delivering a drug to localized disease site. Very high concentrations of chemotherapeutic
or radiological agents can be achieved near the target site, such as tumour, without any
toxic effects to normal surrounding tissue or to whole body. Fig.2 highlights the concept
of magnetic targeting by comparing systemic drug delivery with magnetic targeting. In
magnetic targeting, a drug or therapeutic radioisotope is bound to a magnetic compound,
injected into patient’s blood stream, and then stopped with a powerful magnetic field in
the target area. Depending on the type of drug, it is then slowly released from the
magnetic carriers (e.g. release of chemotherapeutic drugs from magnetic microspheres) or
confers a local effect. It is thus possible to replace large amounts of drug targeted
Dept. of Pharmaceutics, KLES’s College of Pharmacy, Belgaum. 11
Chapter 1 Introduction
magnetically to localized disease sites, reaching effective and up to several –fold
increased localized drug levels (wider et al., 1979; Gupta and Hung, 1989; Hafeli et
al.,1997).Figure 3 shows the principle of magnetic drug targeting.
Systemic Drug Delivery Magnetic Targeting
Fig.2: Concept of magnetic drug targeting
Fig 3: Principle of magnetic drug targeting
Dept. of Pharmaceutics, KLES’s College of Pharmacy, Belgaum. 12
Chapter 1 Introduction
Benefits Offered By Magnetically Responsive Microspheres 4,15
Magnetically responsive microspheres (MRM) are site specific and by the
localization of these microspheres in the target area, the problem of their rapid clearance
by RES is also surmounted.
Linear blood velocity in capillaries is 300 times less i.e. 0.05 cm/sec as compared
to arteries, so much smaller magnetic field, 6-8 Koe, is sufficient to retain them in the
capillary network of the targeted area. Moreover, restricting microspheres to capillary
bed of targeted area offers more benefits.
a) Diffusion occurs maximally in capillary network so efficient delivery of drug to
diseased tissue is achieved.
b) Microspheres can transit in to extravascular space thereby creating an
extravascular drug depot for sustained release of drug within the targeted area.
c) Therapeutic responses in targeted organs at only one tenth of the free drug dose.
d) Controlled release with in target tissue for intervals of 30 minutes to30 hrs. as
desired.
e) Avoidance of acute drug toxicity directed against endothelium and normal
parenchyma.
f) Adaptable to any part of the body.
g) This drug delivery system reduces circulating concentration of free drug by a
factor of 100 or more.
h) Magnetic carrier technology appears to be a significant alternative for the
biomolecule malformations (i.e. composition, inactivation or deformation).
In case of tumor targeting, microspheres can be internalized by tumor cells due to
its much increased phagocytic activity as compared to normal cells. So the problem of
drug resistance due to inability of drugs to be transported across the cell membrane can
be surmounted.
Dept. of Pharmaceutics, KLES’s College of Pharmacy, Belgaum. 13
Chapter 1 Introduction
Limitations 4
However, this novel approach suffers from certain disadvantages also as given
below:
• Drug(s) can’t be targeted to deep-seated organs in the body. So this approach is
confined to the targeting of drugs in superficial tissues only like skin, superficial
tumors or to joints etc.
• Magnetic targeting is an expensive, technical approach and requires specialized
manufacture and quality control system.
• It needs specialized magnet for targeting, advanced techniques for monitoring,
and trained personnel to perform procedure.
• Magnets must have relatively constant gradients, in order to avoid local over-
dosing with toxic drugs.
• A large fraction (40-60%) of the magnetite, which is entrapped in carriers, is
deposited permanently in target tissue.
Magnet Design 13,16
The force exerted by a gradient magnetic field is an important parameter that governs
magnetic targeting of micro carriers. The relationship of magnetic force to field gradient
and magnetic moment of particles is expressed by following equation : -
F=M∇H
Where,
F= Force on particles
M=Magnetic moment of particles after saturation magnetization
∇H= Magnetic field gradient
Dept. of Pharmaceutics, KLES’s College of Pharmacy, Belgaum. 14
Chapter 1 Introduction
This equation explains that spheres with increased magnetic moments will
experience force sufficient for extra vascular migration of proportionately lower field
gradients. The magnetic moments of microspheres can be increased in three ways: -
a) By clustering magnetite at the center of each sphere to produce large macro
domains.
b) By magnetizing the spheres to saturation levels prior to vascular targeting.
c) By substituting one of the newer ferromagnetic materials that has high
susceptibility than Fe3O4.
Techniques Of Preparation2
There are mainly two techniques, which are commonly employed for
microspheres preparation: -
a) Phase separation emulsion polymerization (PSEP)
b) Continuous solvent evaporation (CSE).
Phase separation emulsion polymerization:
Schematic diagram of preparation of magnetically responsive microspheres by
PSEP technique
Dept. of Pharmaceutics, KLES’s College of Pharmacy, Belgaum. 15
Chapter 1 Introduction
Continuous solvent evaporation:
Schematic diagram of preparation of magnetically responsive microspheres by CSE
Factors Affecting Rate Of Drug Delivery 2
The amount and rate of drug delivery via magnetically responsive microspheres
can be regulated by varying size of microspheres, drug content, magnetite content, their
hydration state and drug release characteristic of carrier. Actually all these factors are
interconnected. The size of microspheres is related to their drug content by a direct
proportionality. However, drug content is also governed by the solubility characteristic of
the drug and method of preparation of microspheres. Hydration step of microspheres
affect their body distribution and drug release rate from the microspheres. The magnetic
content and magnitude of applied field governs the retention of microspheres at targeted
sites. In case of microspheres with higher magnetic content, smaller magnetic field are
sufficient for efficient retention of microspheres in the targeted area. But by
incorporating excessive magnetite into the microspheres, the effective space available for
the drug in microspheres is reduced appreciably. So amount of drug and magnetite
Dept. of Pharmaceutics, KLES’s College of Pharmacy, Belgaum. 16
Chapter 1 Introduction
content of microspheres needs to be delicately balanced in order to design an efficient
therapeutic system.
Drugs Generally Used For Magnetized Targeting
Adriyamycin Doxorubicin
5-Fluro uracil Oxantrazole
Cisplatin Hydrocortisone
Dactinomycin Diclofenac sodium17
Dexamethasone18
Employed carriers
Carriers generally used for entrapping drug and magnetite are: -
Poly lactide Ova albumin
Casein Fibrinogen
Ethyl cellulose Chitosen
Calcium alginate Gelatin
Nitrocellulose Polyvinyl alcohol (PVA)
Starch19 Polyalkylcynoacrylates
Agarose Poly ethylene glycol (PEG) 20
Carnauba wax Polystyrene
Human serum albumin (HSA) Bovine serum albumin (BSA)
N-Isopropyl acrylamide and their copolymer
Dept. of Pharmaceutics, KLES’s College of Pharmacy, Belgaum. 17
Chapter 1 Introduction
Applications
a) The most popular applications of magnetic carrier technology are bioaffinity
chromatography, wastewater treatment, immobilization of enzymes or other
biomolecules and preparation of immonological assay 15.
It is also used in the delivery of insulin, nitrates as well as in selective β
blockers, in general hormone replacement immunization and cancer
chemotherapy.
b) Magnetic delivery of chemotherapeutic drugs to liver tumors : -
The first clinical cancer therapy trials using magnetic microspheres were
preformed by Lubbe et.al. in Germany for the treatment of advanced solid
cancer21, 22.
While current preclinical research is investigating use of magnetic particles
loaded with different chemotherapeutic drugs such as mitoxantrone, paclitaxel 23.
c) Magnetic targeting of radioactivity: Magnetic targeting can also be used to deliver
the therapeutic radio isotopes (Hafeli, 2001) 24. The advantage of this method over
external beam therapy is that the dose can be increased, resulting in improved
tumor cell eradication, without harm to adjacent normal tissue.
Magnetic targeted carriers, which are more magnetically responsive
iron carbon particles, have been radio labeled in last couple of years with isotope
such as 188 Re (Hufli et al. 2001) 25, 90 Y, 111 In and 125I (Johnson et al. 2002) 23 and
are currently undergoing animal trials.
d) Treatment of tumors with magnetically induced hyperthermia: Developments by
Jordan and chan led to the current hyperthermia application of single domain
dextran- coated magnetite nanoparticles in tumors (Jordan et al. 1993; chan et
al.1993) 26. The first clinical trials are going on in Germany (Jordan et al. 2001) 27.
Dept. of Pharmaceutics, KLES’s College of Pharmacy, Belgaum. 18
Chapter 1 Introduction
Magnetic hyperthermia is also possible with larger magnetic particles, as shown
by the group of moroz et al. (2002) 28.
e) On going investigations in magnetic hyperthermia are focused on the
development of magnetic particles that are able to self-regulate the temperature
they reach. The ideal temperature for hypothermia is 43˚C - 45˚C, and particles
with a curie temperature in this range have been described by kuznetsov et al.
(2002) 29.
f) Other magnetic targeting application: It can be used for encapsulation of peptide
octreotide and the protein tumor necrosis factor alpha (TNF-α) (Johnson et al.
2002) 23. Advantages of such an approach are target gene transfection at rapid
speed and high efficiencies.
It is also possible to use only the mechanical- physical properties of magnetic
particles or ferrofulids for therapy. One example is the embolization (clogging) of
capillaries under the influence of a magnetic field (Flores and Liu, 2002) 30. In
this way, tumors could specifically starved of their blood supply. Another elegant
example is the use of magnetic fluids to prevent retinal detachment, thus
preventing the patients from going blind (Dailey et al. 1999) 31.
g) Magnetic control of pharmacokinetic parameters and drug release: Langer et al.
embedded magnetite or iron beads into a drug filled polymer matrix and then
showed that they could activate or increase the release of the 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) 32. The microenvironment with
in the polymer seemed to have shaken the matrix or produced “micro cracks ײ and
thus made the influx of liquid, dissolution and efflux of the drug possible. In this
way, it was possible to magnetically activate the release of insulin from a depot
Dept. of Pharmaceutics, KLES’s College of Pharmacy, Belgaum. 19
Chapter 1 Introduction
underneath the skin (Kost et al., 1987) 33. Done repeatedly this would allow for
pulsative drug delivery.
h) Magnetic system for the diagnosis of disease: The most important diagnostic
application of magnetic particles is as contrast agent for magnetic resonance
imaging (MRI). Suini et al. tested 0.5-1μm sized ferrites in vivo for the first time
in 1987 (Suini et al., 1987) 34. Since then, smaller supramagnetic iron oxides
(SPIOs) have been developed into unimodular nanometers sizes and have since
1994 been approved and used for the imaging of liver metastasis (ferumoxide
based feridex I.V, or Endorem) or to distinguish loops of bowel from other
abdominal structures (GastroMark, or Lumirem in Europe).
i) Magnetic systems for magnetic cell separation: The era of using magnetic
particles with surface markers against cell receptors started in 1978 with a seminal
paper by Kronick et al. (1978) 35. Currently, many different kits for the sample
preparation, extraction, enrichment and analysis of entire cells based on surface
receptor, and subcellular/ molecular component such as protein, mRNA, DNA are
available (Bosnes et al., 1997) 36. Analytical procedures such as many different
immunoassays are often based on magnetic separation (Meza, 1997) 37.
CANCER
“The main problem of cancer therapy is not the lack of efficient drugs, but that
these drugs are very difficult to concentrate in the tumour tissue without leading to toxic
effects on neighbouring organs and tissues.”
Cancer is a Latin word meaning a crab. A malignant tumor, like the crab, has a fat
main body with extensions, like the crab’s feet, which invade the surrounding tissues.
Dept. of Pharmaceutics, KLES’s College of Pharmacy, Belgaum. 20
Chapter 1 Introduction
Irrespective of the aetiology, cancer is basically a disease of cells characterized by
the loss of normal cellular growth, maturation and multiplication and thus homeostatis is
disturbed. 38
The main features of cancer are:
1. Excessive cell growth, usually in the form of tumour.
2. Invasiveness, i.e., ability to grow into surrounding tissue.
3. Undifferentiated cells or tissues, more similar to embryonic tissue.
4. The ability to metastasize or spread to new sites and establish new growths.
5. A type of acquired heredity in which the progeny of cancer cells also retain cancerous
properties.
6. A shift of cellular metabolism towards increased production of macromolecules from
nucleosides and amino acids, with an increased catabolism of carbohydrates for
cellular energy39.
Causes Of Cancer
Dept. of Pharmaceutics, KLES’s College of Pharmacy, Belgaum. 21
Chapter 1 Introduction
Pathophysiology Of Cancer40
A healthy individual has trillions of cells that divide at an orderly rate with a
controlled pace. However, as a result of various carcinogens and exposure to ultraviolet
light which cause DNA mutations, three things happen which turn the once normal cell
into a cancer cell:
(1) The conversion of protooncogenes to oncogenes.
(2) The inhibition of tumor suppressor genes.
(3) The inhibition of DNA repair genes.
In the first step protooncogenes, which encode proteins for cell growth and which
are normally tightly regulated, become oncogenes whereby they never stop producing
growth related proteins. In the second step, genes that would normally suppress these
oncogenes get turned off. In the third step the genes which encode the proteins which fix
DNA mutations get turned off stunting the cells ability to regain control. Basically if any
of these three things do not occur then the cell would either remain normal or the cancer
cell would form but not survive. The immune system also contributes to destroying
cancer cells by recognizing abnormalities on the cancer cells membrane.
Cancer Pathogenesis And Cancer Chemotherapy
General principles:
The term cancer refers to a malignant neoplasm (new growth).
Cancer arises as a result of a series of genetic and epigenetic changes, the main
genetic lesions being:
- Inactivation of tumor suppressor genes.
- The activation of oncogenes (mutation of the normal genes controlling cell
division and other processes.)
Dept. of Pharmaceutics, KLES’s College of Pharmacy, Belgaum. 22
Chapter 1 Introduction
Cancer cells have four characteristics that distinguish them from normal cells:
- Uncontrolled proliferation
- Loss of function because of lack of capacity to differentiate.
- Invasiveness
- The ability to metastasize.
Cancer cells have uncontrolled proliferation owing to changes in:
- Growth factors and/or their receptors
- Intracellular signaling pathways, particularly those controlling the cell
cycle and apoptosis.
- Telomerase expression
- Tumor-related angiogenesis.
Simplified Outline Of The Genesis Of Cancer
Chemical, viruses, irradiation, etc Acquired mutations Inherited mutations Altered gene expression Proto-oncogenes Oncogenes Decreased expression of tumor Sis, erbB, ras, myc, gene for cyclin D, etc suppressor genes: p53, Rb1, etc Other factors Uncontrolled cell proliferation, Decreased apoptosis, Dedifferentiation alterations in telomerase Development of primary tumor Production of metalloproteinases etc. Invasion of nearby tissue by tumor cells. Angiogenesis Metastasis Development of secondary tumors
Dept. of Pharmaceutics, KLES’s College of Pharmacy, Belgaum. 23
Chapter 1 Introduction
Various Cancer Therapies And Their Limitations
Conventional cancer therapies include; surgery, chemotherapy and radiation
therapy. Each has its own limitations in providing a complete cure. Surgical resection is
limited by the ability to expose and remove the tumor and can only remove those tumors
detectable by current imaging techniques. Any cells that are not removed by the surgeon
have the ability to proliferate causing a recurrence. Surgery is also not effective against
micrometastases that may have migrated from the site of primary tumor.
Chemotherapy, whether given systemically or by regional perfusion of a
particular organ, is impeded by the lack of specificity of the drugs for cancer cells.
Therefore, therapy is often limited due to systemic toxicity before truly therapeutic drug
levels in the tumor can be achieved. Drug concentrations in the tumor must also be
sustained for prolonged periods of time for maximum efficacy, so as to catch all the
cancer cells during cell cycle. Chemotherapeutic drugs, usually act on rapidly dividing
cells, so cells of the intestinal lining and bone marrow can be extensively damaged during
treatment.
Radiation therapy can be specifically directed to the site of tumor, but is also
limited by the potential for damage to non-cancerous cells. Radiation therapy like surgery
is a local modality used in the treatment of cancer. Its success depends on the inherent
difference in radio sensitivity between the tumor and the adjacent normal tissues.
Radiation therapy for most solid tumors involves the administration of radiation in the
form of X-rays or gamma rays to a tumor site. Radiation therapy is associated with both
acute toxicity and long-term sequel. Acute reactions occur during or immediately after
therapy. Common manifestations include systemic symptoms such as fatigue, local skin
reaction, gastrointestinal toxicity with nausea, vomiting and dysphagia or diarrhea.
Dept. of Pharmaceutics, KLES’s College of Pharmacy, Belgaum. 24
Chapter 1 Introduction
Chemoembolization is an extension of traditional percutaneous embolization
techniques. With Chemoembolization, investigators embolize tumors with Gelfoam or
Ivalon particles soaked with chemotherapeutic agents, providing vascular occlusion, with
sustained therapeutic level of chemotherapy in the tumor areas. Generalize ischemia
would reduce the ability of the cell to relive itself from the toxicity of chemotherapy.
Such therapy is standard therapy for non-resectable primary hepatocellular carcinoma
(HCC). For Metastatic cancer, however, the benefits are less clear except for Metastatic
neuroendocrine tumors. Chemoembolization, especially in patients with liver metastases
should presently only be performed in the setting of clinical trials41.
None of these therapies alone or in combination have achieved complete cure for
all cancer types in all patients. Therefore many researchers have been exploring
controlled release or targeted delivery options for the treatment of this disease.42, 43
Anti Neoplastics44
Antineoplastic drugs (also known as cytotoxic drugs) are used in the treatment of
malignant neoplasms when surgery or radiotherapy is not possible or has proved
ineffective as an adjunct to surgery or radiotherapy, or as in leukemia, as the initial
treatment. Therapy with Antineoplastics is notably successful in a few malignant
conditions & may be used to palliate symptoms and prolong life in others.
The two main groups of drugs used in the treatment of malignant disease are the
alkylating agents and the antimetabolites Nitrogen Mustards, ethyleneimine compounds
and alkyl sulphonates are the main alkylating agents. Other compounds with an
alkylating action are the various nitrosoureas. Cisplatin and dacarbazine appear to act
similarly.
Dept. of Pharmaceutics, KLES’s College of Pharmacy, Belgaum. 25
Chapter 1 Introduction
Classification
A. DRUGS ACTING DIRECTLY ON CELLS (Cytotoxic drugs) 1. Alkylating agents