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By

N. SREE HARSHA

Dissertation submitted to the


Rajiv Gandhi University of Health Sciences,
Karnataka, Bangalore

In fulfillment of the requirements


for the degree of

Doctor of Philosophy

In
Pharmacy

Under the guidance of

Dr. Shobha Rani. R. Hiremath

DEPARTMENT OF PHARMACEUTICS
AL-AMEEN COLLEGE OF PHARMACY
HOSUR ROAD, BANGALORE - 560 027
2005
This is to certify that the dissertation entitled “Drug targeting
to lungs by way of microspheres” is a bonafide research work
done by N.Sree Harsha in fulfillment of the requirement for the
degree of Doctor of Philosophy.

Date: Dr. Shobha Rani. R.H.


Place: Bangalore Professor & Head
Department of Pharmacy Practice
Al Ameen college of pharmacy
Bangalore
I hereby declare that this dissertation entitled
is a bonafide and genuine
research work carried out by me under the guidance of Dr.
Shobha Rani. R. Hiremath, Professor and Head, Department
of Pharmacy Practice, Al-Ameen College of pharmacy,
Bangalore.

Date:
Place: Bangalore N.Sree Harsha
This is to certify that the dissertation entitled “Drug targeting
to lungs by way of microspheres” is a bonafide research work
done by N.Sree Harsha under the guidance of Dr. Shobha Rani.
R.H. Professor & Head, Department of Pharmacy Practice, Al
Ameen college of pharmacy, Bangalore.

Dr. Sarasija Suresh


Prof. B .G. Shivananda
Professor & Head Principal
Department of Pharmaceutics Al Ameen college of pharmacy,
Al Ameen college of pharmacy, Bangalore
Bangalore

Date: Date:
Place: Bangalore Place: Bangalore
I hereby declare that the Rajiv Gandhi University of Health
Sciences, Karnataka shall have the rights to preserve, use and
disseminate this disseratation in print or electronic format for
academic / research purpose.

Date:
N. Sree Harsha
Place: Bangalore

C Rajiv Gandhi University of Health Sciences, Karnataka


Acknowledgement
It’s a great privilege to express my profound gratitude and heartfelt thanks to my
esteemed teacher Dr.Shobha Rani R.Hiremath for her dedicated support and
encouragement, intellectual guidance and affection bestowed upon me which
instilled confidence in completing my work.

I wish to express my profound gratitude to Prof. B.G.Shivananda , Principal.


Al-Ameen college of Pharmacy for providing all the support, encouragement and
facilities during my course of study.

With deep sense of respect, I thank Rajeev D. Hiremath . for timely suggestion
and encouragement throughout my work.

I thank Dr.Sarasija Suresh , Prof and Head, Department of Pharmaceutics, Al-


Ameen college of Pharmacy, for her support throughout the project.

Thanks to the dedicated staff of Al-Ameen College of pharmacy Dr. Shymala


Bhaskaran, Dr. Kusum Devi, Mrs. Asha, Dr. Sanjay Pai, Dr. Kshama
Devi, Dr. G.K. Rao, Dr. Mahesh Attimarad, for their encouragement, advice
and support.

Thanks to Dr. Venugopal Reddy, Mrs Geeta Pradeep and Mr.Samson


staff and all the students of Pharmacy Practice department for their friendly
approach at all times. A word of thanks to Dr. Praveen, Mr. Bharat,
Mrs.Aisha for their moral support and encouragement all through.

Special thanks to my friends Hemant, Avinash, Jagadeesh, Sam, Mr


srinivasan, Mrs. Narmada for all their help rendered whenever required.
Saying thank you is a small word to my friend Mr.Chandramouli who has been
guiding force and essence of intellectual and moral support in completion of this
work.
I am greatly indebted to my friend Rajarajan for being with me at all times
lending a helping hand in all my work.

All the non-teaching staff of Al-Ameen college of Pharmacy have always helped
in a long way, thanks to Mrs Sabiha Banu, Mrs.Sujatha, Ms.Syeda, Mrs
Jayamma, Mr. Shekar, Mr. Gowda, Mr. Irshad (Jr and Sr.), Mr.
Siddaraju, Mr.Shivanna, Mr.Ravi, Mr.Shareef and Mr. Javed.

Its great pleasure to express my thanks to Chairman Aizaz Khan, Secretary Mansur Ali Khan, Director
Tappan Naik and Principal Mr. M. N. Narasimha Rao for all the support.

I express my gratitude to Mrs Akkamma, Mrs Uma, Mrs. Vijay, Mr.


Suresh, Mrs.Roopashree , staff of C.N.K. Reddy College of Pharmacy for the
support and affection bestowed upon me.

Thanks to Dr.Mohan , Principal, PES College of Pharmacy and all the staff of
PES for their encouragement and concern.

With great appreciation, I thank Principal Dr. Krishnamurthy rao and staff
of Milind College of Pharmacy for their support and motivation and also I thank
Mr. Ravi for helping me in lab.

I wish to thank a crew of people for all the technical support provided at all
stages of my research work.
Thanks to Mrs. Sheela Verma of Microlabs, Hosur for the gift sample of
ofloxacin and for determination of drug content by HPLC method.
Thanks to Mr. Santosh SRF for helping me in animal studies.
Thanks to Dr. Suguna, Prof and Head, Veternary College for Histopathological
studies.

Thanks to Mr.Girish , Cipla, Bangalore for determining particle size analysis.

Thanks to Mr.Rajendra , Biocon for carrying out residual solvent analysis.

Thanks to Mr.Vikram.C for helping me with Tachometer at Zydus Cadila,


Bangalore.
Thanks to Mr.Satish , lecturer, GCP for helping me in procuring required
information at the library.
Thanks to Mr N.S.Chandrashekhar for his great help in XRD studies.

Thanks to Mr. Guruling , IISc, for carrying out SEM studies.

Thanks to Dr.Venkateshiah, Prof and Head, Dept. of Dairy Technology, Hebbal,


Bangalore, for spray-drying my formulations and my sincerely thanks Mr. Arun
Kumar, Lecturer, who helped me during the spray-drying process.

Thanks to Mr.Jairali , IISc for carrying out DSC studies at SSCU unit.

Thanks to Mr. Rajesha B.C . Formulator at Apotex Research Pvt. Ltd for all his
intellectual support in optimization studies.

Thanks to Mrs. Vanaja. K for her inspiring words, support and for proof
reading of my thesis.
Thanks to Mr. Narendra , Health scribe for proof reading of my thesis.

I take this chance to express my word of appreciation to my sister Anu and my


brothers Nagesh, Vinod Girish, Guru who always have been a pillar of support
all through.
I also thank my father in-law and my mother in-law, and Nagashree, for
their encouragement and concern.

I consider it a privilege in thanking my wife Sneha for the unrelenting support,


patience, and encouragement given to me to perform my research work.

All in all, my parents are my life’s greatest treasure. Thank you mom and dad.
LIST OF ABBREVIATIONS USED

1. A The zero time intercept associated with the Alpha phase

2. ADME Absorption Distribution Metabolism Excretion

3. ALOME Albumin ofloxacin microspheres emulsion method

4. Alpha Macro rate constant associated with the distribution phase

5. Alpha-HL The half-life associated with the macro constant Alpha,

Sometimes denoted as Alpha-HL

6. API Active Pharmaceutical Ingredient

7. AUC Area Under the Curve

8. AUMC Area under the first moment curve

9. B The zero time intercept associated with the Beta phase

10. Beta Macro rate constant associated with the elimination phase

11. Beta-HL The half-life associated with the macro constant Alpha,

Sometimes denoted as Beta-HL

12. BSA Bovine Serum Albumin.

13. CCD Central Composite Designs

14. CL Total body clearance.

15. C max Peak plasma concentration

16. CSF Cerebro Spinal Fluid

17. DMSO Dimethyl sulfoxide

18. DOE Design of experiments

19. EA Emulsifying Agent

20. R 2 Co-relation Coefficient


21. FDA Food and Drug administration

22. GLOME Gelatin ofloxacin microspheres emulsion method

23. HIV Human Immunodeficiency Virus

24. ICH International conference on harmonization

25. IV Intravenous

26. K10 half-life

27. K10-HL the half-life associated with rate constant K10, sometimes

Denoted as K10-HL

28. MRT Mean Residence time

29. PLA Poly D,L-lactide

30. PLGA Poly D,L-lactide-co-glycolide

31. ppm parts per million

32. r e time-averaged relative drug exposure

33. RES Reticulo endothelial systems

34. RSM Response Surface Methodology

35. RT Retention time

36. SC Subcutaneous

37. t e targeting efficacy

38. T g Glass transition temperature

39. USP United States Pharmacopoeia and National Formulary

40. V SS Volume of distribution at steady state


ABSTRACT

The efficacy of drug candidates is frequently limited by their inability to reach the target
site of action, especially when they are administered through conventional dosage forms
or drug delivery systems. Targeted drug delivery systems has increased the quantum of
drug reaching the site and simultaneously decrease the amount being distributed to other
parts of the body. Microspheres are solid colloidal particles ranging in size from 1 to 1000
µm, particles with diameter 7 – 15 µm when given through IV route are trapped by the
first capillary system encountered. The capillary sizes of the lungs are of dimensions
lesser than that of the injected microspheres. This phenomenon offers a greater scope of
targeting the drug to the lungs in a more efficient manner, which can act as good targeted
drug delivery platforms especially for pneumonia infections. The research work is aimed
at developing microparticulate targeted drug delivery system of ofloxacin employing
albumin and gelatin as macromolecular carriers for passive targeting to the lungs resulting
in the improved therapeutic efficacy of the drug in case of pneumonia. Pre-formulation
studies were carried out to confirm the suitability of the polymers. Ofloxacin
microspheres were prepared by emulsion polymerization method and mathematically
optimized for the desired particle size range (8-12 µm). Optimized formulae were
GLOME -03 and ALOME -03ofloxacin gelatin and albumin microsphere respectively.
The drug and polymer(s) possessed acceptable suitability and compatibility as evidenced
by the pre-formulation studies. Optimization formulations were in the size range of 8 –
12µm. The surface morphology of the particle were spherical. In-vitro release studies
showed 99.3% and 91.62 release for gelatin and albumin microspheres, release kinetics
showed that the drug release followed Peppas model for both gelatin and albumin
microspheres. Differential scanning colorimetry and X-ray diffraction analysis proved
that the drug is entrapped in gelatin and albumin microspheres. Residual solvent was well
below the permissible ICH limits and provides its safety for formulations. The
formulations were found stable. In-vivo tissue distribution studies of GLOME –03 and
ALOME –03 showed the largest value of AUC for lung when compared with other
organs. The drug distribution was 90.98% and 91.7% for GLOME –03 and ALOME –03.
Pharmacokinetics of the formulations after I.V administration followed two-compartment
model.
Keywords : Microspheres, targeting, lungs, ofloxacin, gelatin, albumin, optimization, in-vivo
TABLE OF CONTENTS
Title Page

1. Introduction 1 - 29
1.1. Targeted Drug Delivery 1
1.2. Drug – carrier delivery systems 4
1.3. Colloidal drug delivery systems 9
1.4. Microsphere 11

1.5. Pneumonia infections 26

2. Objectives 30-31

3. Review of Literature 32-61


3.1. Drug data
3.1.1. Ofloxacin 32
3.2. Excipient data
3.2.1. Gelatin 36
3.2.2. Albumin 37
3.3. Review of research paper 39

4. Methodology 62-113

4.1. Preformulation studies 62-76

4.1.1. Estimation procedure of the drug 62


4.1.2. Selection of polymer 69
4.1.3. Selection of method of preparation 70
4.1.4. Sterilization studies 73
4.1.5. Drug-polymer interaction studies (FT-IR) 75

4.2. Formulation studies 77-101


4.2.1. Preparation of ofloxacin gelatin microspheres. 77
4.2.2. Preparation of ofloxacin albumin microspheres. 78
4.2.3. Optimization of ofloxacin gelatin and albumin

microspheres . 79

4.2.4. Final optimized formulae and formulation codes of


Ofloxacin gelatin and albumin microspheres. 85
4.2.5. Sterilization studies by γ-irradiation. 85
4.2.6. Microsphere recovery / Yield. 87
4.2.7. Free drug in microspheres (un-entrapped) drug. 88
4.2.8. Adsorbed drug in microspheres. 89
4.2.9. Entrapped drug in microspheres. 89
4.2.10. Surface morphology. 90
4.2.11. Particle size analysis. 91
4.2.12. In-vitro drug release and release kinetics. 93
4.2.13. Differential scanning colorimeter. (DSC) 97
4.2.14. X-ray diffraction analysis. (XRD) 99
4.2.15. Residual solvent analysis. 100

4.3. Stability studies 102-104


4.4. In-vivo tissue distribution studies 105-112
4.4.1. Quantitative evaluation of targeted drug delivery
systems 107
4.4.2. Compartment modeling 110

4.5. Histopathological studies 113


5. Results and Discussion 114-203

5.1. Optimization studies 114-141


5.1.1. Optimization of ofloxacin gelatin microspheres 114
5.1.2. Optimization of ofloxacin albumin microspheres 128

5.2. Formulation characterization studies 142-183


5.2.1. Microsphere recovery / Yield. 142
5.2.2. Analysis of free drug in microspheres (un-entrapped) drug. 143
5.2.3. Analysis of adsorbed drug in microspheres. 143
5.2.4. Analysis of entrapped drug in microspheres. 143
5.2.5. Surface morphology. 144
5.2.6. Particle size analysis. 151
5.2.7. In-vitro drug release and release kinetics. 152
5.2.8. Differential scanning colorimeter (DSC). 162
5.2.9. X-ray diffraction analysis (XRD). 172
5.2.10. Drug polymer interaction studies (FT-IR). 178
5.2.11. Residual solvent analysis. 182
5.2.12. Sterilization studies by γ-irradiation 183

5.3. Stability studies 184-190

5.4. In-vivo tissue distribution studies 191-197


5.4.1. Quantitative evaluation of targeted drug delivery
systems 192
5.4.2. Compartment modeling 195

5.5. Histopathological studies 198-203

6. Summary 204-213

7. Conclusion 214-215

8. Bibliography 216-235

9. Annexures 236-237
9.1. List of papers presentation 236
9.2. Ph.D. registration letter 237

10. Index 238-243

10.1. List of Table 238


10.2. List of Figures 240
INTRODUCTION

For a large majority of pharmaceutical formulations in contemporary use,

their ‘specificity’ towards appropriate sites of a disease is not based on their

ability to accumulate selectively in the target organ or tissue. Usually, they

are more or less evenly distributed within the body. Moreover, to reach the

target area, the drug has to cross-different biological barriers – organs, cells,

even intracellular compartments, where it can cause undesirable side-

reactions, or be partially inactivated. The best solution to this issue is drug

targeting.

1.1 TARGETED DRUG DELIVERY

It has been almost 200 years since Paul Ehrlich first formulated the idea of

drug targeting. It was after visiting the opera ‘Der Freischtz’ in which the

Freikugeln plays a major role; Ehrlich came upon the idea of Zauberkuglen –

magic bullets. The Freikugeln in the opera could be fired in any direction, yet

still reach their goal. On similar lines Ehrlich imagined that targeted tiny

drug-loaded magic bullets would target to the required site of action, while

non-target sites would be largely exempted from drug effect. 1, 2

Rationale for targeted drug delivery

Most of the drugs introduced to clinical medicine exert their effects by

interaction with cell and cell membrane related structure and functions

through concentration dependent reversible interactions at specific receptor

sites. Obviously, to obtain a desirable therapeutic response, the correct


amount of drug should be transported and delivered to the site of action with

subsequent control of drug input rate. 3 The efficacy of many drugs is often

limited by their potential to reach their therapeutic site of action. In most

cases, only a small amount of the administered dose of the drug reaches this

site, while the major drug amount is distributed to the rest of the body

depending on the physicochemical and biochemical properties of the drug. 1

Most drugs after administration in a conventional or controlled release dosage

forms freely travel throughout the body, leading to uptake by cells, tissues or

organs other than where their pharmacological receptors are located. This

distribution of the drug to other tissues is unnecessary, wasteful and a

potential cause of toxicity.

The lack of target - specificity can be attributed to the formidable barriers that

the body presents to a drug. A drug taken orally must withstand large

fluctuations in p H as well as the action of enzymes before it gets absorbed.

Moreover it also needs to survive the inactivation by first pass effect. To

produce therapeutic effect, the drug must selectively access and interact with

its pharmacological receptors, and the concentration of the drug at the active

site must be adequate. Administration of the drug by parenteral route avoids

the gastrointestinal associated problems, but deactivation and metabolism of

the drug and dose related toxicity is frequently observed. Further, it cannot be

assured that the drug will reach its desired destination in adequate

concentrations. There are many diseases that are inadequately accessible to

drugs such as cancer, rheumatoid arthritis and certain bacterial, fungal


parasitic infections, etc. The treatment of these diseases often requires high

frequent dosing of drugs, which can lead to toxic manifestations,

inappropriate pharmaco-disposition and untoward metabolism. 4

In contrast, a site-specific delivery would not only increase the amount of

drug reaching the site but also simultaneously decrease the amount being

distributed to other parts of the body. Thus, a target oriented drug delivery

system supplies the drug selectively to its site(s) of action(s) in a manner that

provides maximum therapeutic activity (through controlled and predetermined

drug release kinetics), prevents degradation or inactivation during transit to

the target sites and protects the body from adverse reactions because of

inappropriate disposition. For drugs that have a low therapeutic index,

targeted drug delivery provides an effective treatment at a relatively low drug

concentration 5, 6,7

Requirements for Targeted Drug Delivery

• The delivery system should be biochemically inert, nonimmunogenic and

physically & chemically stable in vivo and in vitro.

• The carrier must be biodegradable or readily eliminated without any problem.

• The preparation of the delivery system must be reproducible, cost-effective

and simple.
Different Approaches of Drug Targeting

There are three approaches for drug targeting. The first approach involves the

use of biologically active agents that are both potent and selective to a

particular site in the body.

The second approach involves the preparation of pharmacologically inert

forms of active drugs, that when they reach the active sites become activated

by a chemical or enzymatic reaction (prodrug approach). Although these

approaches are very successful in some cases, most of the times it is not

feasible to synthesize new site-specific drugs for ailments, due to the

formidable cost and the long time it takes for drug discovery.

For this reason, the third approach, i.e., the delivery of the original drug by

specially designed drug delivery systems is the better and the only feasible

solution. This approach utilizes a biologically inert macromolecular carrier

system that directs a drug to a specific site in the body where it is

accumulated and affects its response.

Regardless of the approach, the therapeutic efficacy of targeted drug delivery

system depends on the timely availability of the drug in active form at the

target site(s) and its intrinsic pharmacological activity. The intrinsic

pharmacokinetic properties of the free drug should be the same, irrespective

of whether or not it is introduced into the body attached to a carrier.


1.2. DRUG – CARRIER DELIVERY SYSTEMS

The basic rationale for using particulate carriers in the intravenous

application takes for granted that the drugs included in the system will be

distributed according to the properties of the carrier. Therefore, the carrier is

expected to “seek out” the preferred site and consequently the drug could be

directed to the intended site of action. For this to happen, the particulate

carrier must have an access to the intended site of drug action, and further it
8
must be able to avoid interactions with other sites within the body.

Depending on the specific indication, an ideal particulate delivery system

should facilitate some of the following properties of drugs:

• Prolong drug effect due to longer circulation time as compared to free

drug.

• Increase the drug concentration at the required site of action due to a

preferential sequestering of the particles by the tissue of the site.

• Reduce drug toxicity in the tissue.

• Protect the drug from, metabolism and immune system recognition until it

reaches the desired target site.

• Through an appropriate particle size, confine the drug delivery system to

the chosen anatomical compartment.

• Interact selectively with the cells of the target site, if equipped with

specific, biologically recognized structural units.

• Retain the drug within the particle while ‘in transit’, and release the drug

at the target site at the appropriate rate.

• Through participating in adsorptive endocytosis, deliver drugs to the

appropriate phagocytic cells.


Drug – carrier delivery systems employ biologically inert macromolecules

(polymers) to direct a drug to its target site in the body. Depending on the

carrier system, the drug can be either molecularly entrapped within the carrier

matrix or covalently linked to the carrier molecules. The major advantage of

drug – carrier delivery systems is that the distribution of drugs in the body

depends on the physico- chemical properties of the carrier and not those of

drugs. This implies that targeting can be manipulated by choosing an

appropriate carrier, or by alterations in the physicochemical properties of the

carrier.

The various methods of vectoring the drug to the target site by the drug –

carrier delivery systems can be broadly classified into: 3

• Passive targeting

• Inverse targeting

• Active targeting

• Double targeting

Passive targeting: Here targeting occurs because of the body’s natural

response to the physicochemical characteristics of the drug or drug-carrier

system. It is a passive process that utilizes the natural course of (attributed to

inherent characteristics) biodistribution of the carrier system, through which,

it eventually accumulates in the organ components of the body. The ability of

some colloids to be taken up by the RES especially in liver and spleen has

made them as ideal vectors for passive hepatic targeting of drugs to these

compartments. Passive capture of colloidal carriers by macrophages offers


therapeutic opportunities for the delivery of anti-infective for disease

conditions that involve macrophage cells of the RES.

Inverse Targeting: It is essentially based on successful attempts to

circumvent and avoid passive uptake of colloidal carriers by RES. This

effectively leads to the reversion of biodistribution trend of the carrier and

hence the process is referred to as inverse targeting. One strategy applied to

achieve inverse targeting is to suppress the function of RES by a pre-injection

of a large amount of blank colloidal carriers or macromolecules like dextran

sulfate. This approach leads to RES blockade and as a consequence

impairment of host defense system. Alternative strategies include

modification of the size, surface charge, composition, surface rigidity and

hydrophilicity of carriers for a desirable biofate.

Active Targeting: Active targeting exploits modification or manipulation of

drug carriers to redefine its biofate. The natural distribution pattern of the

drug carrier composites is enhanced using chemical, biological and physical

means, so that it approaches and is identified by particular biosites. The

facilitation of the binding of the drug-carrier to target cells through the use of

ligands or engineered homing devices to increase receptor mediated

localization of the drug and target specific delivery of drug(s) is referred to as

active targeting.

Active targeting can further be classified into Ligand mediated targeting and

physical targeting.
Ligand mediated active targeting: Targeting components, which have

been studied so far, are bioconjugates, which are anchored as ligands

on some delivery vehicle. These can be specifically functionalized

using biologically relevant molecular ligands including antibodies,

polypeptides, oligosaccharides and viral proteins. The ligands afford

specific affinity 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.

Ligand mediated active targeting could be achieved using specific uptake

mechanisms such as receptor dependent uptake of natural low density

lipoprotein (LDL) particles and synthetic lipid microemulsions of

partially reconstituted LDL particles coated with the apoproteins. The

apoprotein coat serves as a ligand for the LDL receptors expressed in the

body.

Physical Targeting (Triggered Release): The selective drug delivery

programmed and monitored at the external level (ex vivo) with the help of

physical means is referred to as physical targeting. In this mode of

targeting, some characteristics of the bioenvironment are used either to

direct the carrier to a particular location or to cause selective release of its

contents. The first such approach was the temperature sensitive

liposomes. The release of drug from temperature sensitive liposomes in

the vicinity of a tumor (temperature status higher or equal to the phase


transition temperature of constitutive lipids) is brought about by serum

components mostly the lipoproteins, which at phase transition induce

release of the entrapped drug. In another approach, the application of

external magnetic field has been suggested for localization of magno-

responsive liposomes and microspheres within a preselected capillary

bed.

Double Targeting: For a new future trend, drug targeting may be combined

with a methodology, other than the 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 controlled drug release (sustained release / stimuli

responsive release / self – regulating release). Spatial control has been

developed in terms of drug targeting (active / passive targeting). This

approach may bring about 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. When these two methodologies are

combined, it may be called double targeting.

A large number of drug - carrier delivery systems have been conceived and

developed for the purpose of drug targeting. Among these systems, colloidal

drug delivery systems (a type of drug-carrier delivery systems) hold great

promise for reaching the goal of drug targeting and because of their small size

(<1 micron), colloidal drug carriers come close to Ehrlich’s idea of magic

bullets.
1.3. COLLOIDAL DRUG DELIVERY SYSTEMS

A colloid refers to a substance that consists of ultra-fine particles that do not

dissolve, but remain suspended in a medium of different matter. These ultra-

fine particles are larger than most molecules but so small that they cannot be

seen by the naked eye. In other words, a colloidal system consists of a

dispersed phase (or discontinuous phase) distributed uniformly in a finely

divided state in a dispersion medium (or continuous phase) 114

Due to the small size of the particles, colloidal drug delivery systems can be

introduced directly into the central circulation by intra-articular or

intravenous injection, or delivered to a given body compartment by injection

into a joint or by administration by an aerosol to the lungs and nose.

There are three types of colloidal systems:

In simple colloids, clear distinction can be made between the dispersed phase

and the dispersion medium.

E.g.: simple emulsions of oil-in-water (o/w) or water-in-oil (w/o).

Multiple colloids involve the co-existence of three phases of which two are

finely divided, e.g.: multiple emulsions of water-in-oil-in-water (w/o/w) or

oil-in-water-in-oil (o/w/o).

Network colloids have two phases forming an inter-penetrating network.

They can be further classified into monolithic (i.e., containing an intimate

matrix of drug and the core material, e.g.: Nanospheres) or capsular (in which

the liquid drug core is surrounded by the carrier material, e.g.: Nanocapsules)
The biodistribution of colloidal particles is dependent on the route of administration

and physicochemical properties such as particle size and surface characteristics (e.g.:

surface charge and surface affinity). If the route of administration is via intravenous

injection, large particles (>7 micron) are retained in the pulmonary region whereas

small particles (<7 micron) are rapidly phagocytosed by macrophages of the

reticuloendothelial system (RES) residing in the liver and spleen9. By manipulating

the physicochemical properties, the biopharmaceutical characteristics (e.g.: drug

release mechanisms) of the carrier systems can be enhanced, in addition to

overcoming the main physiological barriers to achieve drug targeting, namely to the

RES and the vascular endothelium.

The following colloidal dosage forms are commonly used in drug targeting:

• Nanoparticles10

• Liposomes

• Emulsions and microemulsions

• Polymeric microspheres

• Macromolecular microspheres

Among the entire colloidal dosage forms microspheres are popular as they posses

better stability when compared to others.

1.4 MICROSPHERES – A DESCRIPTION

Microspheres can be defined as solid, approximately spherical particles

ranging from 1 – 1000 µm. They are made up of polymeric, waxy

protective substances, where the entrapped substances (the drug) are

completely surrounded by a distinct wall, or the substance is dispersed

throughout the microsphere matrix.


Figure 1.1: Microspheres - schematic representation

Figure 1.2: Microencapsules - schematic representation


The substances preferred in their formulation should be biodegradable synthetic

polymers and natural products like starches, gums, proteins, fats and waxes. The natural

polymers of choice are albumin and gelatin, the synthetic ones being polylactic acid and

polyglycolic acid. The polymers used in their manufacture are chosen according to their

solublities, stability profiles, process safety and economic considerations.

Microspheres - historical, contextual advent and advantages

The concept of packaging microscopic quantities of materials within microspheres dates

back to the 1930s and the work of Bungenberg de Jong and coworkers on the

entrapment of substances within coacervates. The first commercial application of

encapsulation was by the National Cash Register Company for the manufacture of

carbonless copying paper. The technology and applications have advanced over the last

several decades. The agricultural, food, household products, medical, graphics, and

cosmetics industries use this technology. The potential use of microspheres in the

pharmaceutical industry has been considered since the 1960s. Since then this concept

has found a special relevance in the pharmaceutical formulations because of some

mundane and inherent challenges associated with the present-day formulation problems.

The current drug delivery systems in use, in many cases, fails to meet the need of the

efficient drug delivery at the target site / organ and thereby eliciting a less efficacious

pharmacological response. However advances in microspheres have filled this lacuna to

a certain extent. Microspheres have emerged as one of the desired methods of drug

administration since they offer a sophisticated grasp over the control aspects of drug

administration. The administration parameters, which can be satisfactorily controlled,

include: 14
• Taste and odor masking

• Conversion of oil and other liquids facilitating ease of handling

• Protection of the drugs from environment

• Delay of volatilization

• Freedom from incompatibilities from drugs & excipients especially the buffers

• Improvement of flow properties

• Safe handling of toxic substance

• Dispersion of water insoluble substances in aqueous media

• And most importantly, production of sustained release, controlled release, and

targeted medications.

The microsphere drug delivery method thus facilitates accurate delivery of small

quantities of potent drugs, reduced concentration of drug at sites other than the target

organ or tissue, protection of instable drug before and after administration prior to the

availability at the site of its action. The other major advantage offered by the

microspheres is their ability to manipulate the in vivo action of the drug,

pharmacokinetic profile, tissue distribution, and cellular interactions of the drug can

also be influenced. The drugs, which are formulated as microspheres, include

antineoplastic drugs, narcotic antagonists, steroid hormones, leutinizing hormone

analogs, antibiotics and other macromolecules.

In vivo Fate of Microspheres11, 12, 13

The efficient targeting of drug with microspheres is due to its particle size. The size

plays a significant role in controlling the drug delivery to the target organ and the

subsequent uptake of drug in the tissue. This has implications in pharmaceutical,

biopharmaceutical, clinical and commercial aspects. Particles with diameter 7 – 15µm


when given through IV route are trapped by the first capillary system encountered.

The capillary sizes of the lungs are of dimensions lesser than that of the injected

microspheres. This phenomenon offers a greater scope of targeting the drug to the

target tissue (lungs) in a more efficient manner. Studies of importance have

indicated that the numbers of administered particles as well as their size are

crucial. Particle size greater than 10µm are trapped in the lungs with almost 99%

efficiency by mechanical filtration while smaller particles will be cleared by the

reticuloendothelial system, the particles were found to be trapped in the lungs

within 1-2 minutes where subsequent pharmacological response takes place. The

blood-activity profile will depend on the nature of the microspheres and the size

has no role to play. The blood activity- time profile closely follows bi-exponential

pattern of clearance. The initial part is rapid due to the “burst effect” and the

second phase has a much longer half-life and significantly longer release times.

Though the primary seat of activity is observed in the lungs, some activity is also

observed in the liver, spleen, heart and kidneys. The mechanisms of release of

active ingredients from microspheres are attributed to the following factors:

• Liberation of polymers due to erosion or degradation (osmatically driven

burst mechanism)

• Self diffusion through the pore ( pore diffusion)

• Release from the surface of the polymer ( erosion or degradation of

polymer)

The combinations of one or more mechanisms are seen in many cases. The release

profile of the drug from the microsphere depends on the nature of the polymer

used and the nature of the drug, micro-morphology of carrier and the nature of the

drug.
Figure 1.3: Factors influencing the microspheric drug release
The osmatically driven burst mechanism water/moisture diffuses into the core

of biodegradable/ non-biodegradable coating creating sufficient pressure,

which ruptures the membrane. Burst effect is mainly controlled by three

factors namely,

• Macromolecule polymer ratio

• Particle size of the dispersed macromolecule

• Particle size of the microspheres

In pore diffusion method, the waterfront diffuses towards the core, the

dispersed drug gets dissolved thereby creating a water filled pore network

through which the active principle diffuses out in a controlled manner. In case

of biodegradable polymer the release is controlled both by erosion and

diffusion process. In polymer erosion the loss of polymer is accompanied by

the accumulation of the monomer in the release medium. The erosion of the

polymer begins with the changes with the microstructure of the carrier,

leading it to the plasticization of the matrix, which eventually leads to the

cleavage of the hydrolytic bonds. The cleavage process is also facilitated by

the presence of lysozymes in the surroundings.

The erosion of the polymer may be either surfacial or it may be in bulk

leading to the rapid release of the active ingredients. The rate of extent of

water intake determines the release profile of the microspheres, which in turn

depends on the polymer used, porosity of the polymer matrix and protein drug

loading.
Microsphere Preparation

There are some important factors to be considered for the manufacture of

microspheres since they are designed to elicit a targeted release in vivo. The

physiochemical parameters that are to be considered during the manufacture

include: 14

• Particle size distribution

• Ratio of drug to polymer

• Entrapment / encapsulation efficiency

The manufacturing process must consider the aforesaid parameters for an

efficient drug delivery system. The methods used in the microsphere

manufacture include:

1. Wax coating and hot melt

2. Spray coating & pan coating

3. Coacervation

4. Spray drying

5. Solvent evaporation

6. Precipitation

7. Freeze drying

8. Formation of W/O emulsion

Wax coating and hot melt

The aqueous drug solution is dispersed in molten wax to form a W/O

emulsion, which is emulsified in a heated external aqueous phase to form a

water-in-oil-in-water emulsion. The system is cooled and the microcapsules

collected. For highly aqueous soluble drugs, a non-aqueous phase can be used
to prevent loss of drug to the external phase. Wax-coated microcapsules are

inexpensive and often used, release the drug more rapidly than polymeric

microcapsules. Carnauba wax and beeswax can be used as the coating

materials and these can be mixed in order to achieve desired characteristics.

Spray coating & pan coating

Spray coating and pan coating employ heat-jacketed coating pans in which the

solid drug cores particles are rotated and into which the coating material is

sprayed. The core particles are in the size range of micrometers up to a few

millimeters. The coating material is usually sprayed at an angle from the side

into the pan. The process is continued until an even coating is completed.

Coating a large number of small particles may provide a safer and more

consistent release pattern than coated tablets. In addition, several batches of

microspheres can be prepared with different coating thicknesses and mixed to

achieve specific controlled release patterns.

The Würster process, a variation of the basic pan coating method, is an

adaptation of the fluid-bed granulator. The solid core particles are fluidized

by air pressure and a spray of dissolved wall material is applied from the

perforated bottom of the fluidization chamber parallel to the air stream and

onto the solid core particles. The fluidized-bed technique produces a more

uniform coating thickness than the pan-coating method.

Demerits: Problems can arise with inflammable organic solvents because of

the high risk of explosion in the enclosed fluidizer chamber.


Coacervation

Coacervation technique in which solid particles could be entrapped in

coacervate systems. On phase separation by simple or complex methods tiny

coacervate droplets are formed, which sediment or coalesce to form a separate

coacervate phase. The coacervate forms around any core material that may be

present, such as drug particles. Agitation of the coacervate system can prevent

coalescence and sedimentation of the droplets, which can be cross-linked to

form stable microcapsules by addition of an agent, such as glutaraldehyde, or

the application of heat. Cross-linking of coacervate is necessary to stabilize

coacervate emulsion droplets and hence form microcapsules.

Demerits: Although many successful coacervate microencapsulation systems

have been prepared, coacervate microcapsules have a number of limitations.

They can be produced only at specific p H values, they require stabilization by

cross-linking agents or heat, and the retention of the encapsulant depends on

the extent of cross-linking. The p H limitation can be overcome to some extent

by the addition of water-soluble nonionic polymers, such as polyethylene

oxide or polyethylene glycol. The presence of a small amount of these

polymers allows microencapsulation to occur over an expanded pH range.

Spray Drying

Spray drying is a single-step, closed-system process applicable to a wide

variety of materials, including thermolabile materials. This process is often

used commercially, as a closed system; it is ideal for good manufacturing

practice and the production of sterile materials. The drug and the polymer

coating materials are dissolved in a suitable solvent (aqueous or nonaqueous)


or the drug may be present as a suspension in the polymer solution.

Alternatively, it may be dissolved or suspended within an emulsion or

coacervate system. The microsphere size is controlled by the rate of spraying,

the feed rate of the polymer drug solution, the nozzle size, the temperature in

the drying and collecting chambers, and the size of these chambers.

Figure 1.4: Schematic representation of spray drier assembly


Solvent evaporation

This is one of the earliest methods of microsphere manufacture. The

polymer and drug must be soluble in an organic solvent. The solution

containing the polymer and the drug may be dispersed in an aqueous phase

to form droplets. Continuous mixing and elevated temperatures may be

employed to evaporate the more volatile organic solvent and leave the solid

polymer–drug particles suspended in an aqueous medium. The particles are

finally filtered from the suspension.

Demerits: Continuous mixing and elevated temperatures may be employed

to evaporate the more volatile organic solvent and leave the solid polymer–

drug particles suspended in an aqueous medium, this might denature the

thermolabile drugs or the polymers employed in the preparation process.

Precipitation

Precipitation is a variation on the evaporation method. The emulsion

consists of polar droplets dispersed in a nonpolar medium. Solvent may be

removed from the droplets by the use of a cosolvent. The resulting increase

in the polymer drug concentration causes precipitation forming a

suspension of microspheres.

Emulsion Polymerization 129

Emulsion polymerization involves the dispersion of the monomer units in

an aqueous phase to form droplets, a surfactant, in a concentration higher

than its critical micelle concentration, are present in the aqueous phase.

Excess surfactant molecules form micelles whose hydrophobic interiors


take up part of the available monomer, causing them to swell. Initiator

radicals diffuse into these swollen micelles and begin the polymerization

process. As the monomer is consumed, it is replaced by progressive

diffusion of the remaining monomer from its location in the emulsified

droplets to the interior of the micelles. The micelles continue to swell in

size as polymerization proceeds. The enlarging surfaces compete for

available surfactant, thus influencing the number of available micelles that

can participate in the polymer formation. This method yields particles of

very small size and predictable number at low temperatures.

Micelle polymerization differs from emulsion polymerization in that all of

the monomer and the drug are contained within micelles composed of

surfactant. Diffusion of the monomer from the micelles is prevented by the

nonsolvent properties of the outer phase. Therefore, the increase in particle

size is negligible as the polymerization proceeds.

A great deal of attention has been focused on microspheres preparation for

lung targeting in the treatment of pneumonia.


Figure 1.5: Schematic representation of microsphere formation through
emulsion polymerization process
Table 1.1: Microspheres characterization – parameters and methods

Sl # Parameters Characterization method(s)

1 Yield/ Microsphere recovery


Chemical assay employing a suitable UV
spectroscopic method
2 Drug incorporation efficiency

Scanning Electron Microscopy


3 Surface Morphology Transmission Electron Microscopy
Scanning Tunneling Microscopy
Laser Defractrometry
Fourier Transform Raman Spectroscopy
4 Particle size & size distribution X – Ray Photoelectron Spectroscopy

Design-Expert ® version 6 software for


5 Optimization
experiment design

X-ray photoelectron spectroscopy (XPS) or


6 Residual solvent analysis
Gas Chromatography (GC)

Sigma plot 2004 for windows version 9.01


7 In vitro release kinetics
Copy right © 2004 systat software, Inc

Physicochemical status of the


Differential scanning calorimetry (DSC)
8 drug in the microsphere / glass
analysis
transition temperature (Tg)

Crystallography
9 X-Ray Diffraction analysis (XRD)
characterization

7 Drug stability As per ICH Tripartite guidelines

In Vivo tissue distribution in an animal


8 Targeting potential
model (Albino Mice)

9 In-vivo Quantitative evaluation Ofloxacin distributed to lungs (%)

In-vivo Pharmacokinetics of Modeling the data (Two Compartment i.v


10
microsphere bolus)

11 Tissue biocompatibility Histopathological studies


1.5 PNEUMONIA INFECTIONS

Pathophysiology and disease manifestations15

Pneumonia is an infection of the pulmonary parenchyma. Various bacterial species,

mycoplasmas, chlamydiae, rickettsiae, viruses, fungi, and parasites can cause

pneumonia. Thus, pneumonia is not a single disease but a group of specific

infections, each with a different epidemiology, pathogenesis, clinical presentation,

and clinical course. The first line of treatment in managing pneumonia is by

identification of the causative organism since, this is essential in anti-microbial

therapy. However in almost one-third of cases the specific microbial etiology is non-

specific.

Initial antimicrobial therapy is often empirical and is based on the setting in which

the infection was acquired, the clinical presentation, patterns of abnormality on

chest radiography, results of staining of sputum or other infected body fluids, and

current patterns of susceptibility of the suspected pathogens to antimicrobial agents.

After the etiologic agent is identified, specific antimicrobial therapy can be chosen.

Pathology

The pneumonic process involves primarily the interstitium or the alveoli.

Involvement of an entire lobe is called lobar pneumonia. When the process is

restricted to alveoli contiguous to bronchi, it is called bronchopneumonia. In many

instances confluent bronchopneumonia may be indistinguishable from lobar

pneumonia. Cavities develop when necrotic lung tissue is discharged into

communicating airways, resulting in either necrotizing pneumonia (multiple small

cavities, each lesser than 2 cm in diameter, in one or more bronchopulmonary


segments or lobes) or lung abscess (one or more cavities greater than 2 cm in

diameter). As per the pathological convention, classification of pneumonia is based

upon the causative microorganism rather than upon these anatomic characteristics.

Epidemiology

The patient's living circumstances, occupation, travel history, exposure to pet or

animals, and contacts with other ill individuals as well as the clinician’s knowledge

of the frequency of epidemic occurrences in the community provide useful clues

about the microbial etiology of a given case of pneumonia, since the outbreaks are

endemic in certain cases.

The relative frequency of various pulmonary pathogens varies with the setting in

which the infection was acquired e.g., place of dwelling or hospital. In patients

hospitalized with pneumonia, the most frequent pathogens are Staphylococcus

pneumoniae, Heliobacter influenzae, Chlamydia pneumoniae, and Legionella

pneumophila. Microbacterium pneumoniae, which usually causes mild illness, is

common among outpatients. In contrast, enteric aerobic gram-negative bacilli and

Pseudomonas aeruginosa, are estimated to account for more than 50% of cases of

pneumonia, while Staphylococcus aureus is responsible for more than 10%. Enteric

aerobic gram-negative bacilli and P. aeruginosa are more common among who

acquire pneumonia due to environmental settings.

CLINICAL MANIFESTATIONS

Pneumonia is found to have several clinical presentations; the decision on the line

treatment depends upon the type of pneumonia acquired.


Community-Acquired Pneumonia Community-acquired pneumonia is usually

presented in two syndromes; (i) the typical presentation (ii) atypical presentation.

(i) The "typical" pneumonia syndrome is characterized by the sudden onset of fever,

cough productive of purulent sputum, and in some cases pleuritic chest pain; signs

of pulmonary consolidation (dullness, increased fremitus, egophony, bronchial

breath sounds, and rales) anomalies can be found in radiographical diagnosis. The

typical pneumonia syndrome is usually caused by the most common bacterial

pathogen in an endemic acquired condition is S. pneumoniae, but can also be due to

other bacterial pathogens, such as H. influenzae and mixed anaerobic and aerobic

components of the oral flora.

(ii) The "atypical" pneumonia syndrome is characterized by a more gradual onset, a

dry cough, a prominence of extrapulmonary symptoms (such as headache, myalgias,

fatigue, sore throat, nausea, vomiting, and diarrhea), and abnormalities on chest

radiographs despite minimal signs of pulmonary involvement on physical

examination. Atypical pneumonia is caused by M. pneumoniae but can also be

caused by L. pneumophila, C. pneumoniae, oral anaerobes, and P. carinii as well as

by S. pneumoniae and the less frequently encountered pathogens C. psittaci,

Coxiella burnetii, Francisella tularensis, H. capsulatum, and Coccidioides immitis.

Mycoplasma pneumonia may be complicated by with the pre-existing conditions of

erythema multiforme, hemolytic anemia, bullous myringitis, encephalitis, and

transverse myelitis. Legionella pneumonia can also manifest in extra systemic

conditions like deterioration in mental status, renal and hepatic abnormalities, and

marked hyponatremia; similarly in cases of pneumonia due to H. capsulatum or C.


immitis is often accompanied by erythema nodosum. In C. pneumoniae pneumonia,

sore throat, hoarseness, and wheezing are relatively common.

Certain viruses also produce pneumonia that is usually characterized by an atypical

presentation i.e., chills, fever, dry nonproductive cough, and predominance of

extrapulmonary symptoms. Primary viral pneumonia can be caused by influenza

virus infection (usually as part of a community outbreak in winter), by respiratory

syncytial virus infection (in children and immunosuppressed individuals), by

measles or varicella-zoster virus infection (accompanied by the characteristic rash),

and by cytomegalovirus infection (in patients immunocompromised by HIV

infection or by therapy given in association with organ transplantation). In addition,

influenza, measles, and varicella can predispose to secondary bacterial pneumonia

as a result of the destruction of the mucociliary barrier of the airways. Secondary

bacterial infection may either follow the viral infection without interruption or be

separated from the viral infection by several days of transient relief of symptoms.

Bacterial infection may be heralded by sudden worsening of the patient's clinical

condition, with persisting or renewed chills, fever, and cough productive of purulent

sputum, possibly accompanied by pleuritic chest pain.

Patients with hematogenous S. aureus pneumonia may present with fever and

dyspnea only. In these cases the inflammatory response is initially confined to the

pulmonary interstitium. Cough, sputum production, and signs of pulmonary

consolidation develop only after the infection extends into the bronchi. These

patients are usually gravely ill, with intravascular infection as well as pneumonia,

and may have signs of endocarditis.16


2. OBJECTIVES

Pneumonia is one of the widely prevalent diseases of both developed and

developing world. The incidence of pneumonia in most cases are found in

endemic and epidemic proportions currently making this one of the difficult

disease to manage and is rated as the sixth highest disease in terms of contraction

and fatality17. The current therapeutic practices to contain and cure pneumonia

centers around anti-microbial therapy which in most cases is of empirical nature,

since pneumonia is caused by a variety of bacterial flora and hence a low success

rate is achieved in spite of advances in newer molecules available for treatment.

Ofloxacin of late has emerged as the gold standard in the treatment of

pneumonia, nevertheless the conventional approaches adopted to administer

ofloxacin by oral or parenteral routes does not provide the most efficacious mode

of treatment. . Microspheres are solid colloidal particles ranging in size from 1 to

1000 µm, particles with diameter 7 – 15 µm when given through IV route are

trapped by the first capillary system encountered. Exploring alternative routes of

administration like microspheres to develop a targeted drug delivery system to

act locally on the organ of infection will improve the therapeutic efficacy, reduce

side effects and thereby provide patient compliance.

OBJECTIVES OF THE STUDY

 To obtain passive targeting of drug to lungs by way of microspheres,

by increasing the quantum of drug reaching the site and

simultaneously decrease the amount being distributed to other parts of

the body and protects from adverse reactions due to repeated or high

dosing.
 To prepare microspheres using natural, biodegradable and

biocompatible polymers.

 Standardizing the methods of preparation of microspheres

 To obtain the desired particle size range of the microspheres for lung

targeting by mathematical optimization of the variables of

formulation.

 Formulation of microspheres as per the polynomial equations

generated from the optimization studies.

 Evaluation of the formulations for their yield, particle surface

morphology, particle size, in-vitro release, release kinetics,

differential scanning colorimetry, x-ray diffraction analysis, residual

solvent analysis and stability studies.

 To prove the hypothesis of targeted drug delivery of the microspheres

to lungs by studying in-vivo tissue distribution in comparison to

conventional dosage of the drug in an animal (albino mice) model.

 Finally to prove the safety of the formulations by carrying out

histopathological studies.
REVIEW OF LITERATURE

3.1. DRUG DATA

Ofloxacin has a demonstrated activity against a broad spectrum of gram

positive aerobic and anaerobic bacteria it is bactericidal at concentration

equal or slightly greater than inhibitory concentration (2µg/ml); ofloxacin

exerts this effect on susceptible micro organism by inhibiting DNA gyrase, an

essential enzyme catalyst which is critical in duplication, transcription and

repair of bacterial DNA. Since ofloxacin has a proven activity against

microorganisms which causes pneumonia, with a good safety profile, this is

the drug of choice in the current therapy of pneumonia and hence was chosen

to be formulated as microspheres and their activity were studied.

3.1.1. OFLOXACIN 18, 19

Description

Chemically ofloxacin is a fluorinated carboxyquinolone, and is a recemate, (±)-9-

fluoro-2-3-dihydro-3-methyl-10-(-4-methyl-1-piperazinyl)-7-oxo-7H-pyrido[1,2,3-

de]-1,4 benzoxazine-6-carboxylic acid

It has the following structural formula:


Ofloxacin empirical formula is C 18 H 20 FN 3 0 4 and its molecular weight is

361.4. Ofloxacin is off-white to pale yellow crystalline powder. The relative

solubility characteristics of ofloxacin at room temperature as defined by USP

nomenclature, indicates that ofloxacin is considered to be soluble in aqueous

solutions with p H between 2 and 5. It is sparingly to slightly soluble in

aqueous solutions with p H 7 (solubility falls to 4mcg/mL) and freely soluble

in aqueous solutions with p H above 9

Clinical Pharmacology

Pharmacokinetics: The pharmacokinetics of Ofloxacin are linear over the

dose range of 200 to 400 mg administered intravenously. The serum

elimination half-life is approximately 5-8 hours. The absolute bioavailability

of oral Ofloxacin is within a range of 95 –100% with no substantial loss by

first pass metabolism. Following I.V. ofloxacin administration, maximum

serum concentrations are reached at the end of a 30min or 60min infusion.

Compared with oral administration, the maximum serum concentration

achieved after I.V administered may be upto 50% higher, but distribution and

elimination pharmacokinetics are similar, regardless of the mode of

administration. The peak serum concentration after a 400 mg once daily I.V

dose of ofloxacin are 8.1±2.1µg per ml. Steady state is generally reached

within 24 – 36 hours.

Urine: Ofloxacin is mainly eliminated unchanged in the urine, with 70 – 90%

of a dose recoverable after 24 – 48h and only minimal amount (<4%) as

metabolites.
Inactivation in body: Serum protein binding of ofloxacin is about 30%.

Ofloxacin is not metabolized to any large extent, but is excreted unchanged in

the urine. In fact, ofloxacin undergoes less biotransformation than other

commonly available fluroquinolones.

Distribution of Drug in Body: Ofloxacin administered either I.V. or orally

penetrates well into bronchial secretions, lung tissue and pleural fluid.

Toxicity: The overall rate of adverse reactions reported for ofloxacin in

clinical trials is 2.4 – 12.3%, which compares favorably to other

fluroquinolones such as ciprofloxacin. After intravenous administration,

ofloxacin is present in saliva, lung tissue, pleural fluid, blister fluid, bone,

biliary CSF and bronchial secretions

Microbiology: Ofloxacin has activity against a wide range of gram-negative

and gram-positive microorganisms. The bactericidal action of Ofloxacin

results from interference with the enzyme DNA gyrase, which is needed for

the synthesis of bacterial DNA.

Indications and Usage: Ofloxacin is indicated for the treatment of infections

caused by susceptible strains of the designated microorganisms in the

following conditions; urinary tract infections, Pneumonia, Bacterial diarrhea,

enteric fever, sexually transmitted disease, respiratory tract infections,

mycobacterial infections, osteomyelitis, soft tissue and skin, nasal carriage of

Neisseria, meningitidis, prevention of infection in neutropenic patients,


otiitits, sinusitis, ocular infections, intra-abdominal and biliary tract

infections.

Adverse Reactions: The overall rate of adverse reactions reported for

ofloxacin in clinical trials is 2.4 – 12.3%, which compares favorably to other

fluroquinolones such as ciprofloxacin. The most frequently reported adverse

events, without regard to drug relationship, among patients treated with

intravenous Ofloxacin were nausea, dizziness, Neurotoxicity, hypersensitivity

reaction and rashes were also noted in patients treated with the most common

doses of ofloxacin.

Overdosage: In the event of acute overdosage, the patient should be carefully

observed and given supportive treatment. Adequate hydration must be

maintained.

Dosage and Administration: The recommended adult dosage for urinary

tract infections of mild to moderate severity is 200 mg IV every 12 hours. For

severe or complicated urinary tract infections, the recommended dosage is

400 mg IV every 12 hours. The recommended adult dosage for lower

respiratory tract infections, skin and skin structure infections and bone &

joint infections of mild to moderate severity is 400 mg IV every 12 hours. For

severe/complicated infections of the lower respiratory tract, skin and skin

structure and bone and joint, the recommended adult dosage is 400 mg IV

every 8 hours. The recommended adult dosage for mild, moderate and severe

nosocomial pneumonia is 400 mg IV every 8 hours.


3.2 EXCIPIENT DATA

3.2.1 GELATIN

Physical Characters: A faintly yellow to light yellowish-brown, tasteless

solid, usually occurring as translucent sheets, shreds, granules or powder. It is

practically insoluble in common organic solvents; it swells in cold water and

on heating it gives a colloidal solution, which on cooling forms a more or less

firm gel. The isoelectric point of type A gelatin is between pH 6.3 and 9.2 and

that of type B gelatin is between p H 4.7 and 5.2.

Gelatin is one of the preferred biodegradable micro encapsulation polymer

obtained from the partial hydrolysis of collagen derived from the skin,

connective tissue and connective tissue bone and sinews. It obtained either

from a bovine or porcine source. Gelatin is obtained by either an acid

treatment – type-A or an alkaline treatment type-B. Type-B bloom strength

250 is a widely used choice because they offer good particle size range, drug

loading and the entrapment rate, the drug release kinetics is controllable by

the reaction type and concentration of the linking agent usually formaldehyde

or glutaraldehyde. Gelatin microspheres are prepared by cross-linking gelatin

in a w/o emulsion with glutaraldehyde, this method is preferred because the

shape of the microspheres obtained is spherical and the size of the

microspheres can be controlled to an average diameter in the range of 5 - 40

µm. The size and the surface characterizes of the gelatin microspheres are

crucial, since they exert a great influence on the macrophage uptake; and are

susceptible to macrophage interference lesser than this range. However the

susceptibility is less when the microspheres exceed the threshold limit of 5


µm. The other advantages of gelatin microspheres are they are widely used for

controlled drug release, it is biocompatible and the degradation products are

non-toxic and readily excreted. Apart from this, it is a soluble polymer it can

be modified to prepare drug delivery systems. 14, 20, 21, 22, 23, 24

3.2.2 ALBUMIN

Albumin is a widely occurring natural protein. The particulate or the colloidal

form is an ideal carrier of drugs either for a site-specific delivery, or delivery

to anatomically varied sites. Earlier it was confined to be used in diagnostic

procedures since they have a propensity to locate themselves at selected sites

based on their size range to facilitate scopy procedures. In current perspective

they are used to target tumor cells due to their selective uptake of protein

carriers, thereby offering a new scope in targeted drug delivery.

Preparation of Albumin microspheres is easy and particle size range of 15 to

40µm is readily obtainable through various techniques, incorporating the

active ingredients during the preparation or after the preparation of the

microspheres. This offers scope to adjust the process variables, degradation

time and the diameter of the microspheres.

The widely used and the method of choice is emulsion polymerization using

heat denaturation of the particles at elevated temperatures (110 – 165 o C) or

through chemical cross-linking, this process also brings about a

conformational changes due to surface binding of oil to albumin and found to

be least immunogenic. However the microspheres prepared by the aforesaid


process tend to be hydrophobic and a small amount of surfactant is essential

to disperse them for a parenteral formulation. Hydrophobicity is due to the

polar region of the albumin aligning at the water-oil interface to form a

hydrophobic crust or mantle at room temperature; moreover, hydrophobic

microspheres have rapid elimination rate. The Microspheres with enhanced

hydrophilicity are preferred since; this provides enhanced surface and

physical characters in vivo by providing better mechanical entrapment,

absorption and drug release profile which are desirable properties for

microspheres. 13, 14, 23, 24, 25


3.3 REVIEW OF RESEARCH PAPERS

1. To overcome the restriction in using cross-linked gelatin in the pharmaceutical

field, D, L-glyceraldehyde (GAL), a non-toxic crosslinking agent, was proposed.

To evaluate the pharmaceutical properties, an antihypertensive drug, clonidine

hydrochloride, was chosen as drug model and loaded into the microspheres.

Either the increase of the crosslinker concentration or of the crosslinking time

period decreased both the swelling and the in vitro drug release processes of the

microspheres. The biocompatibility studies showed that the microspheres cross-

linked with GAL are well tolerated in vivo. These results suggested the potential

application of gelatin microspheres cross-linked with GAL as a suitable drug

delivery system for the subcutaneous administration.20

2. Transnasal absorption of pharmaceutical drugs has been recognized as an

interesting alternative to the more conventional routes of administration. A

method of administrating L-dopa following the transnasal route was developed.

Gelatin microspheres were prepared by the w/o emulsification solvent extraction

technique: the microspheres had a median particle size of 16.2 +/- 4.2 micro and

were prepared using a stirring speed of 600 rpm for 5 min at 80 degrees C. L-

dopa was incorporated into the microspheres with an efficiency of 65 +/- 6.7%.

L-dopa was released from the microspheres, showing an initial fast release rate,

followed by a second slower release rate.28

3. Lung-targeting cisplatin-loaded poly(lactic-co-glycolic) acid microspheres

(CDDP-PLGA-MS) were prepared by a solvent evaporation method. Uniform

design was used to optimize the technology of preparation, the appearance and
size distribution were examined by scanning electron microscope, and the

aspects such as in vitro release characteristics, stability, drug loading,

loading efficiency, pharmacokinetics and tissue distribution in rabbit were

studied. CDDP-PLGA-MS showed a combination of lung-targeting and

sustained drug release in experiments on rabbits. 41

4. Serum albumins and polylactic acid (PLA) are reported to be as

bioerodable polymers in the preparation of drug-containing microspheres

for parenteral drug delivery, by a process of chemical cross-linking or

heat denaturation of the protein. HPLC analysis of the HSA microspheres

showed that about 37% of MMC was converted to 2,7-diaminomitosene

derivatives in microspheres prepared by heat denaturation at 120 degrees

C. The degradation increased to 82% when the microspheres were

prepared with a denaturation temperature of 170 degrees C. Biacetyl was

found to interact with MMC leading to the formation of 7-aminomitosene

derivatives. In contrast to the albumin system, MMC could be

incorporated into PLA microspheres without degradation. 77

5. Bovine serum albumin nanospheres (BSA-NS), prepared with

glutaraldehyde cross-linking and ultrasonication, were coated with

antibodies by covalent linkage (Schiff's base formation) of aldehyde

groups at the surface of the nanospheres with amino groups of the

antibody. An in vivo binding test showed that monoclonal antibodies

might recognize the target organ (Lewis lung carcinoma). These findings

suggested that, BSA-NS coated with monoclonal antibodies were found to


be trapped in the tumoral tissue of Lewis lung carcinoma-bearing mice

more than in control (BSA-NS coated with mouse IgG) at 24 hr after the

injection. Thus, BSA-NS offered to be potential drug carriers enabling

concentration of drugs at specific target sites. Further, their rapid

elimination from the body and their degradability suggested that side

effects due to long-lasting accumulation in several organs could be

avoided. 78

6. The potential of albumin (bovine serum) microspheres as delivery

systems for haematoporphyrin and/or its zinc derivative was probed at the

molecular level. The microspheres, prepared by heat denaturation, were

stable to detergents and organic solvents but could be degraded by

proteolytic enzymes. The efficiency of encapsulation was found to depend

on the initial drug/protein ratio with a saturable pattern. The population of

the slow-releasing pool was higher, the high-affinity pool being the fast-

releasing one, the low-affinity pool being the slow-releasing one.

Implications for the microsphere-mediated delivery of these drugs in vivo

were also discussed. 79

7. Adriamycin-loaded bovine albumin microspheres were prepared by a

technique that allows preparation and administration to animals on the

same day. In contrast to bolus injection, serum drug levels after

microsphere administration indicated an initial rapid release followed by

a more protracted phase lasting at least 24 h. This latter observation was

consistent with drug release during biodegradation of carrier. 80


8. In order to deliver antitumour agents to target sites, various methods

employed nontoxic and biodegradable drug carriers. Albumin microsphere

is one of those drug carriers. The progress has been made during the last

10 to 15 years in the application of albumin microspheres as drug carriers

to cancer chemotherapy. Preparation and physicochemical properties of

microspheres containing antitumor agents, pharmacokinetics of the

microspheres in experimental animals, and the antitumor efficacy by

intra-arterial use against human primary metastatic liver tumor was also

discussed. 81

9. Gelatin microspheres containing lactic acid were prepared by the

polymerization technique using glutaraldehyde as the cross-linking agent.

The effect of time of cross-linking and the amount of cross-linking agent

on the swelling properties of microspheres and their release profile were

investigated. In vitro release pattern of lactic acid from gelatin

microspheres showed a biphasic profile and the release rates were reduced

upon increasing the amount of cross-liking agent and prolonging the

cross-linking time. 82

10. Gelatin microspheres were prepared by water/oil emulsion polymerization

and by cross-linking with glutaraldehyde. The influence of preparation

compositions on microsphere recovery, particle size and morphology,

swelling and degradation, 5-fluorouracil loading and release, and

cytotoxicity were investigated. It was observed that concentration resulted

in the formation of smaller (140.82-71.47 micron) for gelatin


microspheres. The most rapid 5-fluorouracil release was observed from

uncross-linked microspheres (about 88% in 4 h), whereas a particular

slower release (about 36% in 4 h) profile was obtained for the highly

cross-linked ones. Cytotoxicity tests of free and entrapped 5-fluorouracil

were carried out with MCF-7 breast cancer cell line. Free 5-fluorouracil

produced an immediate effect, whereas entrapped 5-fluorouracil showed a

prolonged cytotoxic effect. 83

11. Pingyangmycin gelatin microspheres: its preparation and drug release

characteristics were reported in vivo and in vitro. The experiment

indicated that the diameter of PYM-GMS was more appropriate for the

application in external carotid artery embolization with PYM-GMS,

which significantly reduces the circulating drug level and the dosage,

prolonged the time period of higher drug concentration, acheiving the

purpose of sustained release and targeted tumor therapy. 84

12. Biologically adhesive delivery systems offer important advantages over

conventional drug-delivery systems. Microspheres intended as a sustained

release carrier for oral or nasal administration were prepared by

associating a known bioadhesive polymer, poly(acrylic acid), in gelatin

microspheres with a model drug oxprenolol hydrochloride. The internal

structure of the microspheres studied by X-ray diffraction, thermal

analysis and optical microscopy showed the absence of drug crystals in

microspheres and a lowering in the glass transition temperature. In vitro

and in vivo experiments in rats showed good adhesive characteristics of


the gelatin/poly(acrylic acid) microspheres, which were greater if the

poly(acrylic acid) content was greater. A significant retardation in gastric

and intestinal emptying time of the beads was observed, which was also

suggested by the bioavailability of the model drug after intragastric and

intranasal administration of the microspheres. 85

13. Monodisperse gelatin microcapsules containing an active agent using

microchannel (MC) emulsification, a novel technique for preparing water-

in-oil (W/O) and oil-in-water (O/W) emulsions was reported. A W/O

emulsion with a narrow size distribution containing gelatin in the aqueous

phase was created as follows. First, the aqueous disperse phase was fed

into the continuous phase through the MCs at 40 degrees C (operating

pressure: 3.9 kPa). The emulsion droplets had an average particle

diameter of 40.7 micron and a relative standard deviation of 5.1%. The

temperature of the collected emulsion was reduced and maintained at 25

degrees C overnight. The reported emulsification technique required only

a low-energy input, creating desirable experimental conditions for

microencapsulation of unstable substances such as peptides and proteins.

This method was promising for making monodisperse microbeads. 86

14. Porous gelatin microcarriers having a diameter of 80-100 micron were

prepared by the suspension method using toluene as the oil phase. Rat

hepatocytes were cultured on gelatin and cytodexIII microcarriers. The

cells retained its spherical shape, which is similar in vivo, and showed no

morphological changes to the flat state. Hepatocyte aggregates on


microcarriers maintained higher metabolic functions than monolayer

cells. Pore size of microcarrier plays an important role in the attachment

and metabolic function of cells in culture. Phase-contrast micrograph and

cell activity showed that hepatocytes cultures on gelatin microcarrier of

<1 micron pore size is better than that of 5-10 micron. 87

15. A study was conducted to compare a series of polymers to PVA in a 2(2)

full factorial design study. The influence of the concentration of PVA and

the polymers tested on particle size and zeta potential value was evaluated

before and after freeze-drying of the prepared particles. The original 2(2)

full factorial design study was further expanded to a central composite

design for poloxamer and carbopol, in order to fit the measured data to a

quadratic model and to calculate response surfaces. 88

16. Preparation of Gelatin microspheres and sponges for delivery of

macromolecules were reported by coacervation and freeze drying

techniques, respectively. Studies demonstrated that release was dependent

on the amount of BSA (Bovine Serum Albumin)present in the system and

crosslinking densities of microspheres. It was concluded that gelatin

microspheres and gelatin sponges are promising carrier matrices for

macromolecules. 89

17. Microspheres based on a poly(hydroxybutyrate-hydroxyvalerate)

copolymer (PHBV) (Mw = 630kD, 21% mol HV) were loaded with

diazepam using different emulsion-solvent evaporation processes. Gelatin


was used as a strategy to alter the release profile of the incorporated drug.

Scanning electron microscopy revealed that the microspheres had

different structures depending upon their method of preparation. 90

18. A fluid mechanics-based correlation for the average size of Bovine Serum

Albumin (BSA) microspheres, prepared using a water-in-oil emulsion

technique, was presented. The correlation was formulated based on the

theory of turbulent dispersion and a non-dimensional Weber number. The

effect of additives on microsphere size was also explored. The average

diameter of the BSA microspheres was doubled by the addition of a

bioadherent vitelline protein B solution. In addition, a Rosin-Rammler

statistical distribution function was shown to accurately represent the

microsphere size distribution obtained at different stirring speeds. 91

19. Preparation and characterization of bovine serum albumin (BSA)

microspheres and the evaluation of the in vitro cytotoxicity of these

microspheres on acute promyelocytic leukaemia (HL-60) cells were

described. Mitoxantrone (MTZ)-incorporated microspheres were

evaluated for particle size, drug loading, release characteristics and

surface morphology. Study demonstrated that BSA microspheres were

well suited for the controlled release of MTZ and were promising for anti-

cancer chemotherapy. 92

20. Controlled-release egg albumin-chitosan microspheres containing

indomethacin as a model drug were successfully prepared by coacervation


method. The proposed method offers a simple method for microsphere

preparation in an aqueous system with the elimination of the use of

organic solvents that are usually needed in conventional techniques of

microencapsulation. The effects of many process variables, such as

amount of formaldehyde as a cross-linking agent, stirring time, final pH

of encapsulation medium, initial drug loading, and albumin concentration

or albumin-to-chitosan weight ratio, on the properties of the prepared

microspheres were investigated. It was also observed that high

concentration of albumin solution and accordingly the increase of

albumin-to-chitosan weight ratio were accompanied with increase in

incorporation efficiency and particle size with improved microsphere

flowability and slow indomethacin release. The proposed microspheres

showed the ability to control the release of indomethacin, and their

properties were highly affected by many process variables that could be

controlled to obtain an optimized system. 93

21. A novel spray-drying technique consisted of feeding a fluid through an

ultrasonic atomiser, drying the spray under reduced pressure and

collecting the particles in a liquid bath was evaluated. The protein bovine

serum albumin as a model (BSA) was encapsulated in poly(lactic-co-

glycolic acid) microspheres. The microspheres were found to be very

porous and exhibited a pronounced 24-h burst release of above 50% of

total dose, probably promoted by the porosity. However, when more

concentrated polymer solutions (8% instead of 5% (w/w)) were employed,

burst release reduced to an average of 16%. 94


22. Terbutaline sulfate has a short biological half-life but a long acting

terbutaline sulfate formulation is desirable to improve patient compliance.

Bovine serum albumin microspheres were prepared by an emulsion

polymerization method using glutaraldehyde as the crosslinking agent. In

the presence of trypsin, a linear relationship was obtained between the

degradation rates and trypsin concentrations, indicating that saturation

was not reached under the experimental conditions. Biodistribution

studies indicated that the degree of uptake by the lungs was higher than

that of the other organs. All these results demonstrated that terbutaline

sulfate loaded microspheres can be used for passive lung targeting. 95

23. Ciprofloxacin albumin microspheres were prepared by the spray drying

technique, with bovine serum albumin as the natural biodegradable wall

material. The spherical microspheres, flowed well, were organic solvent

free and in the size range 1-5 micron. The drug release from the

microspheres could be retarded by further thermal denaturation. The

sustained-release microspheres were suitable for dry powder inhaled lung

drug delivery systems. 97

24. Emulsion spray-drying tecnique for the preparation of albumin-loaded

PLGA microspheres was evluated. Albumin was dissolved in an aqueous

phase (w) in the presence of surfactant and emulsified in an organic phase

containing the polymer (o). Results showed that the emulsion/spray-

drying method is suitable for obtaining small microparticles (2-5 micron)


characterized by high drug payloads (70%-80% encapsulation efficiency).

The type of surfactant affected the microsphere shape and BSA release.

25. Microspheres were prepared by the suspension crosslinking method for

the first time in the absence of any surface-active agent, using paraffin oil

as the dispersion medium and formaldehyde as the crosslinking agent.

The microspheres thus obtained were characterized using a Scanning

Electron Microscope and found that microspheres to be spherical with a

particle size distribution in the range 50-400 microns. This method, being

simple and cost effective, could be a promising technique for the large-

scale manufacture of microspheres. 99

26. Monodisperse albumin microspheres were successfully prepared by both

chemical and thermal hardening methods via membrane emulsification

using microporous glass membranes with uniform pore sizes. The

monodispersity of the microspheres was found to depend strongly on

parameters such as albumin concentration, emulsifier concentration, and

volume ratio of the internal aqueous phase (albumin solution) to the

dispersion medium (organic solvent). The optimum conditions for

obtaining monodisperse albumin microspheres were described. 100

27. Targeted Isoniazid (INH) albumin microspheres were prepared by two

different stabilization processes: chemical denaturation using

glutaraldehyde and heat denaturation. A factorial concept has been

utilized to synthesize microspheres suitable for passive targeting to the


lungs by varying protein concentration, stabilization temperature, time

and aqueous volume. These factors significantly affected the sphere size,

payload and a release profile of the drug. The microspheres carrying

isoniazid were further investigated in experimental animals to validate the

targeting process. 101

28. Albumin nanospheres as carriers for passive drug targeting: an optimized

manufacturing technique was developed to produce albumin particles in

the sub-200-nanometer range with a narrow size distribution and in a

controlled and reproducible manner. A central composite design was used

to evaluate the influence of different process parameters on particle size,

polydispersity and yield. Of all the factors investigated, only the albumin

concentration and the aqueous phase volume showed a significant

influence on response parameters. Albumin nanospheres with sizes below

200 nm in diameter and very narrow size distributions were obtained in

high yields (> 80%). The study concluded that this new preparation

method for albumin nanoparticles was suitable for future drug targeting

studies. 102

29. Preparation of corticosteroid-loaded albumin microspheres were designed

for intra-articular administration with dexamethasone as the model drug

and bovine serum albumin as biodegradable, natural polymer using

spray drying, the effects of both polymer/drug ratio used in the

formulations and the different heat-stabilization conditions were


evaluated on morphology, size, solubility characteristics, drug loading

and 'in vitro' drug release of the microparticles. 103

30. Human serum albumin (HSA) microspheres were produced in the size

range of 200 nm to 10 microns by the controlled addition of acetone to an

aqueous solution of HSA, followed by stabilization of the formed

microspheres at an elevated temperature. The acetone-heat denaturation

method was different from the traditional oil/water technique for

preparation of HSA microspheres, both in terms of production method and

the avoidance of high temperatures (> 100 degrees C) and extended

heating times (> 30 min) for stabilization. This paper describes the

influence of process conditions, such as volumes of acetone and HSA

concentration, on the formation of the microspheres and their

morphological characteristics. 104

31. The preparation of albumin and gelatin microspheres with a

tuberculostatic agent, rifampicin, was studied, and its in vivo distribution

was investigated by providing its accumulation in the target organ lungs.

The purpose was to improve the effectiveness by injecting the

microspheres intravenously with much smaller doses than normally

required with generalized systemic administration, whilst reducing the

systemic side effects. The percentage accumulated was higher in the lungs

than in the other organs for both albumin and gelatin microspheres,

whereas free rifampicin accumulated mainly in the liver. To support the

data of the in vivo distribution studies, the microspheres were


histopathologically investigated after the intravenous injection to Swiss

albino mice. After the microscopic determination of the lungs and liver,

the spherical microspheres were observed after 10,30 and 60 min, and 6

and 24 h post-injection. 105

32. Hydroxy-urea bearing albumin microspheres-preparation, characterization

and evaluation was reported. The process affecting the physical

characteristics with respect to in vitro and in vivo performance of the

prepared microspheres were studied. A remarkable stabilization of

hydroxy urea in the prepared microspheres was recorded when t 10% drug

degradation was compared with the albumin microspheres prepared by a

conventional emulsion polymerization method using water as an internal

phase. 106

33. Albumin microspheres and microcapsules containing cisplatin (CDDP)

were prepared and tested as chemotherapeutic agents for the treatment of

hepatocellular carcinoma by hardening with glutaric aldehyde in

accordance with the method to prepare W/O emulsion. Microcapsules

were also prepared by formation of a coacervate by the phase isolation

method. The blood CDDP concentrations after injection of both

formulations were lower than those noted after injection of CDDP

injectable solution, indicating that CDDP might be accumulated in the

liver at a higher concentration and that use of the two formulations might

result in alleviation of CDDP side effects. 107


34. A new two-step desolvation method for manufacturing gelatin

nanoparticles was developed. After the first desolvation step, the low

molecular gelatin fractions present in the supernatant were removed by

decanting. The resulting particles were purified by centrifugation and

redispersion, analysed by gel permeation chromatography (GPC). Results

concluded that the molecular weight of gelatin has a decisive influence on

the stability of the manufactured gelatin nanoparticles. Two fluorescent

dyes (Texasred and fluoresceinamine) were coupled to the nanoparticles

for cell uptake studies and fluorescent nanoparticles showed a high uptake

into monocytes/macrophages. 108

35. The use of oxidized dextran as a cross-linker for the preparation of

gelatin microspheres was described. Microspheres were obtained by a

thermal gelation method and their dissolution kinetic was examined.

Swelling tests and gelatin microspheres dissolution experiments were

performed, to determine sugar mediated cross-linked. Results indicated

that oxidized dextran can form a cross-linked gelatin network which can

reduce the dissolution of gelatin. Oxidized dextran could be an interesting

means to cross-link gelatin microspheres allowing the use of this delivery

formulation for controlled release of drugs. 109

36. Hyaluronidase is a protein whose enzymatic activity is successfully

employed in extravasation therapy. Microspheres have been prepared

using a water-in-oil emulsification technique, and they have been cross-

linked either by thermal or chemical means. Results showed that


hyaluronidase microspheres with good morphological characteristics can

be obtained by this preparation method. The suitability of this enzyme as

a microparticulate drug carrier was shown by the successful encapsulation

of hydrocortisone sodium succinate. 110

37. Tolnaftate microcapsules and microspheres were prepared by gelatin-

acacia coacervation and emulsion-solvent evaporation methods

respectively. The physical state of the drug in these formulations was

determined by using scanning electron microscopy (SEM), X-ray powder

diffractometry, and differential scanning calorimetry (DSC). High

pressure liquid chromatographic (HPLC) method was used for stability

determination and polymer-drug interactions were evaluated using FTIR.

Powder X-ray diffractometric method was able to demonstrate the

presence of crystalline drug and polymorphic changes, if any, in both

these formulations. HPLC data revealed that the drug was stable in these

formulations for atleast 6 months. The FTIR studies indicated the absence

of any drug interaction with the polymeric matrix materials, during

preparation of these dosage forms. 111

38. The influence of preparation parameters on gelatin microspheres

production, chemico-physical characteristics and drug encapsulation were

investigated. The manufacturing parameters such as amount of polymer,

stirring speed, presence and concentration of surfactants were focussed.

TAPP-Br, clonidine hydrochloride and bromocriptine mesylate were

chosen as model drugs in order to compare their encapsulation and release


characteristics with microspheres. Study of the influence of thermal

treatment on the microspheres is reported, performed with the aim to

possibly modify gelatin dissolution and drug release. The effect of this

treatment was evaluated on microsphere characteristics such as swelling,

porosity and dissolution, and finally on the release profiles of the

encapsulated drugs. 112

39. An attempt to prepare a lung targeting dosage form, ligustrazine

hydrochloride gelatin microspheres (LTH-GMS) by the method of

emulsion process, using 2:1 as the weight ratio of LTH to gelatin was

employed. The preparation technique was optimized and the appearance,

particle size and size distribution, LTH content, in vitro release, stability,

and in vivo distribution of LTH-GMS were studied. The results showed

that the mean diameter of LTH-GMS measured by Coulter counter was

12.65 microns, 87.5% of the microspheres ranging from 5 to 24.9

microns. The release t 1/2 of LTH when given LTH-GMS was about 6 times

as much as that of original LTH. The relative distribution percentage of

LTH-GMS in lung when determined 20 min after iv administration to

mice was significantly higher than those of LTH-GMS in other tissues

and blood, and was about 6 times as much as that of the LTH solution

control group in lung under the same conditions. 113

40. The preparation and characterization of gelatin microspheres (GMs)

containing the antitumour aromatic tetra-amidine TAPP-Br and its release

characteristics from microspheres were described. Spherical gelatin


microspheres, showing a high percentage of drug encapsulation (over 95

per cent) and an average diameter of 16 microns were obtained by a

coacervation method. In vitro studies were carried out and the results

obtained suggested that gelatin-based microspheres offer excellent

potential as carriers for the controlled release of polyamidines in

anticancer chemotherapy. 115

41. The preparation and characterization of biodegradable gelatin

microspheres for the controlled release of peptides and proteins was

investigated. Bovine serum albumin (BSA) was chosen for incorporation

into the gelatin microspheres and the spheres were characterized for the in

vitro release of BSA and other properties. The entrapment efficiency of

FITC-BSA was about 62%. The in vitro release pattern of FITC-BSA

showed that 51% of the entrapped drug was released during the first day

and the release followed approximate zero order kinetics from day 2

onwards. The total release of FITC-BSA lasted for about 8 days. SDS-

PAGE analysis revealed that BSA was not degraded by this preparation of

microspheres. 116

42. A study investigated the organ distribution characteristics of 5-

fluorouracil (5-FU) loaded cross-linked gelatin microspheres (5-FU-MS)

after intravenous (i.v.) injection compared to 5-FU solution in mice, and

to evaluated the targetability of 5-FU-MS. The results showed that the

concentration of 5-FU in the lung was significantly higher than after

application of 5-FU solution, and that the maximum concentration of 5-


FU, 72.8 microgram organ (-1), was reached in the lung at 15 min after

I.V. administration. The percentage of drug distributed to the lung was 2

times as high as after application in solution. So 5-FU-MS were capable

of effectively delivering 5-Fu to the lung and possessed specific

targetability towards the lung compared to 5-Fu solution. 117

43. Incorporation of carboplatin in the gelatin microspheres by the method of

emulsion and the drug content determined spectrophotometrically was

reported. Very high lung-targeting efficiency in vivo was proved by the

results of targeting parameters. The S-180 lung neoplasm models were

established by i.v. cancer cells in mice and the number of pulmonary

nodules examined for evaluation of the treatment effect. The results of

therapeutic tests showed that the antitumour effects were increased by

injection of the microspheres compared with the injection of CPT

solution: half of the dose in the microspheres showed comparable effect

to the original drug. 45

44. The effects of different variables on the preparation of polylactic acid

microspheres (PLA-MS) were studied. The optimized preparation

conditions of rifampicin polylactic acid microspheres (RFP-PLA-MS)

were acquired through orthogonal test. The paddle method was used to

study the drug release properties of RFP-PLA-MS. Stability of RFP-PLA-

MS at different temperatures was also studied. Pharmacokinetic and tissue

distribution of RFP-PLA-MS after intravenous administration were


carried out in rabbits. RFP-PLA-MS showed a combination of lung

targeting and sustained drug release in experiments on rabbits. 118

45. Microspheres are drug carrier system which ensures controlled release

in the shape of solid sphere particles with variable diameter

distributions from a few microns to a millimeter of size. Natural human

serum albumin and bovine serum albumin, which were frequently used

in early microsphere studies and are being used in some studies as

microshpere matrix material, were used. Albumin microspheres

containing clarithromycin were prepared by heat stabilization at

different stirring rate. In the first part of for, drug content, payload,

particle size, surface morphology and release characteristics were

studied from microspheres prepared. 119

46. Rifampicin polylactic acid microspheres for lung targeting were

prepared by a modified emulsion-solvent diffusion method. The

microspheres were free flowing, spherical with regular surface. Drug

content, particle size distribution and in vitro release properties of the

prepared microspheres were evaluated. In vivo experiments on rabbits

showed remarkable accumulation of microspheres in the lung. 120

47. Microspheres of clarithromycin were prepared from human serum

albumin using the emulsion polymerization technique. Albumin

microspheres containing the active substance were injected into the tail

vein of mice. Mice was sacrificed at intervals and microspheres


collected from lungs and livers. The microsphere accumulation began

at 10 min, and increased gradually until 6 h, then a decrease was

observed. The microspheres were still present after 24 h. In the liver

sample, no microsphere accumulation was observed at any time. 121

48. Albumin microspheres of sizes ranging from 1 to 32 am were prepared

by an oil-in-water emulsification method. The effect on mean size and

size distribution of different variables at two levels was studied

according to a factorial design of experiments. The following variables

were studied: (1) type of albumin; (2) albumin concentration; (3) speed

of agitation; (4) chemical cross-linking or heat denaturation; (5)

glutaraldehyde concentration or temperature; (6) addition or absence of

surfactant (Span 85); (7) type of oil; and (8) mixing-cell with or

without baffles. Particle size analysis was performed on the resultant

microspheres using a laser diffraction technique. The effect of each

variable and possible interactions between the variables on

microsphere size are discussed. 122

49. The technology of lung targeting gelatin microspheres of streptomycin

sulphate (SMS) has been reported. The microspheres were prepared

with natural biodegradable gelatin as the load material and castor oil as

the oil phase. The experimental conditions were optimized; the mean

volume diameter obtained being 9.7 microns and the mean rate of

encapsulation 15.69%. In vitro, the SMS release rate was found to


accord with Higuchi equation with t1/2 = 8.6h. In vivo (rabbits) the

gelatin microspheres were proved to be concentrated in the lung. 123

50. Lung targeting gelatin microspheres of mitoxantrone (DHAQ) were

prepared. Release of the drug from the DHAQ-GMS in vitro was much

slower and its t1/2 was 4 times longer than that of pure DHAQ. The

characteristic peak of heat absorption on the differential thermal

analysis curve was at 133 degrees C and almost no change was

observed after the DHAQ-GMS were stored for 3 months at 37 degrees

C (relative humidity 75%). Kinetic behavior of the drug in mouse lung

was described by one open compartment model, and the average

residual time increased by 10 h. 124

51. 125I-labeled bovine serum albumin microspheres loaded cisplatinum

(CDDP-125I-BSA-MS) with the size range of 7-25 microns were

prepared and injected into the tail vein of mice. The results showed

that the microspheres were accumulated almost entirely in the lung

after I.V. injection (about 97.52%-injected dose at the highest

concentration). Photomicrographs showed that microspheres reached

the lung and lodged in precapillary arteriols and capillaries of lung.

The microspheres in lung were eliminated gradually. Based on the

analysis of the models and parameters, it was concluded that the

pharmacokinetics of microspheres in lungs could be described by the

model of "one-compartment of first-order intake and first-order


125
elimination".
52. An optimum procedure was established by orthogonal test for

preparing cisplatinum albumin microspheres (CDDP-BSA-MS) with

emulsion-chemical cross-linking. The results showed that the surface

was regular, the mean size was 13.13 +/- 3.55 microns, embedding

ratio was 21.62% and the release characteristics in vitro were in accord

with "biphase kinetics equation". The microspheres accumulated

almost entirely in the lung 15 minutes after intravenous injection to

mice. The total amount in the lung was about 97% of the injected dose

at the peak concentration. Two-compartmental model was used to

describe the regulation of the pharmacokinetics of albumin

microspheres in lung. 126

53. Factorial concept was utilized to optimize the size and drug loading of

microspheres suitable for targeting of Aminophylline, a bronchodilator,

for targeting to the lung to treat emphysema. They were prepared by

heat denaturation adjusting the manufacturing variables, namely

albumin concentration, drug concentration, water-oil phase ratio and

stirring rate at low and high levels. For two levels of the four variables

the experimentation was performed in 16 batches. The drug loading

was found to be maximum (36.33 per cent) in batch XV of

microspheres prepared with low albumin concentration, high drug

concentration, low water-oil phase ratio and low stirring rate. 127
4.1.1 Estimation Procedures of the drug

Analytical procedures should work reproducibly, when carried out by the

same or different person, in same or different laboratories using different

reagents, different equipment, etc. The objective of any analytical procedure

should be clearly understood since this will govern the validation

characteristics, which need to be evaluated. Typical validation characteristics

that should be considered are listed below: 26

^ Accuracy

^ Precision

• Repeatability

• Intermediate Precision

• Reproducibility

^ Specificity

^ Detection Limit

^ Quantitation Limit

^ Linearity

^ Range

^ Robustness

As per the ICH Harmonized Tripartite Guideline each of these is defined

Accuracy

The accuracy of an analytical procedure expresses the closeness of agreement

between the value, which is accepted either as a conventional true value or an

accepted reference value and the value found. This is sometimes termed

trueness.
Precision

The precision of an analytical procedure expresses the closeness of agreement

(degree of scatter) between a series of measurements obtained from multiple

sampling of the same homogeneous sample under the prescribed conditions.

Precision may be considered at three levels: repeatability, intermediate precision

and reproducibility.

Precision should be investigated using homogeneous and authentic samples.

However, if it is not possible to obtain a homogeneous sample, it may be

investigated using artificially prepared samples or a sample solution.

The precision of an analytical procedure is usually expressed as the variance,

standard deviation or coefficient of variation of a series of measurements.

Repeatability

Repeatability expresses the precision under the same operating conditions over a

short interval of time. Repeatability is also termed intra-assay precision.

Intermediate precision

Intermediate precision expresses within-laboratory variations: different days,

different analysts, different equipment, etc.

Reproducibility

Reproducibility expresses the precision between laboratories (collaborative

studies, usually applied to standardization of methodology).


Detection Limit

The detection limit of an individual analytical procedure is the lowest

amount of analyte in a sample, which can be detected but not necessarily

quantitated as an exact value.

Quantitation Limit

The quantitation limit of an individual analytical procedure is the lowest

amount of analyte in a sample, which can be quantitatively determined

with suitable precision and accuracy. The quantitation limit is a parameter

of quantitative assays for low levels of compounds in sample matrices, and

is used particularly for the determination of impurities and/or degradation

products.

Linearity

The linearity of an analytical procedure is its ability (within a given range)

to obtain test results which are directly proportional to the concentration

(amount) of analyte in the sample.

Range

The range of an analytical procedure is the interval between the upper and

lower concentration (amounts) of analyte in the sample (including these

concentrations) for which it has been demonstrated that the analytical

procedure has a suitable level of precision, accuracy and linearity.


Robustness

The robustness of an analytical procedure is a measure of its capacity to

remain unaffected by small, but deliberate variations in method parameters

and provides an indication of its reliability during normal usage.

Statistics in analytical methods

It is customary to subject calibration curve values of analytical methods to

regression analysis. The general purpose of linear regression (the term was

first used by Pearson, 1908) is to learn more about the relationship between

an independent variable and a dependent variable. The information from a

regression analysis is used to build a regression equation of the form – ‘Y =

mX+ c’. Thus, the Y variable can be expressed in terms of a constant (c)

and a slope (m) times the X variable. The constant is also referred to as the

intercept, and the slope as the regression coefficient or B coefficient

[Statsoft, Inc; 2004].

The regression line expresses the best prediction of the dependent variable

(Y), given the independent variables (X). However, there is bound to be

substantial variation of the observed points around the fitted regression

line. The deviation of a particular point from the regression line (its

predicted value) is called the residual value. The smaller the variability of

the residual values around the regression line relative to the overall

variability, better is the prediction. If X and Y are perfectly related then

there is no residual variance and the ratio of variance would be 0.0. In

most cases, the ratio would fall somewhere between these extremes, that is,

between 0.0 and 1.0. 1.0 minus this ratio is referred to as R-square or the
coefficient of determination. The R-square value is an indicator of how

well the model fits the data (e.g., an R-square close to 1.0 indicates that we

have accounted for almost all of the variability with the variables specified

in the model).

Customarily, the degree to which two or more predictors (independent or X

variables) are related to the dependent (Y) variable is expressed in the

correlation coefficient R, which is the square root of R-square. In multiple

regression, R can assume values between 0 and 1.

Procedure

Ofloxacin

Estimation by spectrophotometry

This method was employed for the determination of ofloxacin

Instrument: Shimadzu 1601 UV visible spectrophotometer.

Reagents: (1) Standard stock – 1mg/ml ofloxacin in 0.1N HCL.

(2) Working stock – 100 mcg/ml ofloxacin in 0.1N HCL.

(3) Working stock – 20 mcg/ml ofloxacin in 0.1N HCL.

(4) 0.1N HCL.

Determination of absorption maxima

The working stock (3) solution was scanned in the range of 200nm to

400nm after correcting the base line with a reagent blank (0.1N HCL). The

λ max was found to be 297 nm. Hence all further investigations were carried

out at the same wavelength.


Determination of Beer’s law range and standard graph

After conducting a number of trials, the concentration range, which obeys

Beer’s law was found to be between 0 – 20 mcg/ml.

From the working stock (3) solution different aliquots of 1.0, 2.0, 3.0…

10.0 ml was taken in series of 10 ml volumetric flasks and volume made up

with 0.1 N HCl. The absorbance was measured against a reagent blank at

297 nm and the standard graph was plotted, which is shown in figure 4.1.1.

Recovery studies

To the pre-analyzed solutions of the above experiment, 4.0 ml of working

stock solution was again added in a 10 ml volumetric flask and the volume

made up with 0.1 N HCl. The absorbance of the resulting solution was

measured at 297 nm against a reagent blank. The recovery result is shown

in table 4.1.1

The calibration curve values of the spectrophotometric methods were

subjected to regression analysis (Microsoft® excel), the results of which

are listed in table 4.1.1


Figure 4.1.1: Calibration curve of ofloxacin in 0.1N HCL

0.7
0.6
ABSORBANCE (nm)

0.5
0.4
y = 0.0319x
0.3 2
R = 0.9986
0.2
0.1
0
0 5 10 15 20 25
CONCENTRATION (mcg/ml)

Table 4.1.1 Statistical (Regression) analysis of calibration curves

R2 Recovery ( % ) Linearity
(mcg/ml)
Spectrophoto- 0.9986 95.08 0 – 20
metric estimation of Standard calibration curve – Absorbance = 0.0319 x
ofloxacin 0.1 N HCl concentration
The coefficient of determination ‘R 2 ’ are within acceptable limits for the

method of spectrophotometric estimation. The spectrophotometric method

was reproducible and hence reliable for routine analysis of drug from

formulations.

4.1.2. Selection of polymer

Natural biodegradable polymers are classified as proteins and

polysaccharides. Proteins include gelatin and albumin, and

polysaccharides include Alginate, Dextran, Chitosan, and Agarose.

Polysaccharides are non-compatible materials, and may cause allergic

reaction, dermatitis, nausea and dizziness; hence protein biodegradable


27
polymers are preferred.

Natural biodegradable polymers remain attractive primarily because they

are natural products of living organisms, ready available relatively

inexpensive and capable of a multiple of chemical modifications. Hence,

albumin and gelatin were found suitable polymers for the preparation of

microspheres.

Procedure

Blank microsphere employing gelatin and albumin as polymers were

prepared by the water in oil emulsion and spray drying method. 28 The

microspheres obtained were subsequently characterized for their surface

morphology and particle size.


Results

Both the polymers were found to be suitable for the formulation of microspheres. The

surface morphology revealed distinct, spherical microspheres of both the polymers,

which were prepared by water in oil emulsion method and shrivel (surface folding)

when prepared by spray drying technique.

Microspheres prepared by water in oil emulsion method as described in section 4.1.3

Using gelatin had an average size of 42µm (1000rpm; gelatin-0.5g; emulsifier-0.5ml)

while albumin microspheres had an average particle size of 37µm, (1000rpm;

albumin-0.5g; emulsifier-0.5ml) section 4.1.3 which was almost near to the specified

range and microspheres prepared by spray drying technique had an average size of

12µm and 10µm respectively, which was within the specified range.

4.1.3. Selection of the methods of preparation:

Many different techniques have been proposed for the production of microspheres and

it was suggested that more than 200 methods could be identified in the patent

literature.29

Many investigations classify microencapsulation process as chemical or mechanical

method. We have selected one chemical and one mechanical process for the

preparation of microspheres.

It has been reported that many scientists have prepared microspheres by water in oil

emulsion technique and spray drying technique for lung targeting.12, 30, 31, 32
Procedure

First blank microspheres of Gelatin and Albumin were prepared by two

different methods of preparation, which have been widely employed for

microspheres preparation from natural polymer, viz. water in oil emulsion and

spray drying method. The blank microspheres formed were characterized for

their surface morphology and Particle size. Thus, two methods of preparation

were feasible for the preparation of microspheres viz.

1. Water in oil emulsion method

2. Spray drying method.

Preparation of the gelatin/albumin microspheres by w/o emulsion method

Preparation of the gelatin microspheres: The microspheres were prepared

by keeping process variables as stirring rate (1000 rpm), concentration of

gelatin (2g) and concentration of emulsifying agent (0.5 ml). Polymer

solution was prepared in 10 ml of water and the dissolution process was aided

by warming the solution. The solution was stirred for 20 min at 25 0 C prior to

this the solution was 80 0 C and added to 30 ml of liquid paraffin maintained at

the same temperature the emulsifying agent tween 85 was added into the two

phase system and was stirred continuously for 5 min then the emulsion was

rapidly cooled to 5 0 C and 100 ml of acetone was added to dehydrate and

flocculate the coacervate particles. The microspheres were isolated through a

filtration process with a sintered glass filter. This process however was

optimized by changing various process parameters as listed above to arrive at

a standardized working procedure.


Preparation of the Albumin microspheres: The albumin microspheres were

prepared by taking 2 gms of albumin. This was solubilized; then the contents

were slowly added to a beaker containing 650 ml of sesame oil and stirred for

1h at 1000 rpm, to this 0.5 ml of tween 85 was added and was stirred

temperature was elevated 40 0 C to ensure the hardening process for 25min.

The microspheres were isolated by filtration and washed with n-hexane and

dried at room temperature. Here to the preparation procedure was optimized

by trying out various changes in the process variables to arrive at a standard

working formula.

44
Preparation of Gelatin/Albumin Microspheres Spray Drying Method

Spray drying constitutes easy and consistent method of preparing

microspheres. The solutions of the polymers viz. gelatin and albumin were

dissolved in 200ml of water with mild warming to solublize the polymers.

Then the solution was spray-dried through a standard nozzle (0.7mm

diameter) 200ml of water by mild warming to solublize gelatin and albumin.

Water in oil emulsion technique produced spherical, discrete, microspheres

with an average particle size of 58µm with gelatin and 42µm with albumin.

Spray drying method also produced shriveled (surface folding), discreet

microspheres with size of 12µm (gelatin) and 10µm (albumin). The shriveled

and bored-out appearance of the spray-dried microsphere was seen during the

SEM surface morphology studies figure 5.1.4 & 5.1.5. Hence sensing upon in

this inelegance of the product and potential failure in case of drug loading,
the spray drying process was discontinued in favor of emulsion

polymerization. 33

However, the size range produced by emulsion polymerization process was

found unsuitable for the intended purpose. The ideal particle size range for

lung targeting and subsequent mechanical filtration in-vivo should ideally be

in the range of 5-15µm. Hence it was decided to optimize the size range and

accommodate in the desired particle size range.

34
4.1.4. Sterilization Studies

Sterilization is a process of ensuring total absence of microorganisms. The

death rate of microorganisms proceeds in a logarithmic scale. A 10 -6 Sterility

Assurance Level (SAL) is required for products coming in contact with

tissues.

The most common form of sterilization is steam sterilization, or autoclaving.

Autoclaving exposes the material to high temperature (greater than 121 o C)

and steam at high-pressure. These conditions destroy microorganisms by

disrupting key metabolic and structural entities, especially membrane proteins

and lipids. Steam sterilization is rapid compared to other methods, requiring

about 30 minutes of exposure. It is also simple, non-toxic, and relatively

inexpensive. However, most polymeric biomaterials degrade under the harsh

process conditions of steam sterilization, which often exceed the glass

transition (Tg) and melting temperatures (Tm). 35 As a result, autoclaving is

generally not used to sterilize polymeric biomaterials.


An alternate sterilization method exposes the biomaterial to ethylene oxide

gas. Ethylene oxide (EtO) kills microorganisms by alkylating the amine

groups on nucleic acids. However, EtO is also toxic to humans, since it

attacks these same amine groups. 35 Any residual gas can induce a toxic

response in vivo. As a result, gas sterilization is generally not used to sterilize

polymeric biomaterials.

In dry heat sterilization, the biomaterial is exposed to 160 – 190 0 C, but the

problem is melting and softening of polymer, so this method is not used to

sterilize polymeric biomaterials.

Aseptic filtration though a popular method for parenterals again could not be

employed for microspheres formulations because they are colloidal in nature

wherein the particle size is around 5 – 15µm.

Gamma (γ) radiation is a type of radiation that sterilizes biomaterials by

ionizing the nucleic acids of the contaminating microorganisms. The use of

gamma radiation has been widely accepted as the sterilization method of

choice for polymeric biomaterials, as it is rapid, non-toxic and leaves no

residue.

Of all the classical methods of sterilization, only γ - irradiation method could

be applied for the sterilization of microspheres.


Surface Morphology

Blank Gelatin and Albumin microspheres were prepared by emulsion

polymerization method. Thus obtained microspheres were characterized for

particle morphology by Scanning Electron Microscopy (SEM) and particle

size (laser diffraction) studies were carried out on these formulations prior to

sterilization. These microspheres formulations were then subjected to γ -

irradiation. After sterilization, the particle morphology and particle size was

determination again carried out.

Results

Scanning Electron Microscopy (SEM) photographs of microspheres prior and

after γ irradiation studies showed no major structural changes. More

importantly the particles did not break and were intact. A slight increase in

the particle size (1.2µm – 3 µm) was observed after sterilization with γ -

irradiation. As the increase was negligible it was concluded that γ - irradiation

was the best-suited method of sterilization for microsphere formulations.

4.1.5. Drug Polymer Interaction Studies

Drug-polymer interactions were studied by FT-IR spectroscopy. The spectra

were recorded for ofloxacin, gelatin, albumin, physical mixture of ofloxacin:

albumin (1:1) and physical mixture of ofloxacin: gelatin (1:1) using. Samples

were prepared in KBr disks (2 mg sample in 200 mg KBr) with a hydrostatic

press at a force of 5.2 τ cm -2 for 3 minutes. The scanning range was 400 –

4000 cm -1 and the resolution was 4 cm -1


Results: The IR spectrum of ofloxacin, albumin and gelatin was employed as

reference with which the spectrums of the physical mixtures were compared.

On comparison of the individual spectrum with that of physical mixtures, no

prominent difference in the spectrums was seen. Thus, it was concluded that

there are no major degenerative drug-polymer interactions and hence the

polymers could be used safely to formulate.


4.2. FORMULATION STUDIES

The formulation studies constitute the phase of the work involving the

optimization of preparation procedures decided during the pre-formulation

phase. This phase of work helps in deciding the right working formulae for

the microspheres, which during this phase of work were finalized upon

conducting optimization techniques like design of experiments involving

changes in various process parameters involved in the preparatory procedure.

4.2.1. PREPARATION OF OFLOXACIN GELATIN MICROSPHERES

Microspheres were prepared by varying parameter such as Stirring rate,

Concentration of gelatin and amount of emulsifying agent as in table - 4.2.1.

A solution of gelatin (Gelatin dissolved in 10 ml of water) was prepared by

warming and the drug was added to the gelatin solution. The concentration of

drug was varied according to the varying concentration of Gelatin (1:1 Drug:

Gelatin). The solution was stirred for 20min at 25 0 C and the solution was pre

heated to 80 0 C and added to 30ml of liquid paraffin previously warmed to the

same temperature. This two-system, plus Tween 85 was added to obtain a w/o

emulsion. The resulting microspheres were stabilized using 0.1ml

glutaraldehyde solution (25%w/v) for a period of 15min. After 5min of

continuous stirring, the emulsion was rapidly cooled to 5 0 C and 100ml of

acetone added in order to dehydrate and flocculate the coacervated particles.

Gelatin microspheres were then isolated by filtering the suspension through a

sintered glass filter. The preparation was carried out according to

optimization procedure table - 4.2.1. The process parameter such as stirring


rate, concentration of gelatin and concentration of emulsifying agent were

varied according to the optimization process. 28

4.2.2. PREPARATION OF OFLOXACIN ALBUMIN MICROSPHERES

Albumin microspheres were prepared by taking varying quantities of albumin

as shown in table - 4.2.2. A solution of albumin was prepared and the drug

was added to the albumin solution. The contents were slowly added to a

beaker containing 650 ml of sesame oil and stirred for 1h. This two systems

plus Tween 85 was stirred. The temperature was raised to 40 0 C for hardening

process and was maintained for 25min.The resulting microspheres were

stabilized using 0.1ml glutaraldehyde solution (25%w/v) for a period of

15min. The microspheres were collected by filtration and washed with n-

hexane and dried at room temperature. The preparation was carried out

according to optimization procedure table - 4.2.2. The process parameter such

as stirring rate, concentration of albumin and concentration of emulsifying

agent were varied according to the optimization process.

Table 4.2.1: Variables operating range for gelatin microspheres

Independent Variable Low Level High Level

Stirring Range (rpm) 1000rpm 5000rpm

Concentration of
0.5g 2g
Gelatin (gms)
Concentration of
0.5ml 2 ml
Emulsifying agent (ml)
Table 4.2.2: Variables operating range for albumin microspheres

Independent Variable Low Level High Level

Stirring Range (rpm) 1000rpm 5000rpm

Concentration of
0.5g 2g
Albumin (gms)
Concentration of
0.5ml 2 ml
Emulsifying agent (ml)

4.2.3. OPTIMIZATION OF OFLOXACIN GELATIN AND ALBUMIN


MICROSPHERES 31, 36

The optimization of pharmaceutical formulations with regard to one or more

features has always been an issue of importance and attention in formulation

research. In a typical pharmaceutical formulation experiment, the focus is on

determining which process variables affect the response the most.

Until recently, formulation and processes were investigated and developed

simply by trial and error while varying one factor at a time. This approach is

both time and energy consuming as well as costly. Furthermore, misleading

conclusions can be drawn, particularly if there are any interaction effects

within the variables.

To over come this, the approach is to optimize - that is, to determine the

region in the factors that leads to the best possible response. In addition to the

experience of the formulator, techniques are available that can aid the
formulator’s choice of formulation components which will optimize one or

more product attributes.

Theoretically, all the parameters that can affect the physicochemical

properties of a final product may be included in the experimental design.

However, in pharmaceutical manufacturing processes, there may be numerous

important parameters due to the large number of ingredients usually required

for formulation. If all the variables are included in the experimental design,

the process may lead to an excessive number of experiments.

The formulator must first identify the potentially dominant parameters that

should be subjected to the screening process in order to limit to a reasonable

extent the number of experiments needed. A delicate equilibrium is

established between what is worth and what is not worth investigating. The

formulator’s skill, knowledge and experience play a crucial role in selecting

the appropriate variables.

Design of experiments (DOE) capabilities provides a method for

simultaneously investigating the effects of multiple variables on an output

variable (response). These experiments consist of a series of runs, or tests, in

which purposeful changes are made to input variables or factors, and data are

collected at each run. This is used to identify the process conditions that

influence best output and then determine the input variable (factor) settings

that maximize results and to draw logical and purposeful conclusions from the

experimental data. The four major types of designed experiments are

factorial, response surface, mixture, and Taguchi (robust) design.


Response surface methods are best suited for this purpose since they are used

to examine the relationship between a response and a set of quantitative

experimental variables or factors. These methods are often employed to

identify a "vital few" controllable factors and to correlate the factor settings

that optimize the response. Response surface methods are employed to:

• Find factor settings (operating conditions) that produce the "best"

response

• Find factor settings that satisfy process specifications

• Identify new operating conditions that produce demonstrated

improvement in product specifications.

• Model a relationship between the quantitative factors and the response

central composite design.

Hence the Optimization of the method of microspheres preparation was

performed by Response Surface Methodology (RSM) (specifically;

randomized rotatable central composite designs – CCD) as it is the most

suitable technique for the modeling and analysis of problems in which the

response of interest is non-linear and influenced by several variables. The

mathematical modeling was carried out by employing the software – Design

Expert Ver. 6.0.5 © Statease Inc., USA.

Experimental Plan

Ofloxacin gelatin microspheres

Variable 1 : Rate of Stirring (RPM) – 1000 (-1) & 5000 (+1)


Variable 2 : Concentration of Gelatin (gms) – 0.5 (-1) & 2 (+1)
Variable 3 : Concentration of Emulsifying agent – 0.5 (-1) & 2 (+1)
Response : Particle Size – range 8- 12µm (100%)
Design : Response Surface methodology – CCD (Central composite design)
A central composite design was created to study the main effects and

interactions of these three factors on the particle size. Fifteen microsphere

formulations were prepared as per table - 4.2.4 to elucidate the effect of the

three independent variables; stirring rate (rpm) concentration of polymer

(Gelatin - mg) and concentration of the Emulsifying agent (Tween 85 - ml)

and the dependent response variable measured was the particle size

distribution. Experiments were conducted in random sequence in a face-

centered manner in order to evaluate the interaction.

Evaluation of experimental data

The obtained results were first fitted into different regression models like

modified, mean, linear, quadratic and cubic. The model with significantly

increased R 2 and increased model sum of squares is always preferred. 37 To

assess the adequacy of the final regression model, Analysis of variance

(ANOVA) was performed [Probability F value less than 0.05 (95% level of

significance) is considered significant]. 38, 39, 40

The contour, response surfaces and interaction graphs were plotted to obtain

visual representation of the dependency of the response on the variables.

Once the relationships of response and variables were known, numerical

optimization studies were performed to obtain the desired response (obtaining

microspheres within the desired size range).

Numerical optimization results solutions predictions, which give different

arrangement of variables (theoretical solution - as per the polynomial


equation) required to obtain the desired response. Only those solutions that

have a maximum desirability (of 1.000) are considered always. 30, 41, 42

Finally, model validation was performed by actually preparing the

formulations as per the probable solutions and subsequent correlation with the

predicted values. That probable solution which gave the desired response and

which was viable was taken as the final optimized formula for the preparation

of microspheres.

Experimental Plan

Ofloxacin albumin microspheres


Variable 1 : Rate of Stirring (RPM) – 1000 (-1) & 5000 (+1)
Variable 2 : Concentration of Albumin (gms) – 0.5 (-1) & 2 (+1)
Variable 3 : Concentration of Emulsifying agent – 0.5 (-1) & 2 (+1)
Response : Particle Size – range 8- 12µm (100%)
Design : Response Surface methodology – CCD (Central composite
design)

A central composite design was created to study the main effects and

interactions of these three factors on the particle size. Fifteen microsphere

formulations were prepared as per table 4.2.12 to elucidate the effect of the

three independent variables; stirring rate (rpm) concentration of polymer

(Albumin - mg) and concentration of the Emulsifying agent (Tween 85 - ml)

and the dependent response variable measured was the particle size

distribution. Experiments were conducted in random sequence in a face-

centered manner in order to evaluate the interaction.


Evaluation of experimental data

The obtained results were first fitted into different regression models like

modified, mean, linear, quadratic and cubic. The model with significantly

increased R 2 and increased model sum of squares is always prefered. 37 To

assess the adequacy of the final regression model, Analysis of variance

(ANOVA) was performed [Probability F value less than 0.05 (95% level of

significance) is considered significant].

The contour, response surface and interaction graphs were plotted to obtain

visual representation of the dependency of the response on the variables.

Once the relationships of response and variables were known, numerical

optimization studies were performed to obtain the desired response (obtaining

microspheres within the desired size range). 38, 39, 40

Numerical optimization results solutions predictions, which give different

arrangement of variables (theoretical solution - as per the polynomial

equation) required to obtain the desired response. Only those solutions that

have a maximum desirability (of 1.000) are considered always. 30, 41, 42

Finally, model validation was performed by actually preparing the

formulations as per the probable solutions and subsequent correlation with the

predicted values. That probable solution which gave the desired response and

which was viable was taken as the final optimized formula for the preparation

of microspheres.
4.2.4. FINAL OPTIMIZED FORMULAE AND FORMULATION CODES
OF OFLOXACIN GELATIN AND ALBUMIN MICROSPHERES

The final optimized formulae of all the formulations and their respective

formulation codes are given in table 4.2.9 and 4.2.16 for gelatin and albumin

microspheres respectively.

All the further studies were carried out using Glome–03 and Alome–03.

Ofloxacin gelatin and ofloxacin albumin microspheres respectively. Since all

the three optimized formulas had the required particle size range 5 – 15 µm

for lung targeting Glome–03 Alome–03 was taken up for further

characterization and in vivo studies.

4.2.5. STERILIZATION STUDIES

Sterilization is a process of ensuring total absence of microorganisms. The death rate

of microorganisms proceeds in a logarithmic scale. A 10-6 Sterility Assurance Level

(SAL) is required for parenteral products which come in direct contact with the

tissues.

The most common form of sterilization is steam sterilization, or autoclaving since it is

simple, non-toxic, and relatively inexpensive. Autoclaving exposes the material to

high temperature (greater than 121oC) and steam at high-pressure. These conditions

destroy microorganisms by disrupting key metabolic and structural entities, especially

membrane proteins and lipids. Steam sterilization is rapid compared to other methods,

however this requires about 30 minutes of exposure this degrades most polymeric

biomaterials under the harsh process conditions of steam sterilization, which often
exceed the glass transition (Tg) and melting temperatures (Tm).37 As a result,

autoclaving was ruled out to sterilize these polymeric microspheres.

An alternate sterilization method exposes the biomaterial to ethylene oxide gas.

Ethylene oxide (EtO) kills microorganisms by alkylating the amine groups on nucleic

acids. However, EtO is also toxic to humans, since it attacks these same amine

groups.35 Any residual gas can induce a toxic response in vivo. Therefore, gas

sterilization was also not used to sterilize the microspheres.

In Dry heat sterilization the polymers are subjected to a temperature 160 – 1900C, but

due to the problem of melting and softening of the polymers, this method is was not

used to sterilize the polymeric microspheres.

Although aseptic filtration is a popular method for parenteral sterilization, again this

could not be employed for these microspheres formulations because they are colloidal

in nature wherein the particle size is in the range of 5 – 15µm.

Gamma (γ) radiation is a type of radiation that sterilizes microspheres by ionizing the

nucleic acids of the contaminating microorganisms. The use of γ radiation has been

widely accepted as the sterilization method of choice for polymeric microspheres, as

it is rapid, non-toxic and leaves no residue. Of all the classical methods of

sterilization, an only gamma irradiation method was found to the best-suited method

for the sterilization of the microspheres.43


Procedure

Drug loaded Gelatin and Albumin microspheres prepared in accordance to the

optimized formulae (formulation codes – ALOME -03 & GLOME -03 – since

they are optimized formulae and were chosen in a random manner to prevent

bias over the particle size) by using the emulsion polymerization method.

Thus obtained microspheres were characterized for particle morphology by

Scanning Electron Microscopy (SEM) and particle size (laser diffraction)

studies were carried out on these formulations prior to sterilization. These

microspheres formulations were then subjected to γ -irradiation. After

sterilization, the particle morphology and particle size was again carried out.

4.2.6. MICROSPHERE RECOVERY / YIELD

The microspheres recovery studies can also be referred as microsphere yield,

since this study accounts for the amount of microsphere obtained at the end of

the preparation; and the polymer and the drug which went into its

preparation 44 table 5.1.1. It is calculated using the following equation.

Recovered weight of microspheres X 100


Microsphere recovery (%) =
Weight of polymer used + drug

Procedure

The equal quantities of polymer and drug (1 gram each) were taken for

microspheres formulation. Thus, formed microspheres were weighed. The

yield excepted by employing the used quantity of excipients and drug

(theoretical yield) and the actual yield got as the results of this formulation

were compared. The percentage yield was obtained as ratio between

theoretical and practical yield. table – 5.1.1


Drug entrapment efficiency

The entrapment efficiency is carried out as a measure of calculating the

amount of drug that is entrapped in the microsphere and the drug, which is

adsorbed in the surface of the polymer. This study carries vital importance in

surface studies since this is indicative of the measure of drug that goes in

forming the microsphere, present in an adsorbed state on the surface of the

polymer or as free drug and as a faction which is found entrapped within the

polymeric matrix.

The experimental method devised to quantify the entrapment efficiency of a

microsphere should take into account the free adsorbed and entrapped

portions of the drug. Experimental plans should be devised in such a way

that, the total drug content of the microspheres should quantify all the three

portions of the drug – free, adsorbed and entrapped and they should be

determinable separately. This determination is an indicative of the efficacy of

the microspheres produced in terms of its active ingredient.

Procedure

a) Free drug

b) Adsorbed drug

c) Entrapped drug

4.2.7. Free drug in microspheres (un-entrapped) drug

The free drug is measured by taking 10mg of accurately weighed

microspheres in a beaker, to this 10ml saline (0.9%w/v) was added, the

mixture was shaken well in order to liberate the free drug present in the
polymeric matrix. This was quantified by measuring the absorbance on

an UV spectrophotometer at 297nm table 5.1.2.

4.2.8. Adsorbed drug in microspheres

In order to measure the amount of drug present in the surface, these

microspheres were digested with 10ml of saline (0.9% w/v) at room

temperature; the solution was then sonicated in an ultrasonic bath for 5

min, and then centrifuged at 3000rpm for 2 min. The supernatant was

filtered through a 0.45µm filter (Millipore) and the absorbance was

measured on an UV spectrophotometer at 297nm table 5.1.3.

4.2.9. Entrapped drug in microspheres 45

The leftover residue from the extraction of free and adsorbed drug was

mixed with 5ml of glacial acetic acid 0.1M the samples were

centrifuged at 5000 rpm for 10mins, and the absorbance of the

supernatant was measured at 297 nm after the filtration process through

a 0.45µm filter (Millipore). The amount of ofloxacin associated with

each phase of the microsphere using a using calibration was

constructed over a range of 0 - 20 µm/ml table 5.1.4.

The free drug in the microsphere was determined using the formula

Qm
L= X 100
Wm

L- Percentage loading of microspheres


Q m – quantity of free drug present in the Wm grams of the microsphere
The adsorbed drug in the microsphere was determined using the formula

Qm
L= X 100
Wm

L- Percentage loading of microspheres


Q m – quantity of ofloxacin present in the Wm grams of the microsphere

The amount of drug entrapped in the microsphere was determined using the

formula

Qp
E= X 100
Qt

E – Percentage of encapsulated drug


Q p – quantity of drug encapsulated in microsphere in gms.
Q t – quantity of drug added for encapsulation in gms.

4.2.10. SURFACE MORPHOLOGY 46

Examining the surface of a polymeric drug deliver system can provide vital

information on the porosity and microstructure of these systems. The

distribution and morphology of the surface and the encapsulated matrix can

also be directly observed.

The most common technique used for characterizing the surface morphology

systems of drug delivery systems is Scanning Electron Microscopy (SEM).

This method offers several advantages, by its versatility of the method,

simplicity of sample preparation and ease of operation. The sample sizes,

which can be analyzed using this method, range from nanometers to


micrometers to centimeters. This method can also use unlimited amount of

samples, offering innumerable viewpoints by means of its translational, tilting

and rotary movements, over various resolving powers of the instrument.

Sample prepared for this method should be sufficiently dehydrated since, a

since vacuum field is necessary for image generation in SEM. Prior to

loading the samples for taking the photomicrograph, samples are coated (20 –

30nm in thickness) with electron-dense coating materials like gold, palladium

or combination of both, to enhance the signal emitted by the sample by

providing heavy metal atoms with incident beam of electron and, to conduct

the accumulated sample charge and heat to the sample holder. The coating

process is either carried through sputter-coating or thermal vacuum

evaporation.

Procedure

The surface morphology of the microspheres (gelatin & albumin formulation

codes GLOME -03 & ALOME-03 respectively) was observed by SEM – SEM

(Joel, Japan). The microspheres were placed on graphite surface and coated

with gold using an ion sputter (Joel, Japan) and were observed at 12KV.

4.2.11. PARTICLE SIZE ANALYSIS

The particle size characterization of microspheres is an important study to

ensure that particle size of the formulation lies in the optimal range. This is

the crucial factor since the target activity as intended in this study exclusively

depends on particle size of the microspheres produced. 33


A wide variety of methods have been developed to measure particle size,

depending upon the size itself. These methods use different physical

principles for the determination of size. For the determination of microsphere

size, laser diffraction method is employed.

In laser diffraction method, the particle size is determined from the

diffraction pattern of the incident light by the particle(s) in question. The

chief attraction of this technique is the quick and easy measurement of size

distribution over a broad range of 0.1 – 1000 µm. The dry powders is sampled

and sized by this method. Helos is an example of the instrument utilizing this

principle of particle sizing.

The only disadvantage in any technique of particle size determination is the

variation in size of the particles if determined by different instruments of

other manufacturers, especially in the lower size range. This is because all the

particles are not spherical always and because each manufacturer uses own

proprietary algorithms, leading to different size results.

The particle size distribution studies of the microspheres were carried out by

Laser Light Diffraction and Ultrasonic techniques; HELOS laser system for

dry powders (Sympatec Inc)

Procedure

The free flowing microspheres were added into sample holder of the HELOS

laser system and average particle size was studied.


4.2.12. IN VITRO RELEASE STUDIES

The in vitro release studies were carried out as per the following procedure to

study the drug release pattern to note the extent and pattern of the drug

release in an in vitro environment.

The in vitro release experiments were performed using dialysis method. In

this method, a weighed quantity of the microsphere is contained in a dialysis

bag, which is immersed in a larger volume of continuous phase acceptor fluid.

The compartment were stirred and the drug which diffuses out of the

microsphere into the continuous phase, was periodically sampled and

assayed. 48, 49

In this study, the in vitro release patterns were studied using conventional

dialysis technique. 50mg ofloxacin gelatin/albumin microspheres were placed

in a dialysis bag and dialyzed against 200 ml of phosphate saline buffer

(PBS), pH 7.4 at 37±1 o c. Sink conditions were maintained and throughout the

course of study and were stirred continuously using a magnetic stirrer.

Aliquots were withdrawn at specific time intervals and the same volume of

dissolution medium was added to the flask to maintain a constant volume. The

withdrawn aliquots were estimated through UV spectrometry method at 297

nm, and the data were used to calculate a cumulative drug release profile from

the microsphere. The accumulated amount of drug released was calculated

using a calibration curve.

The in vitro release studies were carried out for albumin and gelatin

microspheres loaded with ofloxacin and their release at predetermined time


intervals were quantified by UV spectrophotometric method. Since

microspheres are spherical particles composed of a suitable polymer, their

release profile in vitro helps us to understand the behavior of these systems in

terms of drug release, and therefore its efficacy. The initial release is

characterized by a “burst release” due to the release of weakly bound drug at

the particle surface of the microsphere. These are bound by means of

adsorption and do not involve in any chemical bonding with the polymer.

When such microspheres are briefly washed in a sink condition, these drug

particles would be washed, the drug bound to the surface of the microspheres

are rapidly washed, and would lead way to a rapid release of the drug in the

early stages. However, this phenomenon holds good for only a brief period

with maximum drug release. After this time window, the release pattern is

more uniform as the time progresses.

Since the microspheres are heterogeneous systems, this suggests that the drug

release from polymer faction of the microsphere is through a diffusion

process in an in vitro environment. As a result of this, the drug the polymer

matrix are phase separated into two distinct domains a bi-phasic system, the

release of the drug is decided upon the extent degradation of polymeric

microspheres.

The entrapped drug, which separated into a bi-phasic system, upon the

liberation from the polymeric matrix, and through diffusion process the drug,

is released from the microspheres. Several models of release have been

suggested by various workers depending based on the variants like diffusion,


nature of polymer, concentration of drug and extent of cross-linking of the

polymer. The drug release pattern in an in vivo environment follows variety

patterns, various release patterns are suggested by number of workers taking

into account all the variants mentioned above. The loading of drug into the

polymer matrix also plays a significant role in the release profile of the drug

(in this case 1:1); a point of domain separation is reached and the drug

particles come in contact with one another, and the drug diffuses out of the

matrix. Leaving the solvent filled channels open. Through this void channel,

the drug is preferentially released paving way for a controlled drug delivery

state, coupled with the size of the microsphere, the targeting can be achieved

in an in vivo environment and the amount of microspheres reaching the target

organ (in this case –lungs) can be attained satisfactorily with the aforesaid

conditions.

In-vitro release kinetics

However to quantify the drug release pattern, the drug depletion from

microspheres can be explained by means of regression. This is statistical

concept used to investigate:

• How the response variable (drug release pattern) changes as a

particular predictor (dependent variable – such as time etc.)

• Predict the value of the response variable for any value of the predictor

variable, or combination of values of the predictor variables – thereby

predicating the drug release pattern. 40, 50, 51, 52, 53

Since the response and the predicator responses in this case may not follow a

linear pattern (due to formulatory & experimental variants) a non-linear


approach is adopted where the release data is attempted to fit in a pattern and

thereby predicting the release rate (as in the ideal case). Regression equation

is an algebraic representation of the regression line and is used to describe the

relationship between the response and predictor variables. Several workers

have adopted different approaches to curve fit; this study has used the

prominent regression models to study the drug release and their mode of

release. They are:

1. First Order release

2. Higuchi Model

3. Hixon – Crowell Model

4. Peppas Model

5. Baker – Lonsdale Model

The best model of the fit is determined by the R 2 and adjusted R-values. They

represent the proportion of variation in the response data explained by the

predictor factors. R 2 describes the amount of variation in the observed

response values that is explained by the predictor. Higher the R 2 value, best is

the fit offered by the equation / model. The fitted line in the plot is a

regression line, which examines the relationship between the response

variable (y - % drug release) and the predictor variable (x – time of release).

The method used to draw the line using a 95% confidence interval and around

3σ values were used to attain precision in the replicate runs. This was

calculated using Σ Plot® ver. 9.01 © Systat Software Inc 54 .


4.2.13. DIFFERENTIAL SCANNING CALORIMETRY (DSC) ANALYSIS
22, 40, 45, 46, 50, 52, 55, 56, 57, 58, 59
Microspheres characterization through DSC

The DSC technique can provide qualitative and quantitative information about

the physicochemical status of the drug in the microspheres, which involves in

the endothermic or exothermic process; and the related thermal transitions

include melting, recrystallization, decompositions, out-gassing, or a change in

heat capacity of the tested material. DSC is useful to monitor different

samples of the same material to assess their similarities or differences or the

effects of additives on the thermal properties of a material. Using the DSC

analysis of drug, polymer materials and produced microspheres, the nature of

the drug inside the polymer matrix can be assessed, which may emerge in

solid solution, metastable molecular dispersion or crystallization and many

display relevant properties during in vitro release and hence can be utilized as

a characterization tool. There is no detectable melting endotherm if the drug

presents in a molecular dispersion or a solid solution state like in the case of

polymeric microspheres loaded with enough amount of drug. It is because the

drug’s melting point when present in this mixture disappears in the drug

loaded microspheres, and the endothermic polymer under goes a “glass

transition”- T g and shifts the peak to lower temperature with respect to

unloaded microsphere (the ones without drugs). This is indicative of the fact

that preparations lead to a formation of amorphous dispersion of the drug

inside the polymer matrix formation, even if the drug loading is of higher

levels as in this case.


Thermal analysis can be used to investigate and predict any physicochemical

interactions between components in a formulation and therefore can be

applied to the selection of suitable chemically compatible excipients.

Differential Scanning Calorimetry (DSC) is among the most useful tools of

thermal analysis available for the determination of various thermal parameters

of a formulation, which allows for the detection of phase transition of the

samples under study.

The successful formulation of a stable and effective microspheres targeted

drug delivery system depends on the careful selection of the excipients, which

are added to the formulation. Ideally, these excipients should not interact with

the drug leading to its degradation. This fact is indicated by the appearance of

a smothered peak of the drug physical admixture uncharacteristic of its

thermal signature with a broadened peak base, at the end of the thermal

spectra this might not happen in all cases since the thermal activation energy

required to produce such an effect might be more or it might go out of the

range of the instrument as in this case.

Interpretation of DSC

An interaction on DSC will show changes in melting point (peak shift) and/or

the appearance of a transition. However, there is bound to be some change in

transition temperature and peak shape and area by virtue of mixing two

components and this is not due to any detrimental interactions. In general, if

no new thermal events occur or are lost by mixing the two components, no

interaction can be assigned.


Chemical interactions are indicated by the appearance of new peaks whereas

second order transitions produce changes in the baseline. Such observations

may also be indicative of the production of eutectic or solid solution type

melts. 60

The advantages of DSC over more traditional, routine compatibility screening

methods like TLC is that neither long-term storage of the mixture is required

prior to evaluation nor any inappropriate thermal stress required to accelerate

the interactions.

4.2.14. X-RAY DIFFRACTION ANALYSIS (XRD)

X-ray Diffraction is one of crystallography characterization tools. It is

employed by researchers for a wide variety of applications – it is used as an

analytical tool in identification of the constituents of mixtures of crystalline

phases; and for the measurement of lattice parameters. This phenomenon is

used by the pharmaceutical industry to identify changes in the APIs (active

pharmaceutical ingredient), excipient, or methods that might alter the drug

efficacy.

Sample of the microspheres were subjected to XRD analysis, using a Philips

PW 170 system (Philips USA) with Cu-K 2α radiation (40KV, 30 mA; scan

speed 1 o /Min) to investigate the physical state of ofloxacin entrapped in the

gelatin / albumin microspheres. The diffraction patterns obtained for

ofloxacin albumin and ofloxacin gelatin microspheres are given in the figures

1to5.
4.2.15. RESIDUAL SOLVENT ANALYSIS

Residual solvents in pharmaceuticals are volatile organic chemicals that are used in

and/or produced during the synthesis/production of drug substances/formulations.

Many of these residual solvents generally cannot be completely removed by standard

manufacturing processes/techniques and are left behind, mostly at low levels.

Presently in the pharmaceutical industry special importance is given for the residual

solvent testing as they will affect the physicochemical properties of the drug

substances/formulations and mainly because, high levels of these solvents represent a

potential risk to human health due of their carcinogenicity and ototoxicity 20, 25, 33, 61, 62

Residual solvent testing can be carried out by a number of analytical

techniques, important of them being the Gas Chromatographic technique.

Ofloxacin albumin and gelatin microspheres prepared by emulsion

polymerization method were tested for acetone. The acetone levels were

found to be well below the limits prescribed by the ICH.

Procedure
Instrument : Shimadzu Gas Chromatograph GC 17A
Mobile Phase : Nitrogen.
Column : SGE BP-5 capillary column (0.25mm x 30m, 0.25mm).
Column pressure : 30 kpa.
Temperature : Column - 140 0 , Injection port – 220 0 & Detector 250 0 .
Injection volume : 1 µl.
Flow rate : 20 ml/min.
Detector : Flame ionization detector.
Ret. Time : Methanol – 2.5 min, Dichloromethane – 5 min &
Acetone – 7 min.
10 mg of microsphere formulation was extracted with 10-ml portion of

dimethyl sulphoxide (DMSO). The resulting solution was filtered through

0.45 µm filters (Millipore, India) into 10-ml volumetric flasks and the volume

made up with DMSO. From this filtrate, 1.0 µl was injected into the gas

chromatograph. Based on the peak areas recorded in the chromatograms, the

amount of residual solvent present in the formulations was calculated.


4.3. STABILITY STUDIES

The stability study of a pharmaceutical product is done to provide assurance

that the product intended to be used is of adequate quality, and has safety

characteristic intended for a specific use. This may be defined as the

capability of a particular formulation in a specific container, to remain within

its physical, chemical, microbiological, therapeutic and toxicological

specifications. 51, 63

Stability testing is designed to be an integral part of this formulation

development. The purpose of this procedure is to provide evidence on how the

quality of a drug substance or drug product (ofloxacin loaded albumin and

gelatin microspheres in this case) varies with time under the influence of a

variety of environmental factors such as temperature, humidity, and light, and

to establish a re-test period for the drug substance or a shelf life for the drug

product and recommended storage conditions to provide precision to the test

procedure.

Deciding upon stability testing, protocol for a particular product can be

challenging due to the complexity and diversity of the modern pharmaceutical

product such as microspheres. Nevertheless, most emerging and contemporary

guidelines are available today to address these issues.

The de facto guideline for stability testing procedure is ‘The International

Conference on Harmonization (ICH)’ guideline titled “Stability testing of new


drug substances and products (Q1AR)”, which describes the stability test

requirements for drug formulations and new drug substances.

This guideline provides generic directive to study the stability of drug product on a

real time basis – since they provide a more realistic data. The storage conditions

described in the guideline are based on worldwide storage temperature (mean kinetic

temperature- MKT) and percent relative humidity (RH) conditions. The countries of

the world are segregated into four zones depending on their climatic conditions,

namely, Zone I, II, III and IV. India is designated zone III / IV for its tropical

presence and high levels of ambient temperature and humidity levels.64 Minimum

time period to be covered by the stability analysis is at least 12 months for long-term

conditions and at least 6 months for accelerated conditions.

Other significant features of Q1AR guidelines:

• Testing has to be conducted in the final package, i.e., in the containers and

closures proposed for storage and distribution.

• ICH guidelines suggest a sampling frequency of every 3 months during the first

year, every 6 months during the second year and then annually for drug substances

and products stored for real time testing.

• Although the guideline does not propose a testing frequency for samples stored

under accelerated conditions, the FDA guideline has set a frequency of 0, 2, 4 and

6 months for samples stored under accelerated conditions.

• At least two containers are required to be sampled during the stability

study.
The parameter used for evaluation of the drawn samples generally varies with the type

of dosage form. As per the ICH guideline “Specifications: Test procedures and

acceptance criteria for new drug substances and new drug products – Q6A”, one

should consider organoleptic, physical, chemical, biological and microbiological

quality characteristics of the product, which are susceptible to change during storage,

in addition to drug assay and the analysis of degradation products, thus covering the

whole gamut of quality issues in terms of efficacy and elegance.

Procedure

The stability protocol was designed based on the ICH ‘Q1AR’ and ‘Q6A’ guidelines.

The microspheres formulations chosen – gelatin and albumin were stored at 25 ± 20 C

and 60 ± 5% RH for a period of 6 months and at 5 ± 30C for a period of 12 months,

which is the accelerated storage temperature and long-term storage temperature

respectively for products which are intended to be refrigerated. The decision to

refrigerate was taken because of the thermosensitivity and thermolability of the

polymers chosen. Since these polymers were of protein origin and have relatively low

thermal stability profiles – gelatin and albumin degenerate 500C and above 250C

respectively.

The stored samples were tested for their drug content, particle size distribution, and

for any physical change. The drug content was determined through a UV

spectrophotometric method as described earlier.

The testing was carried out at 0, 2, 4 & 6 months (as per FDA guideline) for

accelerated storage condition; and at 3-month intervals for a period of 12 months for

long-term storage condition (as per ICH guideline).


4.4. IN-VIVO TISSUE DISTRIBUTION STUDIES

The in vivo studies are a key component in this study since they provide

tangible evidence on the efficacy of the microspheres, since the properties

exhibited by the microspheres are crucial in understanding the functional

characteristics of formulations in a biological system. As the aim of this

study is to passively, target the microspheres to lungs, it become vital to

study the distribution characteristics in a living system to understand this

parameter effectively. 32, 45, 65, 66

Experimental Procedure

To examine the appropriate properties of the formulations in vivo, thirty six

adult albino mice weighing between 20±2 gms were selected at random and

divided into 6 groups to test the drug release at the following time periods –

10 min, 30 min, 1 hr, 3 hrs, 6 hrs, and 12 hrs. Prior to the administration, the

study groups were randomly divided into 6 groups, with 6 animals in each

group. All the animals were kept on starvation 12 hours before injection, with

free access to water. A dose of 0.52g/kg was administered to each of the

animal as dispersion in saline with 1% of tween 80. At predetermined time

intervals as stated earlier the animals were injected with the microspheres (for

both gelatin & albumin) via the tail route vein; and were sacrificed by

cervical dislocation. The organs, which were studied, for target action – viz.

lungs, liver and spleen were extracted. The tissue samples were stored for 24

hrs at - 20 0 C. Then the concentrations of drug localized in each organ as the

result of targeted release were determined through extraction method as

described below.
Sample preparation

The extracted organs were homogenized by adding saline at the ratio of

0.1mg/ml. The protein in the mixture was precipitated (to prevent interference

during the quantification process) by adding 2M perchloric acid (100µL). The

mixture was then vortexed for 5 seconds to aid the mixing process and was

then centrifuged at 5000 rpm/min. 67, 68, 69, 70


The prepared samples were used

to determine the amount of ofloxacin present in the tissue samples

quantitatively using the above HPLC method.

HPLC quantification method

The method was chosen for its capability to determine ofloxacin in body fluids –

serum and blood as well as tissues. The samples were prepared as follows; the organs

extracted, weighed and were subjected to cutting to tissues with scalpel, homogenized

with 1-3 ml buffer, centrifuged at 9600 g for 5 min, three cycles, injected a 20µl

aliquot of the preparation. HPLC variables were as follows; column used was 200X

45 µm Nucleosil C18. The mobile phase used was a mixture of methanol, methyl

cyanide buffered with pH 3.0 with phosphoric acid in the ratio 13:7:80 the pH

adjusted to 3.0 with phosphoric acid (buffered with was 15mM phosphoric acid
96
adjusted to pH with 3.0 with tetrabutylammonium hydroxide). The HPLC

parameters are given in a nutshell in the following table:


Table 5.3.1 in vivo drug quantification specifications for HPLC method

Parameter Specifications
column C18 type 200 X 45µ m Nucleosil
MeOH:MeCN:buffer 13:7:80
Mobile
adjusted to pH 3.0 with
Phase
phosphoric acid
Flow rate 1 ml/min
injection
20-100µl
volume
Detector F ex 278 em 446
Limit of
10 ng/ml
detection

F ex = Fluorescence excitation wavelength


Em = Emission wavelength

In vivo tissue distribution studies in an animal model (albino mice) were

carried out, to prove the hypothesis of passive targeting of microsphere

formulations to the lungs and compared with the conventional dosage of the

drug.

4.4.1. Quantitative evaluation of targeted drug delivery systems 70

Targeted drug delivery research is an area, which concentrates on the

development and evaluation of systems with precise characteristics. The

characteristics sought may be selective or regional drug delivery, controlled

drug delivery, or the combination of these characteristics. Before any such

delivery system can be made available for routine use, it is important that the

evaluation procedures are critically established and it’s advantageous over a

conventional dosage form.


The evaluation of targeted drug delivery systems indicates that few

investigators have attempted to provide a detailed picture of the time course

of drug, in all major tissues of the body, following its administration via the

test and conventional delivery and data. Some simple mathematical

relationships have been suggested which may be useful in gaining a better

appreciation of the in vivo performance of targeted drug delivery systems.

Where the numerator refers to time-averaged exposure of any tissue i to drug

administered via test targeted drug delivery system, the denominator refers to

the time-averaged exposure of the same tissue following the administration of

an equivalent dose of drug via a conventional drug delivery system, and r e

refers to the time-averaged relative drug exposure. Values of r e > 1 indicate

that the tissue ‘i ’ is exposed to drug to a greater extent following the use of

test targeted drug delivery system, and vice-versa.

Although this relationship provides a good indication about the relative

efficacy of two delivery systems in reference to one tissue, it does not provide

any information regarding the efficacy of a given delivery device in terms of

the target: non-target tissue distribution of drug. This information can be

obtained using the following expression. 70


Where te, refers to the drug targeting efficiency of a delivery system against a given

non-target tissue. Here values of te > 1 indicate greater selectivity of the delivery

system for the target tissue, as compared to the non-target tissue against which this

parameter is estimated. Implicitly, the higher the value of te the greater is this

selectivity.

The drug targeting efficiency of a delivery system against a given non-target tissue, a

composite or an overall drug targeting efficiency of a delivery system against non-

target tissues, Te, can be calculated as

Where the denominator refers to the sum total of drug exposure to all the tissues,

including the target tissue.70

Finally, the time-average distribution of a drug a given tissue, following the

administration of delivery system, can be estimated according to relationship


X 100

Or

Drug distributed to tissue j (%) = Te X 100

In vivo pharmacokinetics of microspheres

4.4.2. Compartmental modeling

Pharmacokinetics in simplest terms is how drugs move in the body and how

quickly or at what rate the movement occurs.

It is concerned with the study and characterization of the time course of drug

absorption, distribution, metabolism, and excretion as well as their

relationship to the time course of therapeutic and adverse effects of drugs.

Pharmacokinetics is also described as ADME processes- Adsorption,

Distribution, Metabolism and Excretion. Once the drug is absorbed, it is

distributed throughout the body depending on the physicochemical properties

of the drug and the physiological properties of the body membranes and

fluids.

The drug then interacts with receptors and causes therapeutic or / and toxic

responses.
Drugs are in a dynamic state within the body. The biological system is very

complex and the kinetics of the drug is very complicated. One can describe

the drug kinetic processes or interpret the data using simple mathematical

models.

Model is noting but a mathematical representation of the data. It is just a

hypothesis.

Types of PK models

There are three types of pharmacokinetic models

1. Compartmental

2. Non-compartmental

3. Physiological

Among all the three, compartmental analysis is the traditional and most

commonly used approach to pharmacokinetic characterization of a drug.

Compartment models describes the pharmacokinetics of drug disposition by

grouping body tissues which is kinetically indistinguishable and describe

transfer of drug between body tissues in terms of rate constants.

Compartmental model

A compartment is not a real physiologic or anatomic region but an imaginary

or hypothetical one consisting a tissue / group of tissues with similar blood

flow and affinity and our body is considered as composed of several

compartments that are connected reversibly with each other. The kinetics of

drugs can be described by a hypothetical model consisting of one, two or three


compartments. It is assumed that the rate of drug movement between compartments

follow first order kinetics.

Advantages of compartment modeling

1. It gives a visual representation of various rate processes involved in drug

disposition.

2. It is possible to derive equation describing drug concentration changes in each

compartment.

3. One can estimate the amount of drug in any compartment of the system after the

drug is introduced into a give compartment.

Types of compartment models

There are two types of compartment models, namely, mammillary and caternary

model. Among two compartment models mammillary model are the most common

compartment model used in pharmacokinetics. Here one or more peripheral

compartments are connected to central compartments like satellites with the central

compartment being in the center.

Classification of compartment models

The compartment models can be classified as

1. One compartment open model.

2. Two compartment open model.

3. Three compartment open model.

Distribution characteristics of the drug can be described by multi-compartment model,

such as two-compartment model.


4.5. HISTOPATHOLOGICAL STUDIES

Histopathology is a discipline of pathology, which concerns the study of microscopic

changes in diseased tissues. Histopathological studies of tissues reveal valuable insight

into the tissue changes that might have occurred due to the pre-existing disease conditions

or due to the administration of drugs.20, 68

Histopathological studies are a method of determining the tissue compatibility with the

formulations. In case of targeted drug delivery systems such as microspheres, major

portion of the drug and excipient are accumulated in specific tissues (such as lung, liver &

spleen) and therefore, determining the compatibility between these tissues and the

microsphere formulations becomes a necessity in order to rectify the safety of the

formulations.

Procedure

After 12 hours of administration of the formulations, animals (including control group)

were sacrificed by excess anesthesia and the lungs, liver and spleen were dissected and

washed with cold saline. The organs were pressed between filter pads and weighed.

Liver and spleen tissues were fixed in 10% neutral formalin using standard techniques

and stained with hematoxylin and eosin (H&E) stain for histopathological examination.

All tissue samples were examined and graded under light microscopy with 500x

magnification71
Table 25 1
5. RESULTS & DISCUSSION

5.1. Optimization studies

5.1.1. Optimization of ofloxacin gelatin microspheres

The particle size optimization data is shown in the table 4.2.4. The results

were fitted to quadratic model of regression as it showed the maximum

values of R 2 and model sum of squares. ANOVA was performed and the

results are shown in table 4.2.6.

ANOVA proved that the model was significant (with a probability F value

of <0.0001). Gelatin concentration most significantly affected the drug

incorporation as indicated by a probability F value of <0.0001.

The polynomial equation giving the mathematical relationship between

each factor was found to be:

The three-dimensional response surface graph along with the contour

graph was plotted figure 4.2.1, 4.2.2 & 4.2.3, followed by numerical

optimization table 4.2.8 and model validation. The 3D response surface

graph substantiated the fact that gelatin concentration most significantly

affects the particle size. From the numerical optimization results, solution

8, 1 & 6 was selected randomly as the optimized formula for the

preparation of ofloxacin gelatin microsphere as it showed maximum

particle size efficiency.


Table 4.2.3: Coded central composite experimental design for three factors

Factor 1 Factor 2 Factor 3


Sl # B: Gelatin C: Amt. of
A: Stirring
Conc. EA
RPM
gms ml
1 0 0 1
2 0 -1 0
3 -1 1 1
4 0 1 0
5 0 0 0
6 -1 -1 -1
7 1 -1 1
8 0 0 0
9 0 0 0
10 1 1 -1
11 1 0 0
12 0 0 0
13 0 0 0
14 0 0 -1
15 -1 0 0
RPM = Rotation Per Minute, EA = Emulsifying Agent
gms = Grams, ml = milliliter

-1: low level; 1: high level; 0: central level


Table 4.2.4: Results for DOE for ofloxacin gelatin microspheres

Factor 1 Factor 2 Factor 3 Response


A:
B: Gelatin C: Amt. Particle
Sl # Run Block Stirring
Conc. of EA Size
Rate
RPM g ml µm
1 1 Block 1 3000 1.25 1.25 2.1
2 5 Block 1 3000 1.25 1.25 2.3
3 6 Block 1 3000 1.25 1.25 2.3
4 13 Block 1 3000 1.25 1.25 2.2
5 11 Block 1 3000 1.25 1.25 2.3
6 3 Block 1 5000 2 0.5 16.2
7 15 Block 1 5000 0.5 2 0.32
8 10 Block 1 1000 2 2 21.2
9 14 Block 1 1000 0.5 0.5 5.1
10 7 Block 1 1000 1.25 1.25 15
11 9 Block 1 5000 1.25 1.25 1.3
12 8 Block 1 3000 0.5 1.25 3.1
13 12 Block 1 3000 2 1.25 22
14 2 Block 1 3000 1.25 0.5 14
15 4 Block 1 3000 1.25 2 1.2

RPM = Rotation Per Minute, EA = Emulsifying Agent, gms = grams,


ml = milliliter, µm=micrometer
Table 4.2.5: Design Summary for DOE runs of ofloxacin gelatin microspheres

Design Summary
Study Type : Response Surface
Experiments: 15
Initial Design: Central Composite
Blocks: Nil
Design Model: Quadratic

Response Units Observatio Minimum Maximum Transformati Model


ns on
Particle Size µm 15 0.32 22 None Linear

Factor Name Units Type Low High Low High


Actual Actual Coded Coded
A Stirring RPM Numeric 1000 5000 -1 1
RPM
B Gelatin Gms Numeric 0.5 2 -1 1
Conc.
C Amt. of Ml Numeric 0.5 2 -1 1
EA

RPM = Rotation Per Minute, EA = Emulsifying Agent, gms = grams,


ml = milliliter, µm=micrometer
The central composite design (a two- level factorial design with central and star

points) was adopted since it is employed to analyze the relationships between multiple

variables with a reduced number of experimental runs. The particle size of the Gelatin

Ofloxacin microsphere was selected as the response to be measured, since this offers a

more tangible set of measurement in an experimental setup. The experimental output

showed the particle size of microspheres to lie in a range of 0.32 – 22µm, and is not

affected by transformations and this followed a linear setup which indicated that the

response measured holds good over a range of other factors viz. stirring, polymer

concentration and the amount of emulsifier used. This would in turn help in fixing the

optimal particle size range, which has the maximum efficacy and the effect of other

factors in arriving at this range.

Table 4.2.6: Anova for ofloxacin gelatin microsphere - quadratic model

Response: Particle Size


ANOVA for Response Surface Quadratic Model
Analysis of variance table [Partial sum of squares]
Source Sum of DF Mean F Value Prob. > F
Squares Square
Model 797.583 9 88.620 6.566 0.0259
A 93.845 1 93.845 6.953 0.0461
B 178.605 1 178.605 13.233 0.0149
C 81.920 1 81.920 6.070 0.0570
2
A 4.293 1 4.293 0.318 0.5971
2
B 84.414 1 84.414 6.254 0.0544
2
C 1.398 1 1.398 0.104 0.7606
AB 55.556 1 55.556 4.116 0.0983
AC 2.823 1 2.823 0.209 0.6666
BC 25.872 1 25.872 1.917 0.2248
Residual 67.484 5 13.497
Lack of 8431.53
67.452 1 67.452 < 0.0001
Fit 1
Pure
0.032 4 0.008
Error
Total 865.067 14
R 2 =0.9220
The polynomial equation giving the mathematical relationship between each
factor was found to be:

Particle Size = (RPM) 5.44755 -1.18010E-003


X (Gelatin Conentration) - 4.90412 X 10.0492
X (Amount of Emulsifying Agent) + 3.20294E-007
X (RPM) 2 +10.09987
X (Gelatin Concentration) 2 +1.29987
X (Amount of Emulsifying Agent) 2 – 4.30333E-003
X (RPM) X (Gelatin Concentration) + 9.70000E-004
X (RPM) X (Amount of Emulsifying Agent) -7.83111

X (Gelatin Concentration) X (Amount of Emulsifying Agent).


Figure 4.2.1. 3D RSM graph of ofloxacin gelatin microsphere – factor: polymer
concentration

DESIGN-EXPERT Plot

Particle Size
X = A: Stirring RPM
Y = C: Amt. of EA

Actual Factor
B: Gelatin Conc. = 1.89
42.48

31.22

19.96

8.71
Particle Size

-2.55

0.50

0.88
5000
1.25
4000
C: Amt. of EA
3000 1.63
2000
A: Stirring RPM 1000 2.00

In this 3D RSM Graph gelatin concentration exerts only a slight influence

upon the particle size, the gelatin concentration increases with that of the

particle size the gelatin microspheres. This can be attributed to the increasing

in viscosity of the gelatin at higher concentration; any change in the viscosity

lowers the subdivision of the gelatin solution into smaller droplets during

emulsification and finally increases the particle size. The increases in drug

concentration therefore had no effect on the particle size and in turn depend

upon the variabilities like stirring rate, concentration of the gelatin and

emulsifier.
Figure 4.2.2: 3D RSM Graph of ofloxacin gelatin microsphere - factor: amount
of emulsifying agent

DESIGN-EXPERT Plot

Particle Size
X = A: Stirring RPM
Y = B: Gelatin Conc.

Actual Factor
C: Amt. of EA = 1.00

42.48
31.22
19.96
8.71
Particle Size

-2.55
0.50

0.88
5000
4000 1.25

3000 B: Gelatin Conc.


1.63
2000
A: Stirring RPM 1000 2.00

In this 3D RSM the increase in the amount of the emulsifying agent decreases

the viscosity present in the system and thereby lowering the particle size, the

decrease in the RPM in interaction with the gelatin concentration increase its

particle size and vice versa. This figure is indicative that optimal particle size

formation depends upon the correct RPM and the concentration of the

polymer.
Figure 4.2.3: 3D RSM graph of ofloxacin gelatin microsphere - factor: stirring
rate

DESIGN-EXPERT
Pl t
Particle
X = B: Gelatin
Si
Y = C: Amt. of
C
EA
Actual
A:
F Stirring
t RPM 42.48
=
4840
31.22

19.96
Pa
8.71
rtic
le
-2.55
Siz
e

0.50
0.88
2.00
1.63 1.25
1.25 C: Amt. of EA
1.63
0.88
B: Gelatin Conc. 0.50 2.00

In the 3D RSM the stir factor is found to have the influence on the

concentration of the gelatin and the amount of emulsifying agent in higher

RPM’s the figure is indicative that the particle size would be lower range due

to the higher dispersion of the emulsifying agent influencing particle size and

vice versa and it also suggests that the optimal particle size formation can be

decided by correct RPM levels.


Table 4.2.7: Index of formulatory abbreviations

Gelatin - Gl -
Polymer
Albumin - Al -
Drug Ofloxacin -O-
Emulsion Polymerization -E-
Method of Preparation
Spray Drying -S-
Microspheres -M-

The Anova for response Surface design showed a quadratic setup, which indicates the

employment of CCD. The higher sum of square values for the factor concentration of

gelatin indicates that this factor is influenced by the other factors – stirring rate and amount

of emulsifier used. The influence exerted by these two values almost similar. The sum of

squares for the gelatin factors as well, is predicted by this model in comparable terms. And

output of interacting factors shows a higher value for factors, which involves gelatin. This

shows that the system is influenced by the inclusion of gelatin – supported by the other two

factors to aid in the formation of microspheres over an optimum range.

Table 4.2.8: Numerical optimization solutions of ofloxacin gelatin microsphere

Solutions Stirring Gelatin Conc. Amt. of EA Particle Size


Number rpm gms ml µm
1 1042.78 1.09 1.00 11.42
2 4242.69 1.82 1.00 11.06
3 2899.73 1.34 1.00 8.38
4 4896.62 1.94 1.00 9.85
5 2238.31 1.29 1.00 10.24
6 4640.52 1.83 1.00 9.13
7 4093.13 1.72 1.00 9.76
8 3551.97 1.58 1.00 9.66
9 3098.70 1.57 1.00 11.61
10 3879.52 1.66 1.00 9.60

EA = Emulsifying Agent
The numerical optimization study was conducted to study the constraints on

the design space and the vulnerability of the experimental model, this is

important since it suggest factors, responses and the goal for each variable

with respect to the measured response. By this study, a list of optimum

solutions can be arrived at in quantitative terms, i.e. the likely behavior of the

measured response in terms of analyzed factors in a random manner within

the design space. The influences of stirring rate, polymer concentration and

the amount of emulsifier on the particle size of gelatin microspheres in such a

manner are shown in the table 4.2.8.

The optimized formulae were selected based on the numerical optimization

data table 4.2.9 suggested the closeness of the predicted results with that of

the ideal results.


Table 4.2.9: Optimized formulae & formulation code of ofloxacin gelatin
microspheres
Emulsi Rate Round Particle
Drug Polymer fier & of ed-up size
Formulati Formul
(g) & its its stirri RPM(t µm
on ation
Ofloxa Concentr concen ng o
Method code
cin ation (g) tration (RPM nearest
(ml) ) -500s)
3551. 9.66 GLOM
1.58 1.58 1 3500
97 E-01
Emulsion
1042. 11.4 GLOM
Polymeriz 1.09 1.09 1 1000
78 E-02
ation
4640. 9.13 GLOM
1.83 1.83 1 4500
52 E-03

RPM = Rotation Per Minute, EA = Emulsifying Agent, gms = grams,


ml = milliliter, µm=micrometer
Figure 4.2.4: Parameters suggestions for GLOME -01

1000.00 5000.00 0.50 2.00

Stirring RPM = 3551.97 Gelatin Conc. = 1.58

1.00

8.00 12.00

0.50 2.00 0.32 22.00

Amt. of EA = 1.00 Particle Size = 9.66

Figure 4.2.5: Parameters suggestions for GLOME –02

1000.00 5000.00 0.50 2.00

Stirring RPM = 1042.78 Gelatin Conc. = 1.09

1.00

8.00 12.00

0.50 2.00 0.32 22.00

Amt. of EA = 1.00 Particle Size = 11.42


Figure 4.2.6: Parameters suggestions for GLOME –03

1000.00 5000.00 0.50 2.00

Stirring RPM = 4640.52 Gelatin Conc. = 1.83

1.00

8.00 12.00

0.50 2.00 0.32 22.00

Amt. of EA = 1.00 Particle Size = 9.13

Table 4.2.10: GLOME predicted Vs actual

Sl.No Predicted Actual


GLOME-01 9.66 9.6
GLOME-02 11.4 11.35
GLOME-03 9.13 9.1

From the numerical optimization solutions 8, 1 & 6 were randomly selected

and further evaluated for the response i.e., particle size. It was seen that the

response was almost similar to the response predicted by the design expert

software.

All the further studies were carried out using GLOME-03. Since all the three

optimized formulae had the required particle size range of 5 – 15 µm for lung

targeting, GLOME-03 was taken up for further characterization and in vivo

studies.
5.1.2. Optimization of ofloxacin albumin microspheres

The particle size optimization data is shown in the table 4.2.12. The results

were fitted to quadratic model of regression as it showed the maximum values

of R 2 and model sum of squares. ANOVA was performed and the results are

shown in table 4.2.14.

ANOVA proved that the model was significant (with a probability F value of

<0.0001). Albumin concentration most significantly affected the drug

incorporation as indicated by a probability F value of <0.0001.

The three-dimensional response surface graph along with the contour graph

was plotted figure 4.2.7,4.2.8 & 4.2.9, followed by numerical optimization

table 4.2.15 and model validation.

The 3D response surface graph substantiated the fact that albumin

concentration most significantly affects the particle size. From the numerical

optimization results, solution 2,1 & 7 was selected randomly as the optimized

formula for the preparation of ofloxacin albumin microsphere as it showed

maximum particle size efficiency.


Table 4.2.11: Coded central composite experimental design for three factors

Factor 1 Factor 2 Factor 3


C: Amt. of
Sl # B: Albumin
A: Stirring Emulsifying
Concentration
(RPM) Agent
(gms)
(ml)
1 0 0 1
2 0 -1 0
3 -1 1 1
4 0 1 0
5 0 0 0
6 -1 -1 -1
7 1 -1 1
8 0 0 0
9 0 0 0
10 1 1 -1
11 1 0 0
12 0 0 0
13 0 0 0
14 0 0 -1
15 -1 0 0
-1: low level; 1: high level; 0: central level

RPM = Rotation Per Minute, EA = Emulsifying Agent,


gms = grams, ml = milliliter
Table 4.2.12: Results of the design of ofloxacin albumin microspheres

Factor 1 Factor 2 Factor 3 Response


C:
A: B: Albumin
Amt. of Particle
Sl # Run Block Stirring Concentrati
Emulsifyin Size
Rate on
g Agent
RPM g ml µm
1 6 Block 1 5000 2 0.5 35
2 2 Block 1 3000 1.25 1.25 19.2
3 5 Block 1 1000 2 2 29
4 3 Block 1 1000 0.5 0.5 3
5 1 Block 1 3000 1.25 1.25 19.2
6 8 Block 1 5000 1.25 1.25 6.8
7 15 Block 1 3000 1.25 1.25 19.3
8 14 Block 1 3000 2 1.25 31
9 9 Block 1 3000 1.25 0.5 16.51
10 4 Block 1 3000 1.25 2 3.32
11 10 Block 1 3000 1.25 1.25 19.12
12 11 Block 1 5000 0.5 2 16
13 7 Block 1 3000 1.25 1.25 19.1
14 13 Block 1 3000 0.5 1.25 8
15 12 Block 1 1000 1.25 1.25 30.2

RPM = Rotation Per Minute, EA = Emulsifying Agent,


gms = grams, ml = milliliter, µm=micrometer
Table 4.2.13: Design summary for DOE runs of ofloxacin albumin microspheres

Design Summary
Study Type : Response Surface
Experiments: 15
Initial Design: Central Composite
Blocks: Nil
Design Model: Quadratic

Response Transformatio
Units Observations Minimum Maximum Model
n
Particle Size Microns 15 3 35 None Quadratic

Low High Low High


Factor Name Units Type
Actual Actual Coded Coded
Stirring
A RPM Numeric 1000 5000 -1 1
RPM
Albumin
B G Numeric 0.5 2 -1 1
Conc.
Amt. of
C Ml Numeric 0.5 2 -1 1
EA

RPM = Rotation Per Minute, EA = Emulsifying Agent, gms = grams,


ml = milliliter, µm=micrometer
The central composite design (a two- level factorial design with central and

star points) was adopted since it is employed to analyze the relationships

between multiple variables with a reduced number of experimental runs. The

particle size of the Albumin Ofloxacin microsphere was selected as the

response to be measured, since this offers a more tangible set of measurement

in an experimental setup. The experimental output showed the particle size of

microspheres to lie in a range of 3 – 35µm, and is not affected by

transformations and this followed a quadratic setup which indicated that the

response measured holds good over a range of other factors viz. stirring,

polymer concentration and the amount of emulsifier used. This would in turn

help in fixing the optimal particle size range, which has the maximum

efficacy and the effect of other factors in arriving at this range.

Table 4.2.14: Anova for ofloxacin albumin microspheres

Sum of Mean
Source DF F Value Prob. > F
Squares Square
Model 1356.29 9.00 150.70 15.30 0.0039
A 273.78 1.00 273.78 27.80 0.0033
B 264.50 1.00 264.50 26.86 0.0035
C 86.99 1.00 86.99 8.83 0.0311
A2 27.97 1.00 27.97 2.84 0.1528
B2 47.69 1.00 47.69 4.84 0.0791
C2 73.88 1.00 73.88 7.50 0.0408
AB 92.85 1.00 92.85 9.43 0.0278
AC 0.08 1.00 0.08 0.01 0.9303
BC 360.80 1.00 360.80 36.63 0.0018
Residual 49.24 5.00 9.85
Lack of Fit 49.22 1.00 49.22 7837.34 < 0.0001
Pure Error 0.03 4.00 0.01
Total 1405.533 14
2
R = 0.9650
The polynomial equation giving the mathematical relationship between each

factors was found to be:

Particle
Size =
( RPM) X -0.004009167
(Albumin Concentration) X 49.60111111
(Amount of Emulsifying Agent) X 50.88444444
(RPM) 2 X 8.175E-07
(Albumin Concentration) 2 X 7.591111111
(Amount of Emulsifying Agent) 2 X -9.448888889
(RPM) X Albumin Conc. X -0.005563333
(RPM) X (Amount of Emulsifying
Agent) X 0.000166667
(Albumin Concentration)
X (Amount of Emulsifying
Agent) X -29.24444444
-34.67555556
Figure 4.2.7: RSM graph of ofloxacin albumin microsphere - factor :
amount of emulsifying agent

DESIGN-EXPERT Plot

Particle Size
X = A: Stirring RPM
Y = B: Albumin Conc.

Actual Factor
56.51
C: Amt. of EA = 1.25
44.49
32.46
20.44
Particle Size

8.42

2.00
5000.00
1.63
4000.00
1.25
3000.00
B: Albumin Conc.
0.88 2000.00
A: Stirring RPM
0.50 1000.00

In this 3D RSM Graph albumin concentration exerts only a slight influence

upon the particle size, the albumin concentration increases with that of the

particle size the albumin microspheres. This can be attributed to the

increasing in viscosity of the albumin at higher concentration; any change in

the viscosity lowers the subdivision of the albumin solution into smaller

droplets during emulsification and finally increases the particle size. The

increases in drug concentration therefore had no effect on the particle size and

in turn depend upon the variabilities like stirring rate, concentration of the

albumin and emulsifier.


Figure 4.2.8: RSM graph of albumin ofloxacin microsphere factor:
polymer concentration

DESIGN-EXPERT Plot

Particle Size
X = A: Stirring RPM
Y = C: Amt. of EA

Actual Factor
B: Albumin Conc. = 1.25
34.59
25.28
15.96
6.65
Particle Size

-2.66

2.00
5000.00
1.63
4000.00
1.25
3000.00
C: Amt. of EA0.88 2000.00
A: Stirring RPM
0.50 1000.00

In this 3D RSM the increase in the amount of the emulsifying agent decreases

the viscosity present in the system and thereby lowering the particle size, the

decrease in the RPM in interaction with the albumin concentration increase its

particle size and vice versa. This figure is indicative that optimal particle size

formation depends upon the correct RPM and the concentration of the

polymer.
Figure 4.2.9: RSM graph of ofloxacin albumin microsphere factor -
stirring rate

DESIGN-EXPERT Plot

Particle Size
X = B: Albumin Conc.
Y = C: Amt. of EA

Actual Factor
A: Stirring RPM = 3000.00
50.93
36.95
22.98
9.00
Particle Size

-4.97

2.00
2.00
1.63
1.63
1.25
1.25
C: Amt. of EA0.88 0.88
B: Albumin Conc.
0.50 0.50

In the 3D RSM the stirring factor is found to have the influence on the

concentration of the albumin. The amount of emulsifying agent in higher

RPM’s the figure is indicative that the particle size would be lower range due

to the higher dispersion of the emulsifying agent influencing particle size and

vice versa and it also suggests that the optimal particle size formation can be

decided by correct RPM levels.

The Anova for response Surface design showed a quadratic setup, which

vindicates the employment of CCD. The higher sum of squares values for the

factors Concentration of albumin indicates that this factor is influenced by the


other factors – stirring rate and amount of emulsifier used. The influence

exerted by these two values is almost similar. The sum of squares for the

albumin factors as well, is predicted by this model in comparable terms. And

output of interacting factors shows a higher value for factors, which involves

albumin. This shows that the system is influenced by the inclusion of albumin

– supported by the other two factors to aid in the formation of microspheres

over an optimum range.

Table 4.2.15: Numerical optimization of solutions of ofloxacin albumin


microspheres

Albumin Amt. Particle


Solutions Stirring
Conc. Of EA Size
Number RPM
gms ml µm
1 3089.31 0.83 1.00 10.49
2 4076.29 0.93 1.00 9.12
3 4553.43 1.19 1.00 10.99
4 4990.31 1.13 1.00 9.23
5 4121.38 0.93 1.00 8.96
6 3630.11 0.91 1.00 10.04
7 2616.28 0.77 1.00 11.29
8 4012.80 0.88 1.00 8.66
9 4999.97 0.68 1.13 8.00
10 4999.99 0.53 1.44 12.00
RPM = Rotation Per Minute, EA = Emulsifying Agent,
gms = grams, ml = milliliter, µm=micrometer

The numerical optimization study was conducted to study the constraints on

the design space and the vulnerability of the experimental model, this is

important since it suggest factors, responses and the goal for each variable

with respect to the measured response. By this study, a list of optimum

solutions can be arrived at in quantitative terms, i.e. the likely behavior of the

measured response in terms of analyzed factors in a random manner within


the design space. The influences of stirring rate, polymer concentration and

the amount of emulsifier on the particle size of albumin microspheres in such

a manner are shown in the table 4.2.15

The optimized formulae were selected based on the numerical optimization

data, table 4.2.16, which suggested the closeness of the predicted results with

that of the ideal results.


Table 4.2.16: -Optimized formulae & formulation codes of ofloxacin
albumin microspheres

Rounded
Emulsifie Parti
Polymer & its Rate of -up
Formulation Drug (g) r & its cle Formulatio
Concentratio stirring RPM(to
Method Ofloxacin concentra size n code
n (g) (RPM) nearest -
tion (ml) µm
500s)
Emulsion 0.93 0.93 1 4121.38 4000 8.96 ALOME-01
Polymerizati 0.83 0.83 1 3089.31 3000 10.49 ALOME-02
on 0.77 0.77 1 2616.28 2500 11.29 ALOME-03
RPM = Rotation Per Minute, EA = Emulsifying Agent,
gms = grams, ml = milliliter, µm=micrometer
Figure 4.2.10:Parameters suggestions for ALOME-01

1000.00 5000.00 0.50 2.00

Stirring RPM = 4121.38 Albumin Conc. = 0.93

1.00

8.00 12.00

0.50 2.00 3.00 35.00

Amt. of EA = 1.00 Particle Size = 8.96

Desirability = 1.000

Figure 4.2.11: Parameters suggestions for ALOME-02

1000.00 5000.00 0.50 2.00

Stirring RPM = 3089.31 Albumin Conc. = 0.83

1.00

8.00 12.00

0.50 2.00 3.00 35.00

Amt. of EA = 1.00 Particle Size = 10.49

Desirability = 1.000
Figure 4.2.12: Parameters suggestions for ALOME-03

1000.00 5000.00 0.50 2.00

Stirring RPM = 2616.28 Albumin Conc. = 0.77

1.00

8.00 12.00

0.50 2.00 3.00 35.00

Amt. of EA = 1.00 Particle Size = 11.29

Desirability = 1.000

Table 4.2.17: ALOME Predicted Vs Actual

Sl.No Predicted Actual


ALOME-01 8.96 9.02
ALOME-02 10.49 10.2
ALOME-03 11.29 11.32

From the numerical optimization solutions 2, 1 and 7 were randomly selected

and further evaluated for the response i.e., particle size. It was seen that the

response was almost similar to the response predicted by the design expert

software.

All the further studies were carried out using Alome-03. Since all the three

optimized formulas had the required particle size range 5 – 15 µm for lung

targeting Alome-03 was taken up for further characterization and in vivo

studies.
5.2. FORMULATION CHARACTERISIZATION STUDIES

Characterisization studies were carried for the formulations:

• GLOME-03

• ALOME-03

5.2.1. MICROSPHERE RECOVERY / YIELD

The recovery / yield studies of microspheres are shown in table 5.1.1. The

recovery of GLOME 03 was observed to be less when compared to the

ALOME 03 microspheres, however the yield in both cases were found to be

higher than 91%. The maximum yield is observed in case of ALOME 03,

prepared by emulsion polymerization method. Though GLOME 03 was also

prepared by same method, factors like nature of polymers, and the extent of

cross – linking, which decides the drug polymer binding, would have

contributed a higher yield in this case.

Table 5.1.1: Recovery / Yield studies of microspheres

Wt. of the Wt. of the


Microsphere
-Sl. Formulation polymeric microspheres
recovery
No. code material + drug obtained
(%)
(g) (g)
2.0
1 GLOME -03 1.83 ± 0.004 91.53
2.0
2 ALOME-03 1.89 ± 0.003 94.8
5.2.2. Analysis of free drug in microspheres (un-entrapped) drug.

Table 5.1.2: Entrapment efficiency - amount of free drug

Sl. Formulation Amount of free Amount of free


No. code drug (mg ± SD) drug (%)

1 GLOME -03 0.94±0.002 9.4

2 ALOME-03 0.75±0.005 7.5

5.2.3. Analysis of adsorbed drug in microspheres.

Table 5.1.3: Entrapment efficiency - amount of adsorbed drug

Amount of Amount of
Sl. Formulation
adsorbed drug Adsorbed drug
No. code
(mg ± SD) (%)

1 GLOME -03 29.07±0.009 29.07

2 ALOME-03 25.05±0.005 25.05

5.2.4. Analysis of entrapped drug in microspheres.

Table 5.1.4: Entrapment efficiency - amount of entrapped drug

Amount of drug
Sl. Formulation Amount of drug
entrapped (mg ±
No. code entrapped (%)
SD)

1 GLOME -03 65.86±0.004 61.05

2 ALOME-03 8.71±0.003 66.95


5.2.5. SURFACE MORPHOLOGY

The surface morphological study of the microspheres revealed a smooth

surface and were spherical to near spherical for all the formulations analyzed.

The microspheres (both gelatin & albumin) produced by emulsion

polymerization showed a perfect sphere, while the microspheres obtained by

the spray drying process (in both polymers) showed a drug dissociated and

shriveled (surface folded) appearance, which might be caused because of the

higher temperature and pressure experienced by the sprayed droplet inside the

cyclone chamber of the spray drying apparatus and shear caused by the spray

nozzle of the spray dryer setup since the formulation was prepared by

dispersing the drug into a polymer solution prior to spraying, this might have

also caused a phase separation between drug and the polymer solution which

could have resulted in drug dissociation from its polymer substrate in the

former case; and shriveled appearance 53, 72


might have caused by the changes

in the temperature - pressure gradient across the height of the cyclone, this

might have caused by relatively quick drying of the outer core of the

microsphere while its inner core is still wet – as the inner core continues to

dry it is shown by a “bored-out” appearance due to the temperature difference

across the core of the microsphere, this in turn causes a shriveled appearance

as the spherical shape is not perfectly formed due to this boring –out effect in

the latter case Fig 5.1.4 & 5.1.5. This however does not happen in the case of

microspheres produced by emulsion polymerization method, this process

showed an enhanced profile for formulation quality and product attributes

Figure 5.1.10 & 5.1.11


Figure 5.1.1: Scanning electron micrograph of ofloxacin

Figure 5.1.2:Scanning electron micrograph of gelatin


Figure 5.1.3: Scanning electron micrograph of albumin

Figure 5.1.4: Scanning electron micrograph of spray dried gelatin microsphere


Figure 5.1.5: Scanning electron micrograph of spray dried albumin microsphere

Figure 5.1.6: Scanning electron micrograph of blank gelatin microspheres by


emulsion polymerization before γ-sterilization
Figure 5.1.7: Scanning electron micrograph of blank albumin microspheres by
emulsion polymerization after γ-sterilization

Figure 5.1.8: Scanning electron micrograph of blank gelatin microspheres by


emulsion polymerization after γ-sterilization
Figure 5.1.9: Scanning electron micrograph of blank albumin microspheres by
emulsion polymerization after γ-sterilization

Figure 5.1.10: Scanning electron micrograph of GLOME by emulsion


polymerization
Figure 5.1.11: Scanning electron micrograph of ALOME by emulsion
polymerization
5.2.6. PARTICLE SIZE ANALYSIS

The particle size of ofloxacin gelatin and ofloxacin albumin microspheres ranged

from 0.32 to 22 µm and 3 to 35 µm respectively table 5.1.5. It was observed that the

size of the microspheres was increased with an increase in the concentration of the

polymer, the size of the microspheres was decreased with increasing with stirring rate

and increase in the concentration of emulsifying agent decreased the particle size.

All these results indicated that the particle size of the microspheres could be modified

by varying the factors such as concentration of the polymer, concentration of

emulsifying agent and rate of stirring.

Furthermore, considering that microspheres lie within the size range of 5 - 15 µm, this

are required for deposition in the lungs all these microspheres can be used for lung

targeting.

Table 5.1.5: Average particle size of ofloxacin gelatin microspheres


and ofloxacin albumin microspheres

Sl.No Gelatin microspheres Albumin microspheres


particle size in µm particle size in µm
1. 2.1 35
2. 2.3 19.2
3. 2.3 29
4. 2.2 3
5. 2.3 19.2
6. 16.2 6.8
7. 0.32 19.3
8. 21.2 31
9. 5.1 16.51
10. 15 3.32
11. 1.3 19.12
12. 3.1 16
13. 22 19.1
14. 14 8
15. 1.2 30.2
5.2.7. IN-VITRO DRUG RELEASE

Table 5.1.6: Drug release profile of ofloxacin from albumin microspheres


(ALOME -03) as a function of time

Release of Ofloxacin
Time (hrs.) from Albumin
microspheres (%)
0.5 26.2
1 42.1
1.5 53.2
2 64.4
3 72.1
4 87
5 92.3
6 99.3

Table 5.1.7: In vitro curve fits for various release systems for albumin
microspheres

Adjusted
Sl# Equation R R2 K P n
R2

1. First Order 0.992 0.984 0.984 0.5099 <0.0001 -

Higuchi, (Square
2. 0.9929 0.9858 0.9858 41.9723 <0.0001 -
Root Time)

3. Hixon and Crowell 0.9781 0.9566 0.9566 0.1375 <0.0001 -

4. Peppas 0.9934 0.9868 0.9846 42.9509 <0.0001 0.4819

Baker and
5. 0.9733 0.9473 0.9473 0.0504 <0.0001 -
Lonsdale
Figure 5.1.12: In vitro release profile of ofloxacin albumin microsphere - First
order release pattern

120

% Drug release Vs Time in hrs


First Order
100 Time (hrs) vs Release (%)

80
Drug Release (%)

60

40

20

0
0 2 4 6

Time in hours

Figure 5.1.13: In vitro release profile of ofloxacin albumin microsphere - Higuchi


release pattern

160

140
% Drug release Vs Time in hrs
Higuchi, Square Root Time
120 Time (hrs) vs Release (%)
Drug Release (%)

100

80

60

40

20

0
0 2 4 6

Time in hours
Figure 5.1.14: In vitro release profile of ofloxacin albumin microsphere - Hixon -
crowell pattern

120

% Drug release Vs Time in hrs


Hixon and Crowell
100 Time (hrs) vs Release (%)
Drug Release (%)

80

60

40

20

0
0 2 4 6

Time in hours

Figure 5.1.15: In vitro release profile of ofloxacin albumin microsphere - Peppas


model

120

100 % Drug release Vs Time in hrs


Peppas
Time (hrs) vs Release (%)
Drug Release (%)

80

60

40

20

0
0 2 4 6

Time in hours
Figure 5.1.16: In vitro release profile of ofloxacin albumin microsphere - Baker -
Lonsdale plot

120

% Drug release Vs Time in hrs


Baker and Lonsdale
100 Time (hrs) vs Release (%)

80
Drug Releae (%)

60

40

20

0
0 2 4 6

Time in hours

The co-relation Coefficient value R 2 is taken into account to decide upon the

relevance of the model /curve fit which will best describe the extent of fit.

The anova (P) values is lesser than that of the 95% confidence interval (0.05)

the fit data are derived form the R 2 values for noting the extent of fit.

According to the R 2 values given by different data fits, the Peppas model

seems to be an ideal fit – 0.9868. This proves that Peppas fit to the ideal

regression pattern as undergone by the microspheres figure 5.1.15.

According to Peppas fit, the release of the drug is decided upon by the

diffusion of the polymeric matrix and the drug release is governed by a

variation of Fick’s law of diffusion. The factors, which control this, are

diffusion coefficient and permeability coefficient of the polymer in a constant


temperature. This would decide upon the concentration of the released drug in

vitro. The drug release is also affected by the morphological and structural

characteristics of the polymers, viz. degree of crystallinity, size of the

crystallites, degree of swelling of the matrix, the size of the mesh formed by

the extent of cross linking, transitional and relaxational phenomenon in the

polymer plays a role in the release of the drug. In light of these facts stated

above, the albumin microspheres releases the drug by a diffusion process

which releases the drug from the polymeric matrix based upon the extent

diffusion, erosion of polymeric matrix and subsequent domain separation of

drug due to diffusion might also be a possibility. Due to this reason the

release profile shows an initial surge in the release pattern due to swelling of

the matrix (above 50% in the first 1.5 hours in this case) - releasing the

adsorbed drug particles on the microsphere surface and there after by

diffusion and there after a more controlled release pattern controlled by the

earlier stated facts. Due to this, the release pattern can be sustained for a

longer time line producing a control over the release profile of the

microsphere in vitro.
Table 5.1.8: Drug release profile of ofloxacin from gelatin microspheres
as a function of time
Release of Ofloxacin
Time (hrs.) from Gelatin
microspheres (%)
0.5 32.27
1 41.95
1.5 48.71
2 53.34
3 58.56
4 67.25
5 71.02
6 74.84
7 78.23
8 83.03
10 88.02
12 91.62

Table 5.1.9: In vitro curve fits for various release systems for
gelatin microspheres

Sl Adjusted
Equation R R2 K P n
# R2

1. First Order 0.8363 0.6995 0.6995 0.3014 <0.0001 -

Higuchi, (Square
2. 0.8863 0.7855 0.7855 30.2577 <0.0001 -
Root Time)

3. Hixon and Crowell 0.7042 0.4959 0.4959 0.0795 <0.0001 -

4. Peppas 0.9985 0.9970 0.9966 42.2149 <0.0001 0.3187

Baker and
5. 0.9861 0.9723 0.9723 0.0278 <0.0001 -
Lonsdale
Figure 5.1.17: In vitro release profile of ofloxacin gelatin microsphere –Baker
and Lonsdale

120

% Drug release Vs Time in hrs


Baker and Lonsdale
100 Time (hrs) vs Release (%)
Drug Release (%)

80

60

40

20

0
0 2 4 6 8 10 12 14

Time in hours

Figure 5.1.18: In vitro release profile of ofloxacin gelatin microsphere – Peppas


model

120

% Drug release Vs Time in hrs


Peppas
100 Time (hrs) vs Release (%)

80
Drug release (%)

60

40

20

0
0 2 4 6 8 10 12 14

Time in hours
Figure 5.1.19: In vitro release profile of ofloxacin gelatin microsphere - Hixon
and Crowell

120

% Drug release Vs Time in hrs


Hixon and Crowell
100 Time (hrs) vs Release (%)

80
Drug Release (%)

60

40

20

0
0 2 4 6 8 10 12 14

Time in hours

Figure 5.1.20: In vitro release profile of ofloxacin gelatin microsphere - Higuchi,


square root Time

120

% Drug release Vs Time in hrs


Higuchi, Square Root Time
100 Time (hrs) vs Release (%)
Drug Release (%)

80

60

40

20

0
0 2 4 6 8 10 12 14

Time in hours
Figure 5.1.21: In vitro release profile of Ofloxacin Gelatin microsphere - First
Order

120

% Drug release Vs Time in hrs


First Order
100 Time (hrs) vs Release (%)

80
Drug Release (%)

60

40

20

0
0 2 4 6 8 10 12 14

Time in hours

The co-relation Coefficient value R 2 is taken into account to decide upon the

relevance of the curve fit which will best describe the extent of fit undergone

by the release pattern of the drug from the formulation. Since the anova (P)

values is lesser than that of the 95% confidence interval (0.05) the fit data

here too were derived form the R 2 values for deciding on the degree of the

curve fit. According the R 2 values given by different data fits, the Peppas

model figure 5.1.18 seems to be an ideal fit in this case too – 0.9970. This

proves that Peppas fit to the ideal candidate regression pattern as undergone

by the gelatin microspheres.

In Peppas fit model as described earlier, the release of the drug is decided

upon by the diffusion of the polymeric matrix and the drug release is

governed by a variation of Fick’s law of diffusion. The factors, which control

this, are diffusion coefficient and permeability coefficient of the polymer in a


constant temperature. This would decide upon the concentration of the

released drug in vitro. The drug release is also affected by the morphological

and structural characteristics of the polymers, thus it can be surmised that the

diffusion coefficient is independent of the temperature in a

thermodynamically ideal condition (maintained constant throughout the in

vitro runs) and the solute concentration. It can be decided from these facts,

the gelatin microspheres releases the drug by a diffusion process which

releases the drug from the polymeric matrix based upon the extent diffusion,

erosion of polymeric matrix and subsequent domain separation of drug due to

diffusion. This results in the manifest the release profile shows an initial

surge – “burst release” (53.34% in the first 2 hours) the release pattern due to

swelling of the matrix - releasing the adsorbed drug particles on the

microsphere surface and there after by diffusion and there after a more

controlled release pattern. Due to this the release pattern is be sustain for a

longer time line producing a control over the release profile of the

microsphere in vitro. 20, 50, 73, 74


5.2.8. DIFFERENTIAL SCANNING CALORIMETRY (DSC) ANALYSIS

In the present study, the DSC analysis were performed to find out the physical

nature of ofloxacin entrapped in the gelatin and albumin microsphere and also

to confirm absence of drug – polymer interaction. Individual thermograms of

the pure drug, raw polymer and drug loaded microspheres (1:1 drug/polymer

ratio), was performed by a computer-interfaced differential scanning

calorimeter (DSC 7 differential calorimeter – Perkin Elmer) equipped with a

thermal analysis data system. Samples (5 - 10mg) in a sealed aluminum pans

were heated from 0 0 C to 25 0 C at a rate of 10 0 /min, using nitrogen as purging

gas.

Procedure

The Ofloxacin gelatin/albumin Microspheres produced by the emulsion were

subjected to DSC analysis. 5 – 10 mg of the microspheres were taken for

analysis. Along with blank, drug loaded, and a physical mixture of Ofloxacin

and blank gelatin/ albumin microspheres in the ratio of 1:1 was analyzed in

this manner. The results are as presented below.

Results and Discussion

Curves of DSC are shown in figure 5.1.22 to 5.1.28 from which one can

conclude that ofloxacin microsphere was not a physical mixture, but exists as

microspheres. They have characteristic exothermic peaks at 545K for

ofloxacin and 386.87K for the blank microsphere, respectively; both almost

disappeared in the ofloxacin microsphere curve, in which a new peak at

372.34K appeared. The DSC curve of the physical mixture also differed from
that of ofloxacin microsphere. In the present study, the DSC analysis was

performed to find out the physical nature of ofloxacin entrapped in the

gelatin/albumin microsphere and also to confirm absence of drug-polymer

interaction. The thermogram of ofloxacin showed peak of 545K. Because of

its higher melting point, which is beyond the scanning temperature,

thermograms of gelatin/albumin microspheres showed no peaks. 50 The

physical mixture of ofloxacin and gelatin microspheres (1:1) produced a peak


45
about the melting point of the drug. The thermogram of unloaded gelatin &

albumin microspheres showed no peak as given in figure 5.1.23 & 5.1.26

indicated that amorphous nature of carrier particles. Ofloxacin peak was

absent in the thermogram of drug-loaded gelatin /albumin microspheres,

which revealed that amorphous nature of entrapped drug in the formulated

microspheres. The thermograms of individual excipients were employed as

reference, with which the thermograms of the physical mixtures were

compared. The physical mixtures of gelatin and albumin thermograms are

shown in figure 5.1.25 & 5.1.28 respectively. The drug, ofloxacin showed an

endothermic peak at 545K, while the polymers albumin and gelatin showed an

endothermic peak at 55.1 0


Figure 5.1.22: DSC thermogram of ofloxacin in API form

API (Active Pharmaceutical Ingredients)

This DSC thermogram shows a characteristic exothermic peak of Ofloxacin at

545K figure 5.1.22. This phenomenon was observed for a plain sample of

ofloxacin without administering in a form of microspheres or a physical

admixture of the drug and unloaded microspheres.

The thermogram shows a feature characteristic of the gelatin. The

thermogram shows no presence of peaks and shows a phenomenon called a

“glass transition”-T g. This phenomenon is due to thermal degradation of


crystalline junctions, which exists between macromolecules of gelatin to an

amorphous form by the application of thermal energy. This transition from

crystalline junctions to amorphous ones thereby adding to the amorphousness

of the polymer, which produces this effect. At the end of this effect a

smothered denatured peak of the loaded drug ofloxacin is noticed – the

uncharacteristic peak of the drug may be produced by the enhanced need of

thermal activation energy which might arise as a result of a thermal

denaturation of the polymer and is also indicative of the fact that the drug is

present in a physical mixture and devoid of any chemical interaction with the

drug, and is indicative of its stability.


Figure 5.1.23: DSC thermogram of gelatin in polymer form

The same Glass transition effect is observed in the thermogram of the plain

gelatin too. The T g is influenced by the level of moisture in the sample –

higher levels of moisture can lead to significantly shorter T g levels and the

thermogram is obtained so is indicative of this effect, since lesser thermal

activation levels can be sufficient to impart an enhanced amorphous character

to an already amorphous polymer.


Figure 5.1.24: DSC thermogram of ofloxacin gelatin microspheres.
Figure 5.1.25: DSC thermogram of ofloxacin – gelatin physical mixture
1:1 ratio
Figure 5.1.26: DSC thermogram of albumin in polymer form
Figure 5.1.27: DSC thermogram of ofloxacin albumin microspheres
Figure 5.1.28: DSC thermogram of ofloxacin – albumin physical mixture
1:1 ratio
5.2.9. X-RAY DIFFRACTION ANALYSIS (XRD)

Throughout the XRD runs conducted for ofloxacin bearing albumin and

gelatin microspheres, the characteristic peak of the crystalline drug ofloxacin

was not visible in the spectra. However, the XRD patterns of drug ofloxacin

produced a characteristic peak when analyzed in API (active pharmaceutical

ingredient) form. It was expected the crystalline drug ofloxacin would show

its specific crystal XRD patterns in a microsphere-formulated form, however

this did not occur. These results indicates that drug present in the polymeric

microsphere formulations developed, is either dispersed / entrapped

molecularly or present in an amorphous form in the polymer matrix. The data

from XRD runs concurs with reported findings of several other workers, and

also supports the DSC studies in terms of parallel results in form of absence

of endotherm peaks pertaining to drug in a microsphere form, which points

out that the drug is indeed present in a entrapped form. 52, 58, 59, 61, 75, 74
Figure 5.1.29: The characteristic XRD pattern obtained for ofloxacin

OFLOXACIN

3000

2500

2000
Relative Intensity

1500 Intensity

1000

500

0
0 5 10 15 20 25 30 35 40 45

The XRD graph shows the presence of the typical XRD pattern observed for

the crystalline drug ofloxacin figure 5.1.29. Due this nature of Ofloxacin, the

peak characterizes the presence of specific crystalline signatures of the drug,

this graph elucidates the purity of the drug by means of its characteristic

peaks which pertains to the crystal lattice of the drug and can act as a

reference spectrum for drug for the microsphere formulated forms of the drug.

The modified XRD pattern which appears in the microsphere formulated

forms of the drug with two polymers helps in understanding the modifications

of the drug in terms of its crystalline structure which is a measure of the

efficiency of the formulatory aspects of the drug by the crystalline

modifications in formulated forms of the drug.


Figure 5.1.30: X-Ray diffraction pattern of albumin

ALBUMIN

1600

1400

1200

1000
Relative Intensity

800 Intensity

600

400

200

0
0 10 20 30 40 50

The XRD pattern of the polymer albumin shows figure 5.1.30 a prominent

peak in the spectra, which may due to the crystalline nature of the polymer.

The prominence of the peak of albumin suggests that the polymer by itself

exist in a crystalline state. As the XRD pattern of albumin microsphere shows

that the crystalline nature of the polymer is further enhanced by loading of

crystalline drug ofloxacin. This also suggests that the crystalline nature of

ofloxacin be decided by the inclusion of the polymer, which shall decide upon

the overall crystalline nature of the formulation.


Figure 5.1.31: X-Ray diffraction pattern of gelatin

GELATIN

1600

1400

1200
Relative Intensity

1000

800 Intensity

600

400

200

0
0 5 10 15 20 25 30 35 40 45

The XRD pattern of the polymer gelatin shows Figure 5.1.31 the presence of

characteristic crystalline pattern of the polymer. Gelatin has limited

crystalline characterization in the high molecular weight macromolecular

junctions of the drug, which forms the structural basis of the polymer.

However, this property of the polymer is not fully exhibited in the spectra;

and the partial crystallinty of the drug is denoted by the presence of

prominent peak, which is indicative of the crystalline nature of the polymer.


Figure 5.1.32: X-Ray diffraction pattern of gelatin ofloxacin microsphere
showing the absence of characteristic crystal diffraction pattern of ofloxacin due
to its entrapment in the polymeric matrix.

Gelatin Microspheres

1600

1400

1200

1000
Relative Intensity

800 Intensity

600

400

200

0
0 5 10 15 20 25 30 35 40 45

The XRD pattern of the gelatin microsphere shows Figure 5.1.32 the characteristic

crystalline signature of the partially crystalline polymer of gelatin. The XRD pattern

which appears in the spectrum is due to the partial crystallanity of the drug – existing

in the macromolecular junctions of the drug. The point of significance in this

spectrum is the absence of the characteristic XRD pattern of ofloxacin. This may be

attributed to the following factors like the denaturation of the crystalline nature of the

drug or partial denaturation leading to amorphousness of the drug – which denotes,

drug is embedded in the polymeric matrix as a physical mixture which would lead to

the denaturation of the primarily crystalline drug, as symbolized in the spectra.


Figure 5.1.33: X-Ray Diffraction pattern of Albumin Ofloxacin microsphere
showing the absence of characteristic crystal diffraction pattern of ofloxacin due
to its entrapment in the polymeric matrix.

Albumin Microspheres

1600

1400

1200
Relative Intensity

1000

800 Intensity

600

400

200

0
0 5 10 15 20 25 30 35 40 45

The albumin ofloxacin microsphere XRD spectrum also shows figure 5.1.33 an

absence of crystalline signature peaks of ofloxacin. This is attributed to change in the

physical nature of the drug due to the changes in the crystalline nature of the drug in

albumin polymeric matrix, which leads to the altered crystal signatures of the

crystalline drug ofloxacin. This suggests that the drug is either molecularly entrapped

in the polymeric matrix or it might be encapsulated by the presence of polymer over

the crystals of the drug which accounts for the absence of the drug peaks in the

spectrum. This infact acts as a pointer for the efficiency of the formulation adopted,

since it acts as a qualitative measure of either the encapsulation efficiency of the drug

or the entrapment capability of the drug in the polymer matrix, which would in turn

contribute to the efficiency of the formulation.


5.2.10. DRUG POLYMER INTERACTION STUDIES

The IR spectrum of ofloxacin, albumin and gelatin was employed as reference

with which the spectrums of the physical mixtures were compared.

On comparison of the individual spectrum with that of physical mixtures, no

prominent difference in the spectrums was seen. Thus, it was concluded that

there are no major degenerative drug-polymer figure 5.1.34 to 5.1.38

interactions and hence the polymers could be used safely to formulate.


Figure 5.1.34: Infrared spectra of albumin

Figure 5.1.35: Infrared spectra of gelatin


Figure 5.1.36: Infrared spectra of ofloxacin: albumin physical mixture

Figure 5.1.37: Infrared spectra of ofloxacin: gelatin physical mixture


Figure 5.1.38: Infrared spectra of ofloxacin
5.2.11. RESIDUAL SOLVENT ANALYSIS

The amounts of acetone in the microsphere formulations were within limits

prescribed by the ICH guideline ‘Q3C’ 26 for the residual solvents. It was

therefore concluded that the formulations were safe to be administered

parenterally. The results are shown in table 5.1.10

Table 5.1.10: Residual solvent analysis of microsphere formulations

Sl. Formulation code Acetone (PPM)


No.

1 GLOME –03 2000

2 ALOME –03 1900

ICH limits 5000


5.2.12. STERILIZATION STUDIES BY γ -IRRADIATION

Scanning Electron Microscopy (SEM) photographs of microspheres prior and

after γ irradiation studies showed no major structural changes. Most

importantly the particles did not break and were intact and withstood the

irradiation process. A slight increase in the particle size (1.2µm – 3 µm) was

observed after sterilization with γ irradiation as shown in the surface

morphology studies figure 5.1.6 to 5.1.9. As the increase was negligible, it

can be concluded that gamma irradiation is the best-suited method of

sterilization for microsphere formulations.


5.3. STABILITY STUDIES

The observations of long-term storage condition and accelerated storage condition are

shown in tables 5.2.1 and 5.2.2. The drug content values of the formulations in long-term

storage condition and accelerated storage condition along with their 95% confidence limits

(Σplot® software) are plotted in figures 5.2.1 to 5.2.4

Depending on the stability of the microspheres formulations with respect to the tested

parameters, a re-test date was arrived upon. After re-test if the formulations are found to

possess acceptable limits, another re-test date is fixed.

Long-term storage conditions

The formulations were stable for the 6-month period in long-term storage temperatures of 5

± 30C. The drug content of the microspheres formulations did not vary to a large extent. A

maximum decrease of 2.47% from the initial concentration observed in GLOME - 03, and

2.14 % in the case of ALOME -03. Minor changes in particle size were noticed during the

storage time while the stability studies were conducted, however the changes were found to

be negligible and found to have no impact on the quality of the formulations.

Accelerated storage conditions

The formulations were stable for the 12-month period in long-term storage temperatures of

25 ± 20C and 60 ± 5% RH. The drug content of the microspheres formulations did not vary

to a large extent. A maximum decrease of 3.5% from the initial concentration observed in

both GLOME - 03 and ALOME -03. Minor changes in particle size were noticed during

the storage time while the stability studies were conducted, however the changes were

found negligible and have no impact on the quality of the formulations.


Table.5.2.1: Observations of stability test studies in accelerated storage
condition (25 ± 2 0 C and 60 ± 5% RH)

Drug Content (%) Particle size (µm) Physical change


Microsphe
res
Months Months Months
Formulatio
ns
0 3 6 9 12 0 3 6 9 12 0 3 6 9 12
ALOME –
99.34 98.56 97.86 96.52 95.85 11.3 11.4 11.3 11.4 11.4 - - - - -
03
GLOME –
99.12 98.68 97.52 96.47 95.68 9.2 9.3 9.3 9.3 9.3 - - - - -
03
- : No physical
change
Table1: 5.2.2: observations of stability test studies in real-time storage
condition (5 ± 3 0 C)

Drug Content (%) Particle size (µm) Physical change


Microspher
es
Months Months Months
Formulatio
ns
0 2 4 6 0 2 4 6 0 2 4 6

ALOME -03 99.02 98.22 97.37 96.88 11.4 11.3 11.3 11.4 - - - -

GLOME -03 99.20 98.12 97.68 96.73 9.5 9.4 9.4 9.4 - - - -
-: No physical
change
The average particle size also did not vary appreciably. More or less it was

around the initial particle size value. No physical changes, like liquefaction or

formation of lumps or discoloration were observed in the microspheres

formulations during this 12-month study period.

Accelerated storage conditions

No problems were observed in the accelerated storage conditions of 25 ± 2 0 C

and 60 ± 5% RH and no physical changes were evident in microspheres of the

drug –ofloxacin.

An action plan was devised and the following considerations were addressed

¾ The both GLOME -03 and ALOME -03 microspheres were fixed with a re-

test date of 2 years from the date of their manufacture, as they were stable

for 6 months in accelerated storage conditions and for 12 months in long-

term storage conditions.

¾ But in the case of ALOME -03 microspheres, the storage conditions were

found to be very critical to maintain the integrity of the samples. At any

time (may it be storage or transit), it should not be stored at temperatures

higher than 5 ± 3 0 C (refrigeration temperature).

¾ The re-test date of ALOME -03, were fixed at 1 year (provided, the

recommended temperature conditions are followed strictly) from the date

of manufacture as the long-term studies were conducted for this period and
the formulations were found to be stable during this period. 64, 26

(Guideline for Submitting Documentation for the Stability of Human

Drugs and Biologics," Food and Drug Administration, DHHS, February,

1987)

Interpretation of stability graphs

The drug activity data shown as solid circles has a linear regression line

superimposed. The lower 95% confidence line was also graphed. The

accepted definition of the shelf life time is the x axis coordinate for the

intersection of the lower 95% confidence line with 90% drug activity

(Guideline for Submitting Documentation for the Stability of Human Drugs

and Biologics," Food and Drug Administration, DHHS, February, 1987). This

intersection is considered the shelf life period of formulation.


Figure: 5.2.1 Drug content plot of ALOME - 03 in accelerated storage
conditions

104
ALOME -03
102 90% reg. line
Concentration of ofloxacin (%)

95% conf. Int.


100

98

96

94

92

90

88

86

0 3 6 9 12 15 18 21 24 27 30

Time (months)

Figure: 5.2.2 Drug content plot of GLOME - 03 in accelerated storage


conditions

104

102 GLOME -03


Concentration of ofloxacin (%)

100 90% reg. line


(5% conf. int.
98

96

94

92

90

88

86

0 3 6 9 12 15 18 21 24 27 30

Time (months)
Figure: 5.2.3 Drug content plot of ALOME - 03 in real time storage
conditions

104
ALOME -03
102 90% reg. line
Concentration of ofloxacin (%)

95% conf. Int.


100

98

96

94

92

90

88

86

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

Time (months)

Figure: 5.2.4 Drug content plot of GLOME - 03 in real time storage


conditions

104
GLOME -03
102 90% reg. line
Concentration of ofloxacin (%)

95% conf. int.


100

98

96

94

92

90

88

86

0 2 4 6 8 10 12 14 16 18 20 22 24

Time (months)
5.4. IN-VIVO TISSUE DISTRIBUTION STUDIES

All the drug concentration data were analyzed using a non-compartmental

method, using WINNONLIN Version to obtain total area under concentration-

time curves (AUC 0 ∞ ). 70

Results

The drug content (µg/ml) with time was determined in blood, liver, spleen and

lung. The results were calculated to express the percentage amount, as shown

in table 5.3.2 & 5.3.3 for GLOME-03 and ALOME-03.

The data of drug content change with time were treated to calculate the

targeting parameters, as shown in table 5.3.2 and 5.3.3.

Table 5.3.2: Percentage drug distributed to various tissues of albino


mice following I.V. administration of GLOME-03

Time (h) Lung Liver Spleen Blood


0.17 27.89 5.12 2.12 3.51
0.5 39.98 4.78 1.89 3.24
1 54.58 4.58 1.75 2.98
3 68.72 4.65 1.65 2.52
6 81.82 4.42 1.54 2.1
12 91.28 4.38 1.47 1.78

Table 5.3.3: Percentage drug distributed to various tissues of albino


mice following the I.V. administration of ALOME-03

Time (h) Lung Liver Spleen Blood


0.17 29.86 5.54 2.35 4.32
0.5 42.85 5.23 2.02 4.12
1 56.32 4.91 1.95 3.24
3 71.85 4.32 1.77 2.86
6 83.69 4.02 1.42 1.98
12 94.89 3.98 1.27 1.56
5.4.1. Quantitative evaluation of targeted drug delivery systems

Tissue distribution studies for GLOME-03

The ofloxacin gelatin microspheres showed the largest value of AUC and r e

for the lung; the targeting efficacy t e of lung increased by a factor of 48

(compared with spleen) ~16.9 (compared with liver) ~33.7 (compared with

ofloxacin), table 35 compares the time-averaged drug distribution to different

tissues, following the I.V. administration of ofloxacin as a solution and

gelatin ofloxacin microspheres (GLOME-03). Drug distribution to the lungs

was 90.98%.

Tissue distribution studies for ALOME-03

The ofloxacin albumin microspheres showed the largest value of AUC and re for

the lung; the targeting efficacy te of lung increased by a factor of 35 (compared

with spleen) ~18.5 (compared with liver) ~25 (compared with ofloxacin), table

36 compares the time-averaged drug distribution to different tissues, following

the I.V. administration of ofloxacin as a solution and albumin ofloxacin

microspheres (ALOME-03). Drug distribution to the lungs was 91.7%.


Table 5.3.4: Lung-targeting parameters of GLOME-03: Ofloxacin after
I.V. administration in mice (n=3)

AUC 1 2 3 4 5
Sample
GLOME Ofloxacin re te Te Te* T e * X 100
Blood 26.74 119.52 0.32 39.06 38.30
Liver 53.21 147.58 0.55 18.32 81.65
Spleen 18.79 9.80 1.92 52.80 28.33 0.90982 90.98
Lung Target
903.25 77.35 12.86 1495.98
tissue
∑ Te* = 1644.26
Table 5.3.5: Lung-targeting parameters of ALOME-03: Ofloxacin after
I.V. administration in mice (n=3)

AUC 1 2 3 4 5
Sample
ALOME Ofloxacin re te Te Te* T e * X 100
Blood 37.34 133.70 0.28 41.51 37.34
Liver 50.05 144.35 0.53 20.42 75.90
Spleen 26.54 9.73 1.84 58.42 26.90 0.91709 91.70
Lung 934.01 114.65 13.52 - 1550.18
∑ Te* = 1690.32
5.4.2. Compartment modeling

In vivo pharmacokinetics of microspheres

The data of GLOME –03 and ALOME –03 was subjected to compartment modeling

using with ‘WINNONLIN Version 1.5 Pharmacokinetic software’. The actual plot

exactly superimposed on the predicted line when two-compartment model was fitted

to the data. The plasma concentration and pharmacokinetical parameters are reported

in table 5.3.6 and 5.3.7 for gelatin and albumin microspheres respectively, 32, 48, 76
.

Thus, the Pharmacokinetics is following a two-compartment model.

Figure: 5.3.1: Two compartment model I.V. Bolus


Table 5.3.6 Pharmacokinetics parameters of GLOME-03

Sl# Parameters Estimation Std.Dev CV%


1. AUC (mcg.hr/ml) 51.37872 15.12995 29.45
2. K10-HL 7.5717 2.506969 33.11
3. Alpha (h -1 ) 1.198388 1.149512 95.92
4. Beta (h -1 ) 0.063424 0.02876 45.34
5. Alpha-HL (hour) 0.5784 0.554495 95.87
6. Beta-HL (hour) 10.92873 4.951178 45.3
7. A (hour) 1.525515 0.673906 44.18
8. B (hour) 3.177922 0.650387 20.47
9. C max (mcg/ml) 4.703437 0.487918 10.37
10. Cl (l/hr) 0.010121 0.002983 29.48
11. AUMC (mcg hr 2 /ml) 791.0708 575.9042 72.8
12. MRT (hour) 15.39686 6.748352 43.83
13. Vss (lts) 0.15583 0.025699 16.49

Figure 5.3.2: Two-Compartment model following I.V. bolus


administration for GLOME-03

Concentration Vs Time for


4.0

3.5

3.0

2.5

2.0
Observed
1.5 Predicted

1.0

0.5

0.0
0 2 4 6 8 10 12
Time in
h
Table 5.3.7: Pharmacokinetics parameters of ALOME-03

Sl# Parameters Estimation Std.Dev CV%


1. AUC (mcg.hr/ml) 82.93261 14.74237 17.78
2. K10-HL 15.87265 2.961216 18.66
3. Alpha 0.730875 0.250712 34.3
4. Beta 0.031682 0.007584 23.94
5. Alpha-HL (hour) 0.94838 0.325122 34.28
6. Beta-HL (hour) 21.87858 5.232947 23.92
7. A 1.039222 0.163694 15.75
8. B 2.582385 0.180955 7.01
9. C max (mcg/ml) 3.621607 0.082527 2.28
10. Cl (l/hr) 0.00627 0.001116 17.79
11. AUMC (mcg hr 2 /ml) 2574.76 1062.537 41.27
12. MRT (hour) 31.04641 7.313496 23.56
13. Vss (lts) 0.194666 0.011891 6.11

Figure 5.3.3: Two-Compartment model following I.V. bolus


administration for ALOME-03

Two Compartment i.v.bolus for ALOME

5.0
4.5
4.0
3.5
3.0
2.5
Observed
2.0 Predicted
1.5
1.0
0.5
0.0
0 2 4 6 8 10 12
Time in hours
5.5. HISTOPATHOLOGICAL STUDIES

Staining showed blue colored nucleus and cytoplasm with various shades of pink with

change in different tissue morphology. The cytoarchitecture photographs of the

organs of control and microsphere formulations are shown in figures 5.4.1 to 5.4.9

below.

Figure 5.4.1: Cytoarchitecture of lung (control)


Figure 5.4.2: Cytoarchitecture of lungs
(Ofloxacin gelatin microspheres – emulsion polymerization method)

Figure 5.4.3: Cytoarchitecture of lungs


(ofloxacin albumin microspheres - emulsion polymerization method)
Figure 5.4.4: Cytoarchitecture of liver (control)

Figure 5.4.5:Cytoarchitecture of liver


(Ofloxacin gelatin microsphere – Emulsion polymerization method)
Figure 5.4.6: Cytoarchitecture of liver
(Ofloxacin albumin microspheres – Emulsion polymerization method)

Figure 5.4.7: Cytoarchitecture of spleen (control)


Figure 5.4.8: Cytoarchitecture of spleen
(Ofloxacin gelatin microspheres – emulsion polymerization method)

Figure 5.4.9:Cytoarchitecture of spleen


(Ofloxacin albumin microsphere – emulsion polymerization method)
On comparison of the microsphere formulations with the control, the

cytoarchitecture of the tissues did not show any major difference

(degenerative changes) as evidenced by the photographs. Based on this

observation, the constituents of the microsphere formulations were declared

safe for parenteral use as a passive targeted drug delivery system.


6. SUMMARY

The efficacy of many promising drug candidates are frequently limited by

their inability to reach the target site of action, especially when they are

administered through conventional dosage forms or drug delivery systems.

However, advances in newer drug delivery systems has tremendously

improved the scope of application of the drugs and has increased the quantum

of drug reaching the site and simultaneously decreasing the amount of drug

being distributed to other parts of the body. Thus, the pharmacotherapy of the

drug is focused, by supplying the drug selectively to its site of action in a

manner that provides maximum therapeutic activity and also prevents

degradation or inactivation during transit to the target sites and protects the

body from adverse reactions due to high dosing or frequent administration.

Microspheres are solid colloidal particles ranging in size from 1 to 1000 µm,

which can act as good targeted drug delivery platforms if the particle size can

be effectively controlled especially for pneumonia infections. They consist of

macromolecular polymeric materials in which the drug is dissolved,

entrapped, or encapsulated, and/or to which the drug is adsorbed or attached

through physical means.

The action of microspheric particles is dependent on the route of

administration and physicochemical properties. After administration via

intravenous injection, large particles (>7 micron) are retained in the

pulmonary region whereas small particles (<7 micron) are distributed in the

RES (liver & spleen). The entrapment of the microspheres in the lungs and
release of drug thereby results in improved therapeutic efficacy of the drug

for diseases of the lungs like pneumonia.

The fluoroquinolones - especially, the current analog ofloxacin is increasingly

employed in the treatment of respiratory tract bacterial infections, due to its

good intracellular penetration and relatively short intracellular residence time.

The pre-formulatory phase studies confirmed the suitability of the polymers

and their compatibility with the drug. The method of estimation of the drug

by UV technique was developed and validated for system suitability and

reproducibility. Drug-polymer (IR) interaction studies were also carried out to

test their suitability in preparing satisfactory microsphere formulations.

The polymers chosen were albumin and gelatin, as they are natural,

biocompatible and biodegradable.

Ofloxacin had a λ max of 297 nm and obeyed Beer’s law in the range of 0 – 20

mcg/ml when 0.1 N HCl was used as solvent. The recovery was found to be

95.08%. Drug-excipient compatibility studies proved that no major

degenerative changes existed as shown by the lack of extra peaks during the

IR runs, and no shift of peaks in the thermograms of the mixtures when

compared to their individual thermograms of the drug and the excipient. This

proved the chemical inertness of the polymer with the drug.


The microspheres of albumin and gelatin were prepared by two methods of

preparation, viz., spray drying process and emulsion polymerization method

from many methods reported in literature. Emulsion polymerization method

was employed as it showed better output in terms size and drug entrapment

whereas spray drying process was discontinued because of shriveling during

the drying process which was cross checked by SEM.

Emulsion polymerization method produced spherical, discrete microsphere

with an average size range of 0.32 to 22 µm for gelatin microspheres and 3 to

35 µm for albumin microspheres in simulated conditions which were diverse

and far apart. However, the size range produced by this process was found

unsuitable for the intended purpose.

The ideal particle size range for lung targeting and subsequent mechanical

filtration should ideally be in the range of 5 - 15 µm. Therefore, all the

methods of preparation of ofloxacin gelatin and albumin microspheres were

subjected to mathematical optimization studies by the “Response Surface

Methodology (RSM)” designed to identify detrimental factors/variables that

influences the particle size. Hence, it was decided to optimize and obtain the

desired particle size range.

Optimization studies helped in obtaining reliable and reproducible procedures

& products, which had desired size 9.10 µm for GLOME-03 (ofloxacin gelatin

microspheres) and 11.32 µm for ALOME-03 (ofloxacin albumin

microspheres).
The optimized formulations were sterilized using γ – irradiation. After

sterilization γ - irradiation studies showed no major structural changes. Most

importantly the particles did not break, were intact and withstood the

irradiation process. A slight increase in the particle size (1.2µm – 3 µm) but

within the required range was observed after sterilization with γ - irradiation.

As the increase was negligible, it can be concluded that gamma irradiation is

the best-suited method of sterilization for microsphere formulations.

The microspheres were characterization by the determination of yield /

recovery, free (un-entrapped) drug, adsorbed drug, entrapped drug, particle

surface morphology, particle size, in-vitro release studies, release kinetics,

differential scanning colorimetry, x-ray diffraction analysis and residual

solvent analysis

The microsphere yield / recovery for GLOME-03 was above 91.5% and

ALOME-03 above 94.48%. Free drug studies revealed that the drug added

during the preparation was not completely incorporated into the microsphere

matrix, there was some amount of un-entrapped drug which varied between

7.5% in case of albumin ofloxacin microsphere (ALOME -03) formulations and

9.4% in case of gelatin ofloxacin microsphere formulations (GLOME -03).

Drug adsorption studies showed that the adsorption efficiency of the

formulation varied from 29.07% and 25.05% in case of GLOME-03 and

ALOME-03 respectively.
Drug entrapment studies showed that the drug entrapment efficiency of the

formulations varied from 61.05% and 66.95% in case of Ofloxacin gelatin

microspheres (GLOME -03) and ofloxacin albumin microspheres (ALOME-

03) respectively.

Particle surface morphological study of the microspheres revealed a smooth

surface and was spherical to near spherical, discreet for the microspheres

prepared by emulsion polymerization process. The microspheres were

shriveled when prepared by spray drying method.

The particle size of ofloxacin gelatin and ofloxacin albumin microspheres

ranged from 0.32 to 22 µm and 3 to 35 µm respectively. It was observed that

the size of the microspheres was increased with an increase in the

concentration of the polymer, the size of the microspheres was decreased with

increasing with stirring rate and increase in the concentration of emulsifying

agent decreased the particle size.

All these results indicated that the particle size of the microspheres could be

modified by varying the factors such as concentration of the polymer,

concentration of emulsifying agent and rate of stirring.

Furthermore, considering that microspheres lie within the size range of 5 - 15

µm, which are required for deposition in the lungs all these microspheres can

be used for lung targeting.


Differential scanning colorimetry (DSC) analysis was performed to find out

the physical nature of ofloxacin entrapped in the gelatin and albumin

microspheres and to confirm absence of drug polymer interaction. Curves of

DSC prove that ofloxacin gelatin and albumin microspheres, which were

prepared, were not just a physical mixture, but existed as microsphere. There

were no new peaks and this confirmed that there was no chemical interaction.

X-ray diffraction analysis (XRD) is one of crystallography characterization

tools. The analysis was performed in order to characterize the physical state

of the polymer and drug in the microspheres. Throughout the XRD runs

conducted for ofloxacin bearing albumin and gelatin microspheres, the

characteristic peak of the crystalline drug ofloxacin was not visible in the

spectra. However, the XRD patterns of drug ofloxacin produced a

characteristic peak when analyzed in API (active pharmaceutical ingredient)

form. It was expected the crystalline drug ofloxacin would show its specific

crystal XRD patterns in a microsphere-formulated form, however this did not

occur. These results indicated that drug present in the polymeric microsphere

formulations developed, is either dispersed / entrapped molecularly or present

in an amorphous form in the polymer matrix. The data from XRD runs also

supports the DSC studies in terms of parallel results in form of absence of

endothermic peaks pertaining to drug in a microsphere form, which points out

that the drug is indeed present in a entrapped form.

Residual solvent analysis was carried out by Gas Chromatography to

determine the concentration of the organic solvent in the microsphere


formulations. Acetone was the organic solvent tested for. The concentration

of acetone was 1900 PPM and 2000 PPM for GLOME -03 and ALOME -03

respectively. The organic solvents were well below the permissible ICH limits

for residual solvents (5000 ppm for acetone), and the formulation are safe.

Stability studies were conducted as per ICH guidelines and accelerated

stability study protocol. The drug content in long-term storage conditions did

not vary to a large extent in the microsphere formulations; the maximum

variation of 2.47% from the initial concentration was seen in case of gelatin

ofloxacin microsphere formulation In case of albumin ofloxacin microspheres

the maximum variation of 2.14% was observed, a re-test date of 2 years from

the date of manufacture was fixed, while a re-test date of 1 year was fixed for

ofloxacin microspheres of albumin and gelatin.

The formulations were stable for the 12-month period in Accelerated storage

conditions of 25 ± 2 0 C and 60 ± 5% RH. The drug content of the microspheres

formulations did not vary to a large extent. A maximum decrease of 3.5%

from the initial concentration was observed in both GLOME - 03 and ALOME

-03. Minor changes in particle size were noticed during the storage time while

the stability studies were conducted, however the changes were found

negligible and have no impact on the quality of the formulations.

The in vitro release studies helps us to understand the behavior of these

systems in terms of drug release. The pattern of GLOME - 03 and ALOME-

03 was observed to be in a bi-phasic manner characterized by a burst effect


followed by a slow release. The drug release profile of GLOME- 03 was

99.3% at 6 h and ALOME- 03 was 91.62% at 12h.

About 42% of the total ofloxacin in GLOME- 03and ALOME- 03 released in

the first one hour, which reflected the significant amount of ofloxacin

adsorbed on or incorporated near the surface of the microspheres. In clinical

practice this would lead to ‘burst effect’, which enables the preparation to

show fast effect to the patients. Although the ofloxacin release from gelatin

microspheres and albumin microspheres was completed during the time

course of the experiment 12 h in case of GLOME- 03 and 6 h in case of

ALOME- 03. During the same period, the released amount was 99.3% and

91.62% respectively. The results indicated that the GLOME-03 and ALOME-

03 had a well-controlled release efficacy.

The data obtained form in-vitro release studies were fitted to various kinetic

equations to determine the mechanism of drug release and release rate using a

computer program sigma plot version 9.01. The co-relation Coefficient value

R 2 is taken into account to decide upon the relevance of the model / curve fit

which will best describe the extent of fit. According to the R 2 values given by

different data fits for ALOME - 03, the Peppas model was to be an ideal fit

having R 2 = 0.9868. According to Peppas fit, the release of the drug is decided

upon by the diffusion of the polymeric matrix and the drug release is

governed by a variation of Fick’s law of diffusion.


The ofloxacin albumin microspheres (ALOME- 03) releases the drug by a

diffusion process which releases the drug from the polymeric matrix based

upon the extent of diffusion, erosion of polymeric matrix and subsequent

domain separation of drug due to diffusion might also be a possibility.

Due to this reason the release profile shows an initial surge in the release

pattern due to swelling of the matrix (above 50% in the first 1.5 hours in this

case) - releasing the adsorbed drug particles on the microsphere surface and

there after by diffusion and there after a more controlled release pattern

In case of ofloxacin gelatin microsphere (GLOME- 03) the ideal fit seems to

be Peppas model having R 2 = 0.9970. The release profile shows an initial

surge – “burst release” (53 % in the first 2 hours) the release pattern due to

swelling of the matrix - releasing the adsorbed drug particles on the

microsphere surface and there after by diffusion and there after a more

controlled release pattern. Due to this the release pattern is be sustain for a

longer time line producing a control over the release profile of the

microsphere in vitro.

In vivo tissue distribution studies in an animal model (albino mice) were

carried out, to prove the hypothesis of passive targeting of microsphere

formulations to the lungs and compared with the conventional dosage of the

drug.
The organ targetability ofloxacin gelatin microspheres showed the largest

value of AUC and r e (time-averaged relative drug exposure) for the lung; the

targeting efficacy t e of lung increased by a factor of 48 (compared with

spleen) ~16.9 (compared with liver) ~33.7 (compared with ofloxacin),

following the I.V. administration of ofloxacin as a solution and ofloxacin

gelatin microspheres (GLOME- 03). Drug distribution to the lungs was

90.98%.

The organ targetability ofloxacin albumin microspheres showed the largest

value of AUC and r e (time-averaged relative drug exposure) for the lung; the

targeting efficacy t e of lung increased by a factor of 35 (compared with

spleen) ~18.5 (compared with liver) ~25 (compared with ofloxacin),

following the I.V. administration of ofloxacin as a solution and ofloxacin

albumin microspheres (ALOME- 03). Drug distribution to the lungs was

91.7%.

Histopathological studies evidence the safety of the formulations after

injecting into the animal. The cytoarchitecture of the tissues (lungs, liver &

spleen) of animals administered with these microsphere formulations did not

show any degenerative changes when compared to that of the control animals.
7. CONCLUSION

Feasibility of formulations of ofloxacin as microspheres was assessed using

biocompatible, biodegradable polymers such as albumin and gelatin from two

different methods, viz. Spray-drying and emulsion polymerization, from

which emulsion polymerization method was selected due to its ease of

preparation and efficiency.

The prepared microspheres were found to posses suitable physico-chemical

properties and the particle size range was ensured by optimization for both

albumin and gelatin microspheres. The microspheres were found to release

the drug to a maximum extent in the target tissue, lungs.

This work has provided an addition to the already significant domain to

targeted drug delivery systems, which holds a very promising alternative over

the conventional means with which the most despondent and perplexive to

treat infections such as pneumonia can be combated.

The study has employed the use of current-generation quinolone – ofloxacin,

in intracellular therapy which otherwise was not favored due to the drawbacks

of low intracellular penetration and higher dosing and subsequent emergence

of resistance. The targeting capability of these microspheres avoids the use of

higher dose, which are known to cause ADRs and the unnecessary exposure of

patients leading to resistance and reduce the frequency of administration

leading to patient compliance.


The study has also created a new vista in pneumonia pharmacotherapy in form

of microspheres - by finding an innovative way for administering one of the

potential drugs of choice in the therapy of pneumonia.

The findings of the work can also be applied for developing effective blue-

print for targeted organ specific drug delivery; and the corollary of this work

can be used as intracellular therapy with other drugs, by a slight modification

in the techniques employed in the preparation of microspheres.

The major conclusion of this work is that microspheres constitutes a useful

means of passive targeting of drugs to the target organ and can be employed

for effective anti-bacterial therapy.


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9.1. LIST OF PAPERS PRESENTATION

International
1. Drug targeting to lungs by way of microspheres. International congress of

Indian Pharmacy Graduates. TAMIL NADU, 2003.

2. Ofloxacin delivery to the lung by way of microsphers, Visakhapatnam,

International Convention of APTI, 2nd & 3rd October 2004.

National

1. Drug targeting to lungs. 54th I.P.C at PUNE from 13th –15th December

2002.

2. Microspheres targeting to lungs, 7th Annual National Convention of APTI on

September 20th & 21st at GOA, 2003.

3. Preparation and in-vitro evaluation of ofloxacin loaded albumin

microspheres by spray-drying technique. 56th I.P.C at KOLKATA from 3rd –

5th December 2004.


LIST OF TABLES

Sl # Table # Title Page #

Microspheres Characterization – Parameters &


1 1.1 25
Methods
Statistical (Regression) analysis of calibration
2 4.1.1 68
curves

3 4.2.1 Variables operating range for gelatin microspheres 78

4 4.2.2 Variables operating range for albumin microspheres 79

Coded Central Composite experimental design for


5 4.2.3 115
three factors
Results for DOE for Ofloxacin Gelatin
6 4.2.4 116
Microspheres
Design Summary for DOE runs of Ofloxacin Gelatin
7 4.2.5 117
Microspheres
Anova for Ofloxacin Gelatin Microsphere -
8 4.2.6 118
Quadratic Model

9 4.2.7 Index of formulatory abbreviations 123

Numerical optimization solutions Ofloxacin Gelatin


10 4.2.8 123
Microsphere
Optimized formulae & formulation code of
11 4.2.9 125
ofloxacin Gelatin Microspheres

12 4.2.10 GLOME Predicted Vs Actual 127

Coded Central composite experimental design for


13 4.2.11 129
three factors
Results of the design of Ofloxacin Albumin
14 4.2.12 130
Microspheres
Design Summary for DOE runs of Ofloxacin
15 4.2.13 131
Albumin Microspheres

16 4.2.14 Anova for Ofloxacin Albumin Microspheres 132

Numerical optimization of solutions of Ofloxacin


17 4.2.15 137
Albumin Microspheres
Optimized formulae & formulation codes of
18 4.2.16 139
Ofloxacin Albumin Microspheres
19 4.2.17 ALOME Predicted Vs Actual 141

20 5.1.1 Recovery / Yield Studies of microspheres 142

21 5.1.2 Entrapment efficiency - amount of free drug 143

22 5.1.3 Entrapment efficiency - amount of adsorbed drug 143

23 5.1.4 Entrapment efficiency - amount of entrapped drug 143

Average particle size of ofloxacin gelatin


24 5.1.5 151
microspheres and ofloxacin albumin microspheres
Drug release profile of ofloxacin from albumin
25 5.1.6 152
microspheres (ALOME -03) as a function of time
In vitro curve fits for various release systems for
26 5.1.7 152
albumin microspheres
Drug release profile of ofloxacin from gelatin
27 5.1.8 157
microspheres as a function of time
In vitro curve fits for various release systems for
28 5.1.9 157
gelatin microspheres
Residual solvent analysis of microsphere
29 5.1.10 182
formulations
Observations of stability test studies in accelerated
30 5.2.1 185
storage condition (25 ± 2 0 C and 60 ± 5% RH)
Observations of stability test studies in real-time
31 5.2.2 186
storage condition (5 ± 3 0 C)
Percentage drug distributed to various tissues of
32 5.3.2 albino mice following the I.V. administration of 191
GLOME-03
Percentage drug distributed to various tissues of
33 5.3.3 albino mice following the I.V. administration of 191
ALOME-03
Lung-targeting parameters of GLOME-03 and
34 5.3.4 193
Ofloxacin after I.V. of mice (n=3)
Lung-targeting parameters of ALOME-03 and
35 5.3.5 194
Ofloxacin after I.V. of mice (n=3)

36 5.3.6 Pharmacokinetics parameters of GLOME-03 196

37 5.3.7 Pharmacokinetics parameters of ALOME-03 197


LIST OF FIGURES

Figure # Title Page #

1. 1.1 Microspheres - schematic representation 12

2. 1.2 Microencapsules - schematic representation 12

3. 1.3 Factors influencing the microspheric drug release 16

4. 1.4 Schematic representation of spray drier assembly 21

Schematic representation of microsphere formation


5. 1.5 24
through emulsion polymerization process

6. 4.1.1 Calibration curve of ofloxacin in 0.1N HCL 68

3D RSM graph of ofloxacin gelatin microsphere –


7. 4.2.1 120
factor: polymer concentration
3D RSM Graph of ofloxacin gelatin microsphere -
8. 4.2.2 121
factor: amount of emulsifying agent
3D RSM graph of ofloxacin gelatin microsphere -
9. 4.2.3 122
factor: stirring rate

10. 4.2.4 Parameters suggestions for GLOME -01 126

11. 4.2.5 Parameters suggestions for GLOME –02 126

12. 4.2.6 Parameters suggestions for GLOME –03 127

RSM Graph of ofloxacin albumin microsphere - factor :


13. 4.2.7 134
amount of emulsifying agent
RSM graph of albumin ofloxacin microsphere factor:
14. 4.2.8 135
polymer concentration
RSM graph of ofloxacin albumin microsphere factor -
15. 4.2.9 136
stirring rate

16. 4.2.10 Parameters suggestions for ALOME-01 140

17. 4.2.11 Parameters suggestions for ALOME-02 140

18. 4.2.12 Parameters suggestions for ALOME-03 141


19. 5.1.1 Scanning electron micrograph of ofloxacin 145

20. 5.1.2 Scanning electron micrograph of gelatin 145

21. 5.1.3 Scanning electron micrograph of albumin 146

Scanning electron micrograph of spray dried gelatin


22. 5.1.4 146
microsphere
Scanning electron micrograph of spray dried albumin
23. 5.1.5 147
microsphere
Scanning electron micrograph of blank gelatin
24. 5.1.6 microspheres by emulsion polymerization before γ- 147
sterilization
Scanning electron micrograph of blank albumin
25. 5.1.7 microspheres by emulsion polymerization after γ- 148
sterilization
Scanning electron micrograph of blank gelatin
26. 5.1.8 microspheres by emulsion polymerization after γ- 148
sterilization
Scanning electron micrograph of blank albumin
27. 5.1.9 microspheres by emulsion polymerization after γ- 149
sterilization
Scanning electron micrograph of GLOME by emulsion
28. 5.1.10 149
polymerization
Scanning electron micrograph of ALOME by emulsion
29. 5.1.11 150
polymerization
In vitro release profile of ofloxacin albumin
30. 5.1.12 153
microsphere - first order release pattern
In vitro release profile of ofloxacin albumin
31. 5.1.13 153
microsphere - higuchi release pattern
In vitro release profile of ofloxacin albumin
32. 5.1.14 154
microsphere - Hixon - Crowell pattern
In vitro release profile of ofloxacin albumin
33. 5.1.15 154
microsphere - Peppas Model
In vitro release profile of ofloxacin albumin
34. 5.1.16 155
microsphere - Baker - Lonsdale plot
In vitro release profile of ofloxacin gelatin microsphere
35. 5.1.17 158
–Baker and Lonsdale
In vitro release profile of ofloxacin gelatin microsphere
36. 5.1.18 158
– Peppas
In vitro release profile of ofloxacin gelatin microsphere
37. 5.1.19 159
- Hixon and Crowell
In vitro release profile of ofloxacin gelatin microsphere
38. 5.1.20 159
- Higuchi, Square Root Time
In vitro release profile of ofloxacin gelatin microsphere
39. 5.1.21 160
- First Order

40. 5.1.22 DSC thermogram of ofloxacin in API form 164

41. 5.1.23 DSC thermogram of gelatin in polymer form 166

42. 5.1.24 DSC thermogram of ofloxacin gelatin microspheres. 167

DSC thermogram of ofloxacin – gelatin physical


43. 5.1.25 168
mixture 1:1 ratio

44. 5.1.26 DSC thermogram of albumin in polymer form 169

45. 5.1.27 DSC thermogram of ofloxacin albumin microspheres 170

DSC thermogram of ofloxacin – albumin physical


46. 5.1.28 171
mixture 1:1 ratio

47. 5.1.29 The characteristic XRD pattern obtained for ofloxacin 173

48. 5.1.30 X-Ray Diffraction pattern of albumin 174

49. 5.1.31 X-Ray Diffraction pattern of gelatin 175

X-Ray Diffraction pattern of gelatin ofloxacin


microsphere showing the absence of characteristic
50. 5.1.32 176
crystal diffraction pattern of ofloxacin due to its
entrapment in the polymeric matrix.
X-Ray Diffraction pattern of albumin ofloxacin
microsphere showing the absence of characteristic
51. 5.1.33 177
crystal diffraction pattern of ofloxacin due to its
entrapment in the polymeric matrix.

52. 5.1.34 Infrared spectra of albumin 179

53. 5.1.35 Infrared spectra of gelatin 179

54. 5.1.36 Infrared spectra of ofloxacin: albumin mixture 180


55. 5.1.37 Infrared spectra of ofloxacin: gelatin mixture 180

56. 5.1.38 Infrared spectra of ofloxacin 181

Drug content plot of ALOME - 03 in accelerated


57. 5.2.1 189
storage conditions
Drug content plot of GLOME - 03 in accelerated
58. 5.2.2 189
storage conditions
Drug content plot of ALOME - 03 in real time storage
59. 5.2.3 190
conditions
Drug content plot of GLOME - 03 in real time storage
60. 5.2.4 190
conditions

61. 5.3.1 Two-Compartment model i.v. Bolus 195

Two-Compartment model following I.V.bolus


62. 5.3.2 196
administration for GLOME-03
Two-Compartment model following I.V.bolus
63. 5.3.3 197
administration for ALOME-03

64. 5.4.1 Cytoarchitecture of lung (control) 198

Cytoarchitecture of lungs (ofloxacin gelatin


65. 5.4.2 microspheres prepared by emulsion polymerization 199
method)
Cytoarchitecture of lungs (ofloxacin albumin
66. 5.4.3 microspheres prepared by emulsion polymerization 199
method)

67. 5.4.4 Cytoarchitecture of liver (control) 200

Cytoarchitecture of liver (ofloxacin microspheres –


68. 5.4.5 200
gelatin emulsion polymerization method)
Cytoarchitecture of liver (ofloxacin albumin
69. 5.4.6 201
microspheres – emulsion polymerization method)

70. 5.4.7 Cytoarchitecture of spleen (control) 201

Cytoarchitecture of spleen (ofloxacin gelatin


71. 5.4.8 202
microspheres – emulsion polymerization method)
Cytoarchitecture of spleen (ofloxacin albumin
72. 5.4.9 202
microsphere – emulsion polymerization method)

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