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This is to certify that the
thesis entitled

ENGINEERED POLYSACCHARIDE CARBOXYLATES
MATRICES FOR OCULAR DRUG DELIVERY

presented by

Laura Marie Fisher

has been accepted towards fulfillment
of the requirements for the

M. S. degree in Chemical Engineering

I Major Professor’s Signatur‘eI

May 14, 2004

Date

MSU is an Affinnative Action/Equal Opportunity Institution

r— -v- f ' “r

 

LIBRARY
Michigan State
University

 

 

 

I PLACE IN RETURN Box to remove this checkout from your record.
To AVOID FINES return on or before date due.
I MAY BE RECALLED with earlier due date if requested.

 

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

6/01 c1CIFIC/DatoDuo.p65-p.15

ENGINEERE

De;

ENGINEERED POLYSACCHARIDE CARBOXYLATE MATRICES F OR
OCULAR DRUG DELIVERY

By

Laura Marie Fisher

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE

Department of Chemical Engineering and Material Science

2004

 

 

ENGINE

Two to th
beta blocl
goal of th
drug deli‘
Sustainec
infection
that has 1
overall a
Product ‘
provide ;
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temperal
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a third 0

ABSTRACT

ENGINEERED POLYSACCHARIDE CARBOXYLATE MATRICES FOR
OCULAR DRUG DELIVERY

By

Laura Marie Fisher

Two to three million Americans suffer from glaucoma. There exist treatments such as
beta blockers, but their ability to deliver drugs to the eye is minimal (<5%). The overall
goal of this project is to develop a biocompatible, biodegradable, in situ- gelling, ocular
drug delivery system using proprietary cellulosic/starch carboxylate copolyrners.
Sustained drug delivery in topical formulations for treating glaucoma, inflammation,
infection and dry eye will be targeted. There is a need for an ocular drug delivery system
that has the characteristics of long retention time, ease of use, ease of manufacture and
overall acceptance by the patient as evident by many review articles on the subject. This
product would be formulated with current topical ophthalmic drug preparations to
provide a longer retention time reducing the overall dosing frequency and increasing
bioavailability of the drugs. It would use the physiological properties of the eye (pH,
temperature and ionic strength) to turn a clear, topical application into a gelling matrix
when placed in the eye. The kinetics of the starch and cellulose oxidation are shown to fit
a third order model. Drug release studies were conducted using ofloxacin and the

matrices are shown follow Higuchi’s model for diffusion from a hydrogel.

 

I would IiI
on such an
guidance ‘
Ian McLe
Sunder B
to thank I
business

me in thc
Sunder, :

all of thc

ACKNOWLEDGEMENTS

I would like to first thank my advisor, Dr. Ramani Narayan, for the opportunity to work
on such an interesting project both in the lab and in the business world. For giving me
guidance with understanding the medical aspects of the project, I would like to thank Dr.
Ian McLennan and Dr. Charles Ngowe. For help in the lab, I would like to thank Dr.
Sunder Balakn'shan and Anna Wolfson. For help with all business aspects, I would like
to thank Dr. Karen Bantel who supported me through the exercise of writing a full
business plan. I would also like to thank all of my friends in the department for helping
me in the many ways that you did, especially Chisa, Christina, Dana, Dina, Phuong,
Sunder, and Yogi. And finally I would like to thank my parents and my fiance, Joe, for

all of the support they have provided me.

iii

List OI T:
List Of F i

Chapter l
1.1 Ob.
1.2 Org

Chapter 1
2.1 DI'I
2.1.
2.1.2

2.2 Oc

TABLE OF CONTENTS

 

 

 

 

 

 

 

 

List Of Tables vi
List 01' Figures vii
Chapter 1. Introduction 1
1.1 Objectives ............................................................................................................. 1
1.2 Organization Of The Thesis .................................................................................. 2
Chapter 2. Drug Delivery Systems 3
2.] Drug Delivery Principles ..................................................................................... 3
2.1.1 Drug Delivery Systems ................................................................................. 7
2.1.2 Mechanisms and mathematical models for drug release from
gel-forming matrices ....................................................................................... 9
2.2 Ocular Delivery Systems ...................................................................................... 12
2.2.1 Bicavailability ............................................................................................... 13
2.2.2 Materials ....................................................................................................... 20
Chapter 3. Engineering Carboxy Containing Flexible Chain Segment
Polysaccharides 25
3.1 Synthesis ............................................................................................................... 25
3.2 Kinetics of the periodate reaction ......................................................................... 31
Chapter 4. Analytical Methods 33
4.1 FTIR ...................................................................................................................... 33
4.2 Titration ................................................................................................................. 33
4.3 In vitrc studies UV-Visible spectroscopy ............................................................. 33
4.4 ESEM .................................................................................................................... 36
Chapter 5.Dicarboxy Matrix Synthesis and Characterization 37
5.1 Oxidation Methods ................................................................................................ 37
5.2 Polysaccharide Choice .......................................................................................... 39
5.3 Titration ................................................................................................................. 40
5.4 Periodate Oxidation Kinetics Data ........................................................................ 44
5.5 Design of Experiments .......................................................................................... 47
5.6 ESEM .................................................................................................................... 52
Chapter 6. Synthesis of Grafted Dicarboxy Starch and Characterization ............... 54
6.1 Background ........................................................................................................... 54
6.2 Results ................................................................................................................... 57
Chapter 7. Drug Release Profiles 58
7.1 Drug Release Results ............................................................................................ 58
7.2 Modeling the Diffusion Method ........................................................................... 63

iv

Chapter
8.1 Cc
8.2 Ki

Chapter
9.1 O\
9.2 Pa

Referenc

66

 

Chapter 8. Conclusions and Recommendations

 

8.1 Conclusions ........................................................................................................... 66
8.2 Kinetic considerations and scale up ...................................................................... 67
Chapter 9. Examination of business potential 70
9.1 Overview of market .............................................................................................. 70
9.2 Path to commercialization ..................................................................................... 80
References 89

 

 

Table 1.
Table 2.
Table 3.
Table 4.
Table 5. '
Table 6. '
Table 7. I
Table 8. I
Table 9. I
Table 10.
Table 11.

Table 12,

 

LIST OF TABLES

Table 1. Glaucoma Treatments And Their Method Of Action ...................................... 17
Table 2. Common Ocular Treatments .............................................................................. 18
Table 3. Ocular Drug Delivery Systems ......................................................................... 23
Table 4. Explanation Of Oxidation Methods .................................................................. 37
Table 5. The Results Of The Oxidation Of Different Saccharides .................................. 40
Table 6. Titration Results ................................................................................................. 42
Table 7. Calculated Rate Constants For The Periodate Oxidation Of Starch .................. 46
Table 8. Reaction Specifications ...................................................................................... 67
Table 9. Competitors In The Area Of Ocular Drug Delivery .......................................... 76
Table 10. Customer Profile .............................................................................................. 79
Table 11. Path To Commercialization ............................................................................. 84
Table 12. Projected Product Development Timeline ...................................................... 86

vi

I:

m."

Figure 1. I
Figure 2. I
Figure 3. E
Figure 4. I
Figure 5. '
Figure 6.

Figure 7. '
Figure 8. I
Figure 9. I
Figure 10.
Figure 11.
Figure 12.
Figure 13.
Figure 14.
l:igure 15_
Figure 16_
Figme 17,
Figure 18.
Figure 19.
Figure 20.
Figure 21.

FIguI-e 22 _

LIST OF FIGURES

Figure 1. Drug Pathway ................................................................................................... 4

Figure 2. Generalized Drug Release Profile .................................................................... 6

Figure 3. Structures Of Some Natural Polysaccharides ................................................... 8

Figure 4. Structure Of The Eye ........................................................................................ 13
Figure 5. Tear Fluid Composition .................................................................................... 13
Figure 6. Drug Pathway In The Eye ............................................................................... 19
Figure 7. Timolol Concentration in Rabbit Aqueous Humor .......................................... 21
Figure 8. Structure Of Starch And Cellulose ................................................................... 26
Figure 9. Oxidation of Starch Reaction Scheme .............................................................. 27
Figure 10. Copolymer Starch Structure ........................................................................... 28
Figure 11. 3D Structure Of 50% Dicarbcxy Starch ......................................................... 29
Figure 12.Possible Structures of Periodate Oxidized Starch ........................................... 30
Figure 13. Stir Plate Dissolution System ......................................................................... 34
Figure 14. USP Dissolution Bath For Drug Release Studies ........................................... 35
Figure 15. FTIR Comparison Of Oxidation Methods ...................................................... 38
Figure 16. FTIR Of A Possible Oxidized Hemialdol Structure ...................................... 39
Figure 17. Carboxyl Content Versus Periodate Ratio ...................................................... 43
Figure 18. Periodate Oxidation Of Cellulose Kinetics .................................................... 44
Figure 19. Periodate Oxidation Of Starch Kinetics ......................................................... 45
Figure 20. Comparison Of Actual And Theoretical Kinetic Data ................................... 46
Figure 21. Relationship Between The Periodate Ratio And The Yield ........................... 48

Figure 22. Relationship Between The Periodate Ratio And The Final Material Acid
Content ........................................................................................................... 49

vii

 

'3'"

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Figure 23. R
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Figure 24. 0

Figure 25. E1
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Figure 26. ES
Figure 27. M
Figure 28. Tr
Figure 29. C b
Figure 30. FT
Figure 31. Str
Figure 32. U\
Figure 33. Cal
Figure 34. Co;
Figure 35. Dn
Figure 36. Re]
FiEuro 37. Re]
Figure 38. sql
Figure 39. Call
Figure 40' Lar
Figure 41. Dr

 

Figure 23. Relationship Between The Periodate Ratio And The Dispersibility Of The

Material .......................................................................................................... 50
Figure 24. Optimization Of The Periodate Concentration ............................................... 51
Figure 25. ESEM Images Of Starch, Air Dried Dicarbcxy Starch And Hydrated

Dicarbcxy Starch ........................................................................................... 53
Figure 26. ESEM Of Native Starch Compared To Maleated Thermoplastic Starch ....... 55
Figure 27. Maleation Of Starch ....................................................................................... 55
Figure 28. Transesterfication Reaction With Maleated Starch ........................................ 56
Figure 29. Chemical Structure Of Polycaprolactone ....................................................... 56
Figure 30. FTIR Of Starch And MTPS ............................................................................ 57
Figure 31. Structure Of Ofloxacin ................................................................................... 58
Figure 32. UV-VIS Curve for Ofloxacin ......................................................................... 59
Figure 33. Calibration Curve For Ofloxacin .................................................................... 60
Figure 34. Comparison of dicarboxy starch to gelrite ..................................................... 60
Figure 35. Drug release of various materials ................................................................... 61
Figure 36. Release profiles using USP dissolution system .............................................. 62
Figure 37. Release with varying drug concentrations ...................................................... 62
Figure 38. Square Root Time Dependence Of The Ofloxacin ......................................... 63
Figure 39. Calculation of the diffusion coefficient .......................................................... 64
Figure 40. Large scale reaction scheme ........................................................................... 66
Figure 41. Drug delivery growth .................................................................................... 69
Figure 42. Largest pharmaceutical companies with ocular drugs .................................... 77

viii

' 5.

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Chapter 1. Introduction

Like many current areas of scientific research, the development of drug delivery
matrices lies at the intersection of two traditional disciplines. Applying traditional
material science and chemical engineering knowledge to pharmaceutical knowledge has
led to the development of many new drug delivery systems within the last decade.
Pharmaceutical companies have found that they can reformulate current pharmaceutical
compounds to have better efficacy, bioavailability and fewer side effects by improving
the method of delivery. The many methods of drug delivery include transdermal,
subcutaneous, intravenous, and intramuscular delivery (Saltzman). One specific current
area of interest to pharmaceutical companies is ocular drug delivery because of the wide
gap between the amount of drug administered and amount of drug that reaches the

targeted site.

1 .1 Objectives

The primary objective of the thesis is to evaluate the potential of using a
carboxylated polysaccharide as an ocular drug delivery matrix that provides a controlled
release gel when placed in the eye. The overall goal is increasing the bioavailability of
the drug in the anterior region of the eye. It is known that carboxylated polysaccharides
have the properties of being water dispersible and biocompatible. This is why materials
containing carboxylated flexible chain segments were chosen to be studied. The
objective the work presented here is attained by:

0 Carboxylating starch and cellulose using different methods of oxidation;

0 Characterizing the materials synthesized using titration, FTIR; and,

12 Organ

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need of new
regarding mi

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0 Conducting in vitro release studies of ofloxacin using the developed

matrices.

1.2 Organization Of The Thesis

The thesis is divided into three main parts. The first part, Chapter 2, reviews the
need of new drug delivery systems and reviews overall advances. It proceeds into detail
regarding the need for new ocular delivery systems.

The second part, Chapters 3 through 8, details the experiments performed to
evaluate a newengineered carboxylated polysaccharide system, in which the carboxy
groups are on flexible chain segments. Flexible chain segments are important to design
into the system because we want to use the carboxy groups to chelate with Ca ions and
form in-situ clear gels.

The third part, Chapter 9, explores the commercial aspects of developing an

improved ocular drug delivery system.

 

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Chapter 2. Drug Delivery Systems

Therapeutics can only be effective if they reach their intended site of action. For
treatments such as topical cortisones applied to a rash on the skin, this is not difficult; but
for many other treatments it can be very difficult due to such factors as absorption,
distribution, binding, biotransformation and excretion. This chapter will give an overview
of general drug delivery principles and techniques in the first section and then focus on

issues peculiar to ocular drug delivery in the second section.

2. 1 Drug Delivery Principles
Drug delivery involves getting a treatment to the site of action. This can be as

simple as applying a cream transderrnally, with the skin being the site of action, or as
complicated as having a chemotherapy agent injected intravenously and reaching the
intended cancerous lung cells. In all cases the drug must pass through membranes, and
then be carried to the site of action. Cell membrane transport can be either passive or
active. In passive transport systems, the approach is diffusion through a membrane due to
a concentration gradient and is influenced by size and concentration; while active
transport involves some type of biological assistance such as ‘tricking’ the body into
thinking a drug is another needed structure.

Figure 1 schematically describes the relationship among the factors affecting
drugs in the body. All of these factors are influenced by the characteristics of the drug
including molecular size and shape, solubility at the site of absorption, degree of

ionization, and relative lipid solubility of its ionized and nonionized forms which will

affect mcmi
the bioavail;
of action or
example, if .-

the site of ac

ABSOR

 

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affect membrane transport (F echner, Teichmann et al. 1998). All of these factors affect
the bioavailability of a drug and describe the extent to which a drug itself reaches its site
of action or a biological fluid transporting the drug can reach the site of action. For

example, if a drug first reaches the liver where it is mostly metabolized before it reaches

the site of action, it would have very low bioavailability.

 

 

 

 

 

 

 

 

 

SITE OF TISSUE
ACTION RESERVOIR
h J

 

l/

r ABSORPTION 7 -————p Free Drug 1:2 BOUND

/ Ventral Compartment

I METABOLISM bl Elimination

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 1. Drug Pathway
Solubility plays an important factor in drug administration because of the cellular

transport required. A cell membrane consists of a lipid bilayer, while cellular fluid is
more polar. Most drugs are weak acids or bases that are present in solution as both the

nonionized and ionized species. The nonionized molecules are usually lipid soluble and

diffuse across the cell membrane, while the ionized forms are usually unable to penetrate

the cell membrane because of their polarity.

If a drug has a low bioavailability, it may have to be administered in a large dose
to ensure the required dose will reach the site of action. This high dose is problematic if
the drug has other known side effects. Therefore, a constant steady-state dose is
preferred over a large one-time (bolus) dose. This steady-state can be reached by
frequent repeated administrations. Once a drug is administered, its half—life will
determine how long it will remain in the body and indicate how frequently a drug must be
administered to reach a steady-state concentration. Figure 2 illustrates the
pharmacokinetic relationships between dosing and steady-state. The average steady-state
concentration, C5,, can be found using the following equation:

C,,=F*dose/(C1*T) (1).
where:

F = the fractional bioavailability

T = the dose interval (time)

C1 = the clearance rate
Dose = the concentration

 

   

 

Steady-state concentrations

 

 

 

Concentration in the body

 

 

 

Time (multiples of elimination half-life)

Figure 2. Generalized drug release profile

The concentration of the drug that is required at steady state is dependent on the dose-
response effect of the drug which is the relationship between the amount of drug applied
and the measured effect (Jose, Polse et al.). Typically, as the drug concentration is
increased, the therapeutic benefit increases until a certain point called the maximum
effective dose is achieved. Another important property necessary for correct dosing
procedures is the time-response curve of a drug. Certain drugs may have a lag time before
they become effective, while others may instantaneously act. Knowing these
pharmacodynamic parameters of a drug is necessary to determine when to expect the
desired effect and whether additional doses are necessary. Those properties are found

experimentally in clinical trials and are not examined in this research.

2.1.1 Drug Delivery Systems

Briefly, this section will describe some current methods and materials being used
to obtain a sustained delivery. Hydrogels and other biodegradable polymers will be
discussed briefly. The use of biodegradable polymers for site-specific drug delivery has
attracted much research.

Natural and modified natural gums (Bhardwaj, Kanwar et a1.) have been widely
used in pharmaceutical applications because of their wide use in the food industry and
their general recognition as safe. Materials that have been widely studied include sodium
alginate, carrageenans, cellulose ethers, chitosans, guar gum and modified starches. In
general, these polysaccharides have significant quantities of oxidized groups in addition
to their normal polyhydroxy format. Please see Figure 3 for representative structures.
Simply put, they function by hydrating and forming a gel when in contact with water.
The drug contained in the matrix is expected to release through the gel layer providing a
constant-rate sustained release. Some polysaccharides, such as sodium alginate, form
gels under more specialized conditions such as the addition of calcium ions and

sensitivity to gelling at certain pH levels.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

OCH 0
Alginic acid
Ho‘“
OH
C 00” c 00 CH3
0 H / Pectic Acid
0H 0 H
l.— _ n
— CH20H 000' M+ CH20H
_n
OH OH

 

 

 

Figure 3. Structures of some natural polysaccharides
Another interesting area that affects distribution of a drug subsequent absorption

is drug targeting. A drug delivery matrix can actively target a site such as “cancer”-
labeled chemotherapy agents or passively target an area, such as enteric coated drugs that
resist destruction in the stomach and that will release in the intestines where they can be
absorbed. This is a large area of active research, but will not be reviewed further in this
paper.

Hydrogels (Peppas) have been studied because of their ability to swell in water or
biological fluids. Hydrogels that have been studied for drug delivery applications include
ones related to poly(2-hydroxyethylmethacrylate) or HEMA, poly(ethyleneoxide), and
various cellulose derivatives. Hydrogels are particularly useful in areas where there is a
high moisture area such as in wounds, for vaginal treatments and drugs that act in the

stomach. Peppas did an extensive review of water-swollen cellulose derived hydrogels

and their applications, specifically related to prolonged and controlled release
applications. Specific to this research, Peppas notes that treated cellulose with higher
carbonyl and carboxy] content are claimed to be more stable, to exhibit more regular and
sustained release properties and to be effective at a much lower concentration. It is also
noted that carboxyl-containing derivatives have stronger muco-adhesive capacity which
is important in this research with regards to increasing retention time in the eye as the eye
contains a mucin layer on the surface. Data from a controlled release study of diazepam
in a 15-mg tablet show that the controlled release formulation administered once daily
was as effective as five doses of a S-mg unfonnulated tablet. This reduction of dosing
frequency from five times per day to one time a day can increase patient compliance.
This study also showed the ability of a controlled release system to lower the side effects
of the drug, in this case minimizing sedation caused by the rapid raise in the plasma level.

2.1.2 Mechanisms and mathematical models for drug release from
gel-forming matrices

Peppas describes the release of drugs from a gel-forming matrix as a three step
process. The first is the initial burst when the liquid dissolves the drug present at the
immediate surfaces of the matrix, creating a small “burst” effect. At this time the water
or biological fluids begin to penetrate the gel at a rate that is dependent on the porosity of
the matrix. The second phase is classified as the stationary phase, where the water
continuously penetrates the matrix at a constant rate. This penetration is accompanied by
an expansion of the gel layer in the direction of the external medium. This phase
accounts for the majority of the drug release. It is generally accepted that the release of

the drug is controlled by diffusion process, not by the rate of drug dissolution or the rate

 

of penetratio
period WhiCI
the drug C0111
the release r;
Thestil
suspended d1
been develor
dissolved drt
the system is

layer present

the dIUg in t1

 

= MW, Wht
time) and V,

valid for 0 S

M

I

The:

(1114

Wher
M,c =

‘ tl
Co: t

‘

m\

of penetration of the front for hydrophilic matrices. The third phase is the exhaustion
period which begins when the penetration fi'ont has reached the center of the matrix and
the drug concentration has dropped below its solubility limit in water. During this stage
the release rate rapidly falls.

These controlled release systems can either be classified as systems with
suspended drugs or systems with dissolved drugs. Mathematical models for both have
been developed and adapted by various authors, and Higuchi’s model for systems with
dissolved drugs can be used for this research. This model is presented for the case that
the system is homogenous, there is one plane of diffusion, there is no diffusion boundary
layer present and there are sink conditions. In this situation, the initial concentration of
the drug in the hydrated matrix is less than the drug solubility in it (Co < Cm). Setting Co
= Moo/V, where M... is the initial drug loading (the total amount of drug release at infinite

time) and V, the effective volume of the hydrated matrix, the following expressions are

valid forOS Mt/ M00506:
1/2
Dm l/2
M, ‘-"—" 2AC0(7] .t (2)

The rate of release is:

1/2
dM, =2ACO(&) .t-l/Z (3)
dt 7:
where:

Mt = the amount of drug released at any time

Moo = the initial drug loading

A= the diffusional area

Co= the initial concentration of the drug in the system
D.In = the apparent diffirsion coefficient

10

These equations are for the planar case, but can be modified for other shapes. All
of these cases will also show a ‘/t dependency. Peppas brings up faults with this model
due to the following assumptions:

1. This model was not developed for systems undergoing dimensional
change.

2. A pseudo steady-state analysis was used which ignores the external mass
transfer resistance and is only valid when the solute loading is in great
excess of it solubility limit.

3. The countercurrent solvent diffusion was not considered

4. Drug diffusion in the gelled matrix was assumed to be the rate limiting
step.

However, the shortcoming of these assumptions are not as much a problem with
the ocular system being described because the gel is already hydrated when applied, will
not undergo dimensional change, and solvent diffusion can be neglected.

Matrix formulations play an important role in the drug release profiles. Release
profiles can usually be modified by type and viscosity of the polymers, the polymer
concentration, and the drug particle size. The type of polymer is ofien determined by the
solubility characteristics of the drug. For example, hydrophilic matrices are generally
used to prolong the release of highly water-soluble drugs. The viscosity of the polymer
appears to play a role in the initial burst period of drug release, but have no effect in the
stationary phase where the majority of release takes place. Polymer concentration
follows the general rule that increasing the proportion of hydrophilic material decreases

the rate of release. Drug particle size affects the dissolution rate of the drug with the

11

smaller particles having larger surface/volume ratios and, therefore, faster dissolutions

rates.

2.2 Ocular Delivery Systems
The specific focus of the paper is drug delivery to the eye, which has been

identified as a crucial area because of the unique physiological and anatomical properties
of the eye. With current topical treatments, only 1%-5% of the drugs even reach the site
of action (Mindel), which is counterintuitive considering the short distance between the
site of application and the site of action. There is a need for an ocular drug delivery
system that has the characteristics of long retention time, ease of use, case of manufacture
and overall acceptance by the patient as evidenced by many review articles on the subject
(Urtti 1995),(Kaur and Kanwar 2002) (Jarvinen, Jarvinen et al. 1995) . Complicating
these research efforts is the fact that there are unique pharmacodynamic,
pharmacokinetic, and drug delivery issues for ocular drug therapy owing to the eye’s
distinctive anatomical and functional properties. Treatment of ocular diseases, especially
diseases of the retina, is often a drug delivery problem. Improving topical applications
would include not only drugs for treatment of retinal degenerative diseases and
glaucoma, but also anti-bacterial agents, corneal wound repair, and intraocular

treatments .

12

Cornea

 

Conjunctiva

Figure 4. Structure of the eye

LIPID LAYER-0.1 urn
consisting mainly of
waxy and cholesteryl
esters and some polar
lipids

Outer

AQUEOUS LAYER ~7
jun containing salts,
glucose, urea, and
proteins in a water base

MUCIN LAYER -~0.02-
0.05 pm containing a
hydrated layer of
mucoproteins

 

Figure 5. Tear fluid composition

2.2.1 Bicavailability
To understand the unique drug delivery issues associated with the eye, the structure of the

eye is the first consideration. Reasons for low bioavailability include the blinking reflex,

13

tear turnover, and low corneal permeability (Burrows, Tsibouklish et al.). Figure 3
shows a schematic cross section of the eye. Treatments for glaucoma, infections and
allergies target the anterior section of the eye, while treatments for retinal diseases must

reach the posterior section of the eye.

Anatomy of the eye
The unique anatomy of the eye must be understood before attempts at drug delivery can
be made. The three areas of interest in the eye with respect to topical application of drugs
are the cornea, the conjunctiva and the nasolachrymal drainage system (Burrows,
Tsibouklish et al.). The cornea is considered to be the main pathway for the permeation
of drugs into the eye. It is approximately 0.5 mm thick in the central region and up to 0.7
mm thick at the periphery. It is an optically transparent tissue that conveys images to the
back of the eye and covers about one-sixth of the total surface of the eyeball. In terms of
drug delivery, the cornea can be considered to comprise three distinct layers, which
accounts for the poor permeability characteristics. First, the outer epithelium is lipophilic
in nature consisting of 5-6 layers of cells and tight junctions, thereby making it the most
significant barrier to drug delivery. The second area is the stroma, which accounts for
approximately 90% of the corneal thickness and which is a relatively open hydrophilic
region. The final important region is the inner endothelium, which consists of a single
layer of flattened cells and which is in direct contact with the anterior chamber. Because
of both hydrophilic and hydrophobic regions, the cornea provides an effective barrier to
drug transport.

The second major region of the eye with importance to drug delivery is the

conjunctiva - a thin, vascularized mucous membrane that lines the inner surface of the

14

eyelids and the outer region of the cornea. It is involved in the formation and the
maintenance of the tear film that coats the outer region of the cornea. The precomeal
region of the human eye has a surface area about 17 times greater than (Jarvinen,
Jarvinen et al.) the cornea and provides an alternative absorption path for drugs applied
topically. Intercellular spaces of the conjuctival epithelium are wider than those in the
corneal epithelium, making permeabilities of hydrophilic drugs typically an order of
magnitude greater than corneal permeabilities. This order of magnitude difference does
not occur for moderately lipophilic, small molecules. Also considered part of the
precomeal region is the tear film which covers the cornea (Figure 5). This region is of
importance because the applied drops will mix with tear film and must be compatible
therewith to reach the cornea. The tear film is approximately 9-10 pm thick and consists
of a superficial oily layer, an aqueous layer with proteins, electrolytes and other small
molecules, and a mucin layer.

The nasolachrymal drainage system accounts for most of the drug loss. It consists
of the secretory, distributive and excretory systems. The excretory part of the
nasolachrymal drainage system includes the lachrymal puncta, the superior, inferior and
common canaliculi, the lachrymal sac and the nasolachrymal duct. It is thought that tears
are largely absorbed by the mucous membranes of the ducts and that only a small amount
reaches the nasal passages (Davies 2000). This leads to another route of systemic
absorption of the applied drugs. The cul-de-sac of the eye normally holds 7-9 pl of tears

and can contain up to 20-30 ul if care is taken not to blink. The normal tear flow rate is 1

ul per minute and the pH is maintained at 6.5-7.6. The high turnover of tear fluid

15

(approximate 15% per minute) and the limited capacity of the sacs lead to a high
clearance rate of drugs once applied.
Eye diseases

Glaucoma, eye allergies and irritation, and eye infections are generally treated
topically. Topical application of drugs is preferred because (Davies): 1)drug effects are
localized and less drugs enter the systemic circulation, 2) it facilitates drug absorption
into the eye that is otherwise hard to target, 3) it avoids hepatic first-pass metabolism and
4) it is a relatively convenient, simple and painless method of administration. Glaucoma
is a group of disorders characterized by progressive damage to the eye at least partly due
to intraocular pressure damaging the optic nerve. It is the second most common cause of
blindness in the USA, with roughly 2 million Americans affected . Because glaucoma
comes in many forms, there is not a universal treatment for it. In general, glaucoma is
caused by disruption of the aqueous outflow of the eye through the anterior chamber
angle by either a physical obstruction or by other factors, such as hypertension or
diabetes. Treatments can be categorized by either increasing outflow or by decreasing
aqueous production. Table 1 shows a list of drugs that are commonly used to treat

glaucoma and their mechanism of action.

16

Table 1. Glaucoma treatments and their method of action

 

 

 

 

 

 

 

 

 

Type Drug Examples Mechanism of Action
Miotics, direct and Pilocarpine Causes miosis, increase
indirect acting Carbachol aqueous outflow, cause
Physostigmine accomodation
Neostigmine
Carbonic anhydrase Acetazolamide Decrease aqueous
inhibitors Dorzolamide production
Non-selective Epinephrine Cause mydriasis,
adrenergic agonists Dipivefiin increase outflow and
decrease fluid
production
org-selective Apracionidine Decrease aqueous
adrenergic agonists Brimonidine production, increase
uveoscleral aqueous
outflow
B-blockers Timolol Decrease aqueous
Betaxolol production, does not
Levobunolol affect pupil size
Prostaglandin analogs Latanoprost Increase uveoscleral
outflow rather than
altering conventional
aqueous outflow
Osmotic diuretics Glycerin Hypertonic plasma
Mannitol(IV) draws fluid from eye

 

 

 

Other common diseases of the anterior section of the eye include (Burrows,

Tsibouklish et al.):

Conjunctivitis - an inflammation of the conjunctiva that may be caused by bacterial and

viral infection, pollen and other allergens, smoke and pollutants.

Dry eye syndrome - the inadequate wetting of the ocular surface.

Keratitis - an inflammation of the cornea, caused by bacterial, viral or fungal infection.

17

 

Iritis (anterior uveitis) - commonly having acute onset with the patient suffering pain and

inflammation of the eye. Other rare conditions include the ophthalmic complications of

rosacea, blepharitis (inflammation of the lid margins) and chalazia (Meibomian cysts of

the eyelid).

Table 2 lists some of the common treatments for these conditions.

Table 2. Common ocular treatments

 

 

 

 

Classification Examples Typical Indications
Antibacterials Chloramphenicol, Conjunctivitis, keratitis,
gentamicin, fusidc acid blepharitis
Anitvirals Aciclovir, idoxuridine Viral infections such as
dendritic corneal ulcers,
keratitis
Corticosteroids Betamethasone, Uvetis, scleritis

prednisolone,
hydrocortisone

 

Local anaesthetics

Amethocaine, lignocaine

Anesthesia during
treatments

 

 

Amt-inflammatory agents

 

Cromoglycate, nedocromil,
antihistamines

 

Inflammation and allergic
conjunctivitis

 

Retinal disorders encompass a group of diseases that are not currently treated

topically but potentially could be if the drug delivery vehicle was improved. Age-related

macular degeneration is the leading cause of visual loss in the elderly.

Reasons for not reaching

While there are many topical applications, all of them in common have very low

bioavailability. The most common method of ocular drug delivery is the instillation of

30-50 pl drops into the lower cul-de-sac. The concentration of the drug in the precomeal

area provides the driving force for its transport across the cornea via passive diffusion.

18

 

Therefore, efficient ocular drug absorption requires good corneal penetration as well as
prolonged contact time with the corneal tissue (Burrows, Tsibouklish et al.). These are
hindered by the rapid solution drainage, systematic absorption through the conjunctiva,
and the limited surface area of the corneal barrier. Figure 6 shows schematically the

competing factors related to topical ocular drug administration.

Drug in dosage Release Drug Corneal Reaches site
form in Tear Fluid absorption of of action
drug

 

 

 

 

 

 

 

 

 

 

Removal by. Removal by:
drainage, drainage, blinking.
blinking. induced lacrimation
induced lacrimation tear tumover and

and tear turnover Conjuntival absorption

 

 

..... .1,

Leads to systemic
absorption and
elimination

 

 

95% of the drug is
lost in the 1m two
steps

 

 

 

 

 

 

 

 

Figure 6. Drug pathway in the eye

Drug release in vivo and precomeal kinetics

An ocular drug must reach its action site in adequate concentration to be efficacious.

The steady-state concentration of a drug in the precomeal tear fluid is a function of drug
release rate in viva, rate of drug clearance via tear turnover, drug clearance from the
lacrimal fluid to the cornea, drug clearance from the lacrimal fluid to the conjunctiva,
drug permeability in the conjunctiva, conjunctiva] surface area, corneal permeability of

the drug and the corneal surface area.

19

If the dosage form flows partly or completely from the eye after administrations, as
viscous vehicles, gelling systems, nanoparticulates and liposomes do, it is very important
that the drug release rate and the rate of dosage form drainage from the eye match each
other. If the release rate fi'om the matrix is too slow and not compensating for the
drainage of the free drug, the overall bioavailability could be reduced. The mechanism
for the increased rate of drug release in lacrimal fluid is not known exactly, so it is

important to collect both in vivo and in vitro data to correctly design a system.

As can be seen by Figure 6, there are many paths leading to systemic absorption. The
main routes of absorption are the nose and conjunctiva (Urtti). It has been shown that by
using a viscous carboxymethyl cellulose vehicle without release rate control, the systemic
peak concentrations of timolol were decreased in rabbits 2-3 times, probably due to the
longer precomeal retention and the slower vehicle spread to the nasal mucosa. This is a
very important finding for some ocular drugs such as beta blockers which can lead to
heart attacks in patients with heart conditions and even for other glaucoma drugs which

may have side effects such as drowsiness

2.2.2 Materials

Natural Polymers
Natural polymers and gums have been used in pharmaceutical formulations of

sustained-release carriers, and modified celluloses; carboxy methylcellulose (CMC) and
MMC are found in a large number of ocular formulations as viscosity enhancers.
Because of the wide acceptance of these modified natural polymers, pharmaceutical

companies are interested in the use of modified natural polymers for their ocular drug

20

delivery systems. Natural polymers with gelling properties that have been successfully
used in ocular topical formulations include gellan gum and carrageenans. Ocular topical
formulations with gelling properties afford increased ocular bioavailability of certain
drugs. Figure 7 below is work done by Vrbanac that shows the increased bioavailability

of timolol, a glaucomic agent, in rabbits with a formulation including Gelrite.

[3-H]T irnolol Concentration
in Rabbit Aqueous Humor
Effect of XE Formulation
50004

—- Timoptic
-V— Timoptic XE

4000‘

 

U

8

‘i’
—>

Cmax
2000-

 

[3-H]Timolol
(pg eqlmL; :1: SEM)

1000‘

 

 

 

o , f

0 1 2

Time (h)

w.
b

Figure 7. Timolol concentration in rabbit aqueous humor

A review of literature and patents shows that much of the focus in this area is
centered around natural polymers. Gelrite®, a registered trademark of Monsanto, is used

by Merck in a preparation of timolol, Timoptic XE. This is the only known in situ

21

gelling drug delivery system currently on the market. According to the package insert it
requires half of the doses as the standard Timoptic. It is a low-acetyl gellan gum which
would have a structure similar to Figure 3 and can ionically crosslink in the presence of
a divalent cation such as calcium. Rozier et al has shown that the in in viva testing,
Gelrite behaved similar to HEC (hydroxyethylcellulose), a known viscosity enhancer. It
significantly reduced intraocular pressure over the HEC, which was determined to be
caused by an increased residence time at the surface of the eye.

Another natural gel-forming polysaccharide is alginate. Cohen et al. describe an
alginate system that gels in the presence of calcium ions in the eye. Alginate is a mixture
of guluronic and mannuronic acids as seen in Figure 3. They suggest using a mixture
with the guluronic acid concentrations higher than 65% to form a suitable gel. When
testing pilocarpine, a common glaucoma treatment, the alginate formulated system
demonstrated a correlation between the gelation capability of the alginate formulation,
the speed at which it occurs and the sustained release properties. It was also claimed that
there was excellent ocular tolerance in the test rabbits; even though redness of the
conjunctivae was reported for 1-2 hours after instillation of the drops.

A final natural polysaccharide that can form gels in situ is pectin (see Figure 3). A
patent filed by Ni and Yates claims that pectin isolated fi'om Aloe Vera, which contains a
higher galacturonic acid ratio will form a gel when subjected to mono- or divalent ions at
a low pectin of concentration of 0.25% w/v. It will also form a gel in the presence of
small organic compounds, proteins, nucleic acid, and live cells.

Gelfoam ® is a structured matrix of gelatin has been studied for the release of

pilocarpine. The matrix is a structured water-insoluble sponge prepared from purified

22

 

 

 

the;
pro

Ho

pork skin gelatin that will biodegrade. Because this simple matrix released most of the
drug within 15 minutes, retardants had to be added. This matrix embedded in cetyl ester
wax demonstrated zero-order release kinetics while the matrix impregnated with
polyethylene glycol 400 monostearate exhibited close to first—order kinetics. The results
show that gelatin itself does not provide for good sustained release. The following table

summarizes work in ocular drug delivery systems.

Table 3. Ocular drug delivery systems

 

 

 

 

 

 

 

Matrix Material I Method of action I Author
Natural Polymers
Alginate Ionic concentration Cohen (Cohen 1998)
Gellan Gum Ionic concentration Rozier(Rozier, Mazuel et a1.
1989)
Pectin Ionic concentration Ni(N i and Yates 2002)
Gelatin Not in situ gelation Nadkami(Nadkami and
Yalkowsky 1993)
Cyclodextrins Not in situ gelation

 

 

 

 

 

 

 

 

 

 

 

 

Synthetic Polymers
Poloxamer Temperature change Lin(Lin and Sung 2003)
Pluronic Temperature change Lin (Lin and Sung 2003)
Carbopol pH change Lin (Lin and Sung 2003)
Cellulose pH change Gurney (Gurney 1986)
acetophthalate
Synthetic polymers

Many synthetic polymers have been tested for sustained release in the eye. While
they have the advantage of being engineered to specific applications, their breakdown
products are not always known potentially leading to extended FDA testing. A patent by
Hong-Ru Lin explains a formulation approach of combining Carbopol and Pluronic.

Carbopol is a high molecular weight carboxy vinyl polymer and Pluronic is a class of

23

block copolymers containing polyoxyethylene and polyoxypropylene. This formulation
claims to be free-flowing at non-physiological conditions (pH 4.0 and 25° C), but
forming a gel at physiological conditions (pH 7.4 and 37° C). A disadvantage to this
system is the high amount of Pluronic (14%) required for optimal gel formation. Again
there are many disadvantages of synthetic polymers including high polymer
concentration, irritancy and potentially harmful breakdown products.

Dicarbcxy polysaccharides were chosen for this work because of their water
dispersibility, their potential muco-adhesive capacity and because their breakdown
products are known and safe. Experimental data by Singh et al. has shown that similar
dicarboxy cellulose matrices are biocompatible and can be broken down in the body by

metabolism into 2-carbon and 4— carbon intermediaries.

24

Chapter 3. Engineering Carboxy Containing Flexible

Chain Segment Polysaccharides

3. 1 Synthesis

The objective of the work is to find a flexible, water dispersible, biocompatible
material that would have the ability to encapsulate a drug in the physiological conditions
of the eye. The natural polysaccharides listed in the previous chapter exhibit some of
these characteristics such has biocompatibility and water dispersibility but they were not
optimized for the conditions of the eye. The carboxyl content of these polysaccharides
were improvements could be made. It was determined that engineering carboxy groups
onto a natural polysaccharide backbone would be the best method to achieve this. Starch
and cellulose were chosen as the polysaccharide backbone because of their abundance
and their current acceptance in other pharmaceutical applications. The oxidation of
starch and cellulose is not a new science, but the application of these oxidized
polysaccharides to ocular drug delivery systems is novel. Different methods of oxidation
are known including the use of sodium periodate, hypochlorite, or ozone. All of these
methods can be used separately or combined until the desired material properties are
achieved. Oxidation by sodium periodate was studied most extensively in this research

because it has the best method to control the position and extent of oxidation.

Cellulose and starch both consist of repeating glucose units with only the
glycosidic bond differing as seen in Figure 8. The oxidation methods could be applied to

either structure theoretically, though there would be differences in the kinetics because of

25

the structure of the materials. Starch is composed of amylose that forms a helical

structure. When the material is hydrated the helices open and water can penetrate the

material easily. Cellulose on the other hand forms a tight crystalline structure that is not

 

as easily hydrated.
Starch
or 1-4 linkage (EHZOH CH2OH
H H H _ H
C‘"O C O
é/TH T\‘|3 (I/pH INCL /
\
/o/ \C‘C/ \O/ \C‘C/ O
I I I
H OH H OH
Cellulose . ,
B 1-4 linkage CHQOHEM
/
/O

 

 

 

 

Figure 8. Structure of starch and cellulose
In the periodate method the starch/cellulose ring is opened between the C-2 and

C-3 using NaIO4 in the first step (Floor, Kieboom et al.) which forms an aldehyde

structure. Secondly, that dialdehyde is oxidized using any oxidizing agent (i.e. NaOCl)

and carboxyl groups will be formed at the C-2 and C-3 (see Figure 9).

26

\

T
/ O WZOOH
_ Na|O4 0 0'02
O/<O_H2\O —-> [ 7‘0/—_—_1’O\7‘O/
0
II II chl) Cl)| H
_ _ X _ o o — X 0 _I1

O
I
O\
0
:r:
0
:r:

 

 

 

 

 

 

Figure 9. Oxidation of starch reaction scheme

In this method, by controlling the amount of ring opening, the total amount of
carboxylation can be controlled. Floor (Floor, Kieboom et al.) described a process where
the second step oxidation uses hydrogen peroxide as an inexpensive HOCl scavenger that

will reduce the HOCl. The reaction is as follows:

R-CHO + 00'2 —. R—COO' + HOCI

HOCL + H202 -> HCI + H20 + 02

This was important improvement over previous methods which used ClO '2 as a
scavenger. Besides being less toxic and less expensive, Floor reports that this method
gives higher yields of the dicarboxy polysaccharide with superior calcium sequestering

properties as compared to the reactions using chlorite as the scavenger.

By controlling the amount, nature, and conditions of oxidation or hydrolysis, the
percent carboxyl groups incorporated, the position of attachment, and the molecular

weight can be controlled. By effectively controlling the first periodate oxidation step,

27

. l .

Drill-Ll. .
.h. .

copolymers can be formed that contain both the structure of the glucose ring and the

flexibility of the open ring structure with —COOH groups on them (structure IV).

Structure IV

p—II—

CHZOH

O
f w
/ /
OH 0‘

c 0
0H /11 ”\OH
x O 0 Y

_ d— —

 

 

 

 

Figure 10. Copolymer starch structure

A completely flexible copolymer structure can be engineered by partial oxidation
of the —CH0 groups to —COOH and reducing the remaining aldehyde groups to —OH
using sodium borohydride. Polycaprolactone is a biodegradable polymer that can be
grafted on the backbones allowing for a method of increasing the hydrophobicity of the
drug delivery system as required by certain hydrophobic drugs. The controlled grafting of

polycaprolactone onto polysaccharide backbones has been patented by Narayan

(N arayan)

28

     

w . *
. , g . a"
9 O Q‘ 0.3 it. a
o
i a ‘3” c

Figure 11. 3D Structure of 50% dicarboxy starch

Also, the dicarboxy polysaccharides are stable at the alkaline pH of the washing
process but are degraded under acidic wastewater (pH 4-5) conditions due to their
polyacetal structure. The resulting mono- and oligomeric fragments are readily
biodegradable but will not form the structure needed for this application. F loor(Floor,
Kieboom et al.) shows that at a pH = 3 the dicarboxy starch can degrade up to 80% in 24
hrs, while at a pH=7 it will only degrade 20% over a 24hr period. This is important to
note since ophthalmic solutions are usually formulated around pH=7.4. Also this brings
to light how easily hydrolyzed this material is. Erythronic and glyoxylic acids are the
principal acidic hydrolysis fragments with minor amounts of glycolic, oxalic and formic
acids. This indicates the C2-C3 dicarboxy polysaccharide structure stays intact. (Floor,

Kieboom et al.)

These carboxylated cellulosic derivatives can then form gels with the addition of
divalent cations, such as Ca”, which is naturally found in the eye. The rate of gelation,
the gel strength and the release profile are controlled by percent carboxyl group
engineered onto the polymer chain, its position on the polymer chain, and the molecular
weight of the polymer chain. Floor (Floor, Kieboom et al.) has also shown that the

calcium complexing properties does not differ with respect to the type of glycosidic bond

29

(i.e the B-l-4 linkages of cellulose compared to the or 1-4 linkages of starches) . It is also
important to note that the calcium complexing ability is strongly dependent on the
molecular weight in the region Mw 104 to 105 and at least a Mw of 105 is required for

superior calcium complexation.

The dialdhyde reaction may lead into other formations of including a hydrated aldehyde,

an hemiacetal, or a hemiadol (Fan, Lewis et al. 2001) . The structures of these are below

in Figure 12.
CHZOH
O /
o
\\0
CH CH
11 11
o o
CHZOH
o CHZOH
o’/ o
\ 0 /
0 CH1 C” \\o
H CH
th \OHh T\o/I
OH OH
qHz
O o /
o
\\o \7§
CH CH

I |
OH

0....

Figure 12. Possible structures of periodate-oxidized starch. 1) free aldehyde 2)
hydrated aldehyde 3) hemialdol 4) hemiacetal

30

3.2 Kinetics of the periodate reaction

The periodate reaction is light sensitive and, therefore, care was taken to exclude
light. While, some authors (Besemer, deNooy et al.; deNooy, Besemer et al.; Kim, Kuga
et al.) suggest running the reaction at room temperature or colder, Narayan (Engineering,
1984) et al reported the reaction could be run at slightly elevated temperatures with little
interference from side reactions. For the following work, the periodate reaction was run
at 40° C. Concentrations were used that were similar to earlier work by others. A full

description of the reaction conditions is listed in Chapter 5.

It was first proposed (N arayan (Engineering), 1984) that the periodate oxidation of

cellulose follow the rate law:

_dIPI _K1[P][Cl
dt —K“+[P]

 

(4)
This rate law was explained by being consistent with a mechanism involving the
formation of an intermediate cellulose-periodate complex, most likely a cellulose-

periodate cyclic diester which would then slowly decompose to the final products
Later an improved explanation of the starch oxidation by periodate was proposed. It has

been suggested that the kinetecs follow a 2“° order dependence at t=0, then change to

another model at approximately t=10 minutes (V eelaert, Dewit et al. 1994). This work

31

was conducted using granular potato starch and HPLC for analysis an improvement

method over previous papers which used titration to analyze the dialdehyde formed.

Veelaert proposes that after 5 to 10 minutes the reaction deviates from second order
kinetics because of the polymeric structure of the material and the possibility of hemi-
acetal or acetal formation. The following two rate laws are defined for free and inhibited

anhydroglucose units (an acetal neighbor):

flfl=k,u2[Sol([El-[XD (s)

5’59 = 162/10 — miSntiP. 1 -[X1)
1 (6)

where [X]=the erythritol concentration at any time

[S0] = the initial starch concentration expressed as total initial anhydroglucose
units

[P0] = initial periodate concentration
u = l-degree of oxidation (l-X/g)
These two equations are combined and from experimental data they observed that kg was
much smaller than k,_ The previous formulas then can be simplified into:
21X; z k.
dt [5,]

 

(ISOI-[Xszlfil-[XD

(7)

32

Chapter 4. Analytical Methods

4. 1 FT IR
A Perkins Elmer System 2000 FTIR was used to characterize samples. The samples were

pressed in KBR pellets and run for 16 scans. The wavelength range was 4000 cm’1 to 400

cm"

4.2 Titration
Sodium hydroxide was used to titrate against the COOH groups. The sodium hydroxide

was standardized against potassium acid pthalate to obtain its normality. It was titrated to
an endpoint indicated by phenolphthalein. A concentration of approximately l-5wt %
was used. Because of the viscous nature of the material, the indicator did not react very
quickly and a false endpoint would show up. The protocol used was the indicator staying

pink (acid) for 15 minutes without lightening to be considered the endpoint.

4.3 In vitro UV- Visible Spectroscopy

The drug release profiles were conducted using two different set-ups as more
equipment became available.

Stir plate method

In the first set up a 15 m1 polystyrene centrifuge tube was modified by cutting the
tip off and placing a dialysis membrane (Sigma) with a molecular cut-off of 12,000 over
the open end. The membrane was secured by wrapping Teflon taping tightly around the
tube. The diffusion surface with this setup was 15 mm and 5 ml of the formulated drug

was placed in the tube. Twenty-five milliliters of release medium were placed in a

33

polyethylene cup which was modified by cutting a hole the diameter of the tube in the top
along with another hole for sampling and temperature measurements. A 1” stir bar was
placed in this cup and the cup was placed in a water bath kept constant at 37° C on a stir
plate. One-milliliter samples were taken at time varying intervals and 1 ml of flesh
release medium of was added to keep the volume constant at 25 ml. The composition of
the release medium, simulated tear solution was as follows:

Simulated Tear Solution I

Sodium Chloride 0.67 g
Sodium bicarbonate 0.2 g
Calcium chloride dihyrdate 0.008 g
Water to 100 g

This method led to variability because of the variations in the rpm of the stir bar

between stir plates. Below is a schematic of the system (Figure 13).

Dissolution Apparatus
Sampling and
temperature port

  
  

Dialysis
membrane \
- 12,000 ‘ \

MW cutoff Water bath
Kt e 1

i FBI

]

 

 

 

 

 

 

 

Figure 13. Stir plate dissolution system

USP dissolution method
The second method for obtaining release profiles was using a Hanson EZ-lift

dissolution system which had 6 separate chambers that were all kept in the same constant

temperature bath which was regulated by a feedback loop. Each chamber had a rotating

paddle attached to the same drive motor. One-liter beakers were used to hold the release

medium and they were filled with a specified amount ranging from 400 ml-650 ml. The

formulated drug was place in a well 5 ml with a diameter of 5 cm which was covered

with the same dialysis membrane. Again 1 ml samples were taken at varying intervals;

however the release medium was not replaced in these experiments since the volume

difference was considered negligible. Below (Figure 14) is a schematic of the system:

 

 

 

 

<::>

   

 

Membrane Support System
V=5 ml
Stainless Steel

 

 

. L

 

 

 

 

 

Motor
Controller

 

 

 

 

r

 

 

Temperature
Controller

 

 

 

UUUUI ( 1‘1/

 

C:
C:
c:
<2:
#

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 14. Membrane support (above) and dissolution bath for drug release studies

35

A Perkins-Elmer Lambda 900 Ultraviolet-Visible Spectrophotomer was used to
determine the concentration of the drug in the drug release profiles. The strongest peak
was at 290 nm and the absorbance there was used to determine the concentration. The
UVNIS integration time was 0.3600 s, and the slit width was set to 2.00 nm. Deionized
water was used as the reference, since the tear solution did not contribute to the peak at
290 nm. The software used to obtain the data was UV Winlab for Lambda 900, version

2.90.02.

4.4 ESEM

An environmental scanning electron microscope was used to characterize the structure of
the material. The instrument is an Electroscan 2020 environmental scanning electron
microscope. For these samples, there was a beam voltage of 15 kV with an emission

current of 49 uA. The water pressure was varied from 2 Torr to 9 Torr.

36

Chapter 5.Dicarboxy Matrix Synthesis and
Characterization

5.1 Oxidation Methods:

First the method of oxidation was examined.

The following three methods were used with the native starch.

Table 4. Explanation of Oxidation Methods

 

 

 

 

 

 

Reaction Time Results
Method 1 l-step oxidation ,24 hours Completely
with sodium water soluble
hypochlorite product that is
extremely
hydroscopic in
the presence of
air. Also yellows
when exposed to
air.
Method 2 l-step oxidation 6 hours Non-water
with ozone soluble product
that shows very
little carboxyl
peaks in IR.
Method 3a 2-step oxidation 6 hours + 12-24 Gummy product
with sodium m- hrs that is soluble in
periodate water. Swells
followed by quickly when
sodium chlorite rewetted. Low
yield because of
over-oxidized
starch.
Method 3b Same as above, 3 hours + 6 hrs Gummy product

 

except that
special care was
taken to keep the
dialdehyde from
drying out in
between
reactions

 

 

that is soluble in
water. Swells
quickly when
rewetted.

 

37

 

 

A

W

 

I From Top to Bottom
-— Method 33 (Rxn 45) Carboxyl Stretch
— Method 3 A (Rxn 29) ~1730 cm-1
— Method 2.

   
   
     

— Method 1
— Control

Absorbance

 

 

 

 

r l I I I I I

3900 3400 2900 2400 1900 1400 900 400
Wavenumber cm-1

 

 

Figure 15. FTIR Comparison of oxidation methods

From the FTIR in Figure 15, the carbonyl stretch around 1740 cm'1 shows that the
different methods had different impacts on oxidation. While not quantitative, a
comparison can be made by referencing it to the neighboring 1620 cm" (C-OH) peak.
The ozonated starch, shows a very slight shoulder around 1740 cm'1 indicating that there
was some reaction. The hypochlorite method and Method 3a show that there is slightly
more carbonyl present, but the peak is much smaller than the 1620 cm". This leads to
the possibility that the water solubility of the material may be due to hydrolysis of the
starch as opposed to high carboxyl presence. As seen in the top peak, there is a high level
of carboxyl and the peak is stronger than the 1620 cm'1 peak. The difference between
the 3a and the 3b method, which in this case had the exact same reactant concentrations,

indicates that the structure of the dialdehyde product before the second oxidation plays a

38

 

very important role in the subsequent oxidation. As seen in Figure 16, the additional
peak at 1784 cm'1 indicates the presence of an anhydride which suggests the presence of
the hemi-aldol structure. Specifically a strong anhydride of the structure R-COOCO-R
shows a carbonyl stretch at 1790-1740 cm", This would be consistent of the oxidization

of the hemi-aldol structure.

 

i x . _ ,
0.251 1ct110d3B

0.225%

.->—' 1625.5

0.2%

“ 1040.5

0.175%

0.15%

1428

0.125%

1308.5
1224

0.1%

0.075%

 

810.5

0.05%

0.025%

 

 

v ' f i v I I v v I T r I v I v 'V v I v I v I 1

'24'00 2200 '20'00' 18.00 - 1600' 1400 1200 1000 800 .
Figure 16. FTIR 01' a possible oxidized hemialdol structure

 

 

 

5.2 Polysaccharide Choice

Oxidation Method 3a was tried on different saccharides including native corn starch,
waxy starch, cellulose, pretreated cellulose, xlyans and glucose. The native and waxy
starch produced the best results. The cellulose produced similar results; but the reaction

time was longer and required pretreatment with a strong acid. Accordingly, the starch

39

was used in subsequent reactions. The following chart summarizes the resultant products

from each of these materials.

Table 5. The results of the oxidation of different saccharides

 

 

 

 

 

 

 

 

Material Comments Periodate Chlorite Results
Oxidation Oxidation
Reaction Reaction
time Time
Native Starch 6 hours Good results, high
dicarboxy content,
material swells
Waxy Starch Waxy pearl 6 hours Good results, high
1108 dicarboxy content,
material swells
Cellulose Sigmacell 24 hours Only small percentage
from Si gma- was oxidized
Aldrich
Pretreated Sigmacell 24 hours Good results, high
Cellulose pretreated dicarboxy content,
with material swells
phosphoric
acid and
sodium ‘
hydroxide
Glucose 24 hours Material was over
oxidized
Xylans 7 hours No change in the
material

 

 

 

 

 

5.3 Titration

Titration with sodium hydroxide was used to measure the amount of carboxyl

groups present in the samples. All of the values reported are in terms of carboxyl

groups/anhydrogluco ring. For example, 100% would indicate that every anhydrogluco

ring has one carboxyl group present. Theoretically, the maximum value would be 300%

since the C-2, C-3 and C-6 carbon could potentially contain a carboxyl group. Besides

40

 

actual content, the titration also could be used to quantify the reproducibility of the
reaction.

Because of the heterogeneity of the material produced using Method 3a for
oxidation, titration of those samples was not reproducible. A single sample would have
values ranging from 10%-30%. This proved that the material was not being produced in
a consistent manner and further confirms that other structures, such as hemi-aldols, were
being formed. Table 4 shows the titration results for the material produced by Method
33. The standard deviation of the titrating a sample in duplicate was from 0.01%-3.1%,
which were acceptable values. Also it can be seen from the table that materials produced
using the same periodate-to-starch ratio showed consistent carboxyl content. All of the
data presented here were for reactions using 3 hours for the periodate reaction followed
by 6 hours for the chlorus acid oxidation with waxy cornstarch as the starting material.
Figure 14 graphically shows the relationship between the periodate ratio used and the
resulting carboxyl content. A logarithmic dependence is can be explained by the fact that
as more dialdehyde is the polysaccharide becomes more susceptible to acid hydrolysis
breaking the chain into smaller molecular weight chains. These chains are removed

during the washing of the material and therefore do not show up in the titration.

41

Table 6. Titration results

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Periodate
Sample ratio% COOH/ring Std. Dev.
44 50 46.7% 0.01%
45 50 45.0% 0.02%
46 30 29.1% 0.03%
47 10 0.8% 0.1%
483 30 27.8% 2.1%
48b 30 24.2% 1 .8%
49a 20 15.3% 3.1%
49b 20 12.7% -
50 50 42.7% 1.9%
51 a 20 14.3% 1.5%
51 b 20 13.6% 2.0%
523 80 54.7% 1.5%
53a 100 58.2% 0.0%

 

 

 

Titration results

 

70%
y = 0.2771Ln(x) - 0.6655

0 ._______ _
6° /° /° R2 = 0.9784
40% —~ .~—— ——-

“T ‘ o COOH/ring I
-—Log. (COOH/ring”

 

 

 

 

 

30% ,
20%

/ _- --..
-_./.

0 20 40 60 80 100 120
Theoretical % dicarboxy

% COOI-llring

 

 

 

 

 

 

 

Figure 17. Carboxyl content versus periodate ratio

This data deviates from the data presented by Veelart, which shows a linear dependence
as the stoichiometric amount is increased. This data may be explained by the fact that
high amylopectin starch is being used. This highly branch material may be sterically

hindering the oxidation as higher concentrations of periodate are used.

43

5.4 Periodate Oxidation Kinetics Data

Samples were taken during the periodate oxidation of starch and of cellulose at different
varying time intervals. The UV spectrophotometer was used to analyze the samples since
the periodate has a maximum peak at 223 nm. Data was used from previously obtained

periodate data with new data and compared to the model presented by Veelaert and

 

 

 

 

 

 

 

 

 

 

 

 

Narayan.
Periodate oxidation of cellulose
+Conc. of periodate
1: 250 (mmole/liter)
g I + Cone. of oxycellulose
Ed 200 I units (mmoIe/liter)
E
E
5
E
5
8
8
0 20 Time (”$40 60 80

 

 

 

Figure 18. Periodate oxidation of cellulose kinetics
A close fitting relationship was found using this Veelaert’s model. A Runge-Kutta

differential equation solver set up on Excel was used to solve for the rate constants. Two

separate reactions one for cellulose and one for starch were compared to the model.

 

 

 

 

 

 

 

 

 

 

 

Periodate oxidation of starch

120—
c 100
.9
‘6' .
g Ago- I 0 experimental I
8 :1 value
c 2 l—II—model values
0 O 604
0 E
:2 e
8 40 ~
.9
'6
0. 20a

0 l I I I T
0 100 200 300 400 500 600

Time(mins)

 

Figure 19. Periodate Oxidation of Starch Kinetics

45

 

 

 

 

 

 

 

 

 

 

Periodate oxidation of cellulose
250
c 200 I + model amount of
% periodate
*E' A 150 a I—I—experimental
Q)
g E
o E 100 —
3 E
m v
E 50 -
d)
n.
O 1 I l
0 20 40 60 80
Time(mins)

 

 

 

Figure 20. Comparison of actual and theoretical kinetic data

As can be seen in Figures 19 and 20, the models show a close relationship.
The rate constants for each are using the endpoint of three hours and the carboxyl content
at that point. This introduces error because the assumption is that the dialdehyde is fully

oxidized to carboxyl groups.

Table 7. Calculated rate constants for the periodate oxidation of starch

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

k(ave.)
Sample %dialdehyde k(calculated)L/mmole/min L/mmole/min +/-
47 1 0 1 .50E-08 1 .50E-08
49b 20 1 .40E-07
51a 20 1 .25E-07 1.33E-07 8.0%
46 30 1.30E-07
48a 30 1 .20E-07 1 .25E-07 5.7%
44 50 3.49E-07
45 50 3.21 E-07
50 50 2.80E-07 3.17E-07 1 1 .0%
52a 80 8.30 E-07 8.30E-07
53a 100 1.35E-06 1.35E-06

 

 

 

46

5.5 Design of Experiments Optimization

Stat-Ease software, Design-Expert 6.0 was used to create a design of experiments to see
how the initial periodate ratio affected the product. This was used to optimize the
reaction to predict the most desirable product. These final results were used as the case
that was scaled up in Chapter 8. Acid content, overall reaction yield and dispersibility
were used to qualify the product. The titration results were used for the acid content.
Because of the logarithmic relationship show in Figure 17, the exponential values of the
carboxyl content were used. The yield was calculated by the looking at the percentage
of the polymeric material left at the end of the reaction compared to the theoretical
amount that could be produced, and dispersibility was rated on a scale of 0 to 3. On this
scale 3 indicated that within 10 minutes of adding the material to water it appeared
completely dispersed; 2 indicated that in that time frame the majority of the material was
swollen and dispersed; 1 indicated that a majority of the material was not dispersed, but
at the least the material had swollen considerably; and, 0 indicated that there was no
visible hydration of the material within the 10 minute timeframe. This is an important
factor to consider for manufacturing and formulating of a final product and to ensure that
the drug can be unifome distributed in the matrix. Carboxyl contents and reaction yield
showed a statistically significant relationship to the periodate ratio used and the

dispersibilty show a relationship with a p=0.0761.

47

Response: Yield

AN OVA for Response Surface Linear Model
Analysis of variance table [Partial sum of squares]

 

Sum of Mean F
Source Squages DF Square Value Prob > F
Model 0.48 1 0.48 40.98 0.0002
significant
A 0. 4 8 I 0. 4 8 40. 98 0. 0002
Residual 0.094 8 0.012
Lack of F it 0.066 4 0.016 2.30 0.2204 not significant
Pure Error 0.029 4 7. 16615-003
Cor Total 0.58 9

The Model F-value of 40.98 implies the model is significant. There is only
a 0.02% chance that a "Model F-Value" this large could occur due to noise.

Values of "Prob > F" less than 0.0500 indicate model terms are significant.
In this case A are significant model terms.

Values greater than 0.1000 indicate the model terms are not significant.

Final Equation: Yield =+0.97444 -0.81743 * periodate ratio

DESIGN-EXPERT Pl 1
° One Factor

 

Yield 1.00425 —

X = A: periodate ratio

0 Design Points 016455, _

Yield 0.524853 -<

0.285156 “

 

0.0454577 "‘

 

 

 

0.10 0.33 0.55 0.78 1.00

A: periodate ratio

Figure 21. Relationship between the periodate ratio and the yield

48

Response:COOH %/ring

ANOVA for Response Surface Linear Model
Analysis of variance table [Partial sum of squares]

 

 

Sum of Mean F
Source Squares DF Sfiqugre Value Prob > F
Model 0.56 1 0.56 70.70 < 0.0001
significant
A 0.56 I 0.56 70. 70 < 0.0001
Residual 0.063 8 7 .864E-003
Lack of F it 0.061 4 0.015 28. 71 0.0033 significant
Pure Error 2.] I 7E-003 4 5. 293E-004
Cor Total 0.62 9

The Model F-value of 70.70 implies the model is significant. There is only
a 0.01% chance that a "Model F-Value" this large could occur due to noise.

Values of "Prob > F" less than 0.0500 indicate model terms are significant.
In this case A are significant model terms.

Values greater than 0.1000 indicate the model terms are not significant.

Final Eguation : COOH %/ring = +1 .03430 + 0.87633 * periodate ratio

 

DESIGN-EXPERT

 

 

 

 

Plot One Factor
COOH %Il‘lng 2.00137 —1
X = A: periodate ratio
0 Design Points 1.75321 _ .
8
0
COOH 1.50478 ‘1
%lring
1.2563 —‘
I
1.00734 — O
l l W l I

0.10 0.33 0.55 0.78 1.00
A: periodate ratio

Figure 22. Relationship between the periodate ratio and the final material acid content

49

 

Irv .. ILFIEJ
..

Response: Dispersibiligy

ANOVA for Response Surface Linear Model
Analysis of variance table [Partial sum of squares]

 

 

Sum of Mean F
Source Squares D Squage Vglue Prob > F
Model 3.45 1 3.45 4.15 0.0761
not significant
A 3.45 1 3.45 4.15 0.0761
Residual 6.65 8 0.83
Lack of F it 3. 49 4 0.87 1.10 0. 4641 not significant
Pure Error 3.1 7 4 0.79
Cor Total 1 0. 10 9

The Model F-value of 4.15 implies there is a 7.61% chance that a "Model F-Value"

this large could occur due to noise.

Values of "Prob > F" less than 0.0500 indicate model terms are significant.
In this case there are no significant model terms.

Values greater than 0.1000 indicate the model terms are not significant.

Final Eguation: Dispersibility = +1.33978 + 2.18232 * periodate ratio

 

 

 

 

 

 

DESIGN-EXPERT Plot One Factor
dispersibility 4.45325 —
X = A: periodate ratio
0 Design Points 334369 _

2.22913 ~

dispersibility
L11456 —‘
o —‘ O
T T l I I

0.10 0.33 0.55 0.78 1.00
A: periodate ratio

Figure 23. Relationship between the periodate ratio and the dispersibility 0f the material

50

Optimization Results
The following constraints were set to fine the optimal periodate ratio used.

 

 

 

Constraints
Lower Upper
Name Goal Limit Limit Importance
periodate ratio is in range 0.1 1 3
Yield maximize 0.136 1 3
COOH %lring is target = 1.398 1.007 1.789 3
Solution
periodate ratio Yield COOH %lring
9_.4_2_ 0.634683 1.39854
DESIGN-EXPERT Plot One Factor
Desirability 1000 —
X = A: periodate ratio 1 Max 1
0 Design Points 0150 _
0.500 ‘-
Desirability
0.250 -‘
0.000 —

 

 

 

0.10 0.33 0.55 0.78 1.00

A: periodate ratio

Figure 24. Optimization of the periodate concentration

51

This design of experiments could be be expanded in the future for to incorporate the
release data and calculated difussion coeffients to develop a predictive model with

reactant molarity as the input and diffusion coefficients as the output.

5.6 ESEM
As seen in the ESEM images below (Figures 25), there is a difference between native

starch and the dicarboxy starch. It can be observed that the oxidation process destroys
the granular structure of the starch, releasing the amylose and amylopectin fiom the
structure creating a smooth and flexible material. The ESEM is run under vacuum so it
impossible to observe the hydrated structure. However, the swelling and subsequent
dehydration of the material can be observed while the material is first wetted and the

vacuum chamber comes to equilibrium.

52

 

Air Dried Dicarbcxy Starch

 

Rehydrated Dicarbcxy Starch

Figure 25. ESEM images of starch, air dried dicarboxy starch and hydrated dicarboxy starch

53

Chapter 6. Synthesis of Grafted Dicarbcxy Starch and
Characterization

6. 1 Background
Starch grafted with polycaprolactone (PCL) was made by the above synthesis to show

that a similar material with different hydrophobic groups could be made. This has the
potential of increasing the solubility and delivering drugs that are poorly water-soluble.
This is very important as there are many gene therapies being developed currently that

are high effective for their intended purpose but have very low bioavailability.

Polycaprolactone is approved for medical uses for sutures and is being investigated in
many other medical applications including tissue engineering, bone imaging, drug
delivery, and cardiac grafts(Domb, Kost et al. 1997) . PCL was tested in long-term
release studies such as the release of contraceptives and the release of cancer therapies.
To fully improve the functionality of the drug delivery system the PCL must be grafted to
the starch allowing for a flexible hydrophilic backbone chain with the PCL attached at
random intervals. The thermodynamic incompatibility between starch and synthetic
polymers makes it difficult to graft these polymers on the backbone using a mechanical
extrusion process. To improve the compatibility between starch and the polyester phases,
starch is plasticized using common plasticizers such as glycerol, ethylene glycol or
sorbitol in a twin-screw extruder to form thermoplastic starch (TPS). The plasticization
process breaks the hydrogen bonds and disrupts the granular crystalline organization. It
further releases the amorphous polymer chains with 0.1—)4 and 0: 1 —>6 linkages. Figure

26 below shows the ESEM pictures of starch and plasticized starch.

54

 

Starch TPS

Figure 26. ESEM of native starch compared to maleated thermoplastic starch

To be able to graft the copolymer to the backbone, grafiing maleic anhydride to the starch
backbone forms a reactive carboxyl group at the end of the C-6 carbon (Figure 27). This
allows for the polyester to be added in transesterfication reaction to that carbon (Figure
28). For this application PCL was chosen as the polyester because of its accepted use in

medical applications.

CH2§H
0H 0 H<|3=<|3H
C c\
o o— 0% \O/ \0
OH

O H H
—O O —
O H
Figure 27. Maleation of starch
55

'/ _il

CHzo—C—CII=C|)—C—OH
O H H
+ MOI-I
_0 o— ‘-
11 ii
CHzO—C—'C=(|3—-C—Oaww
O H H
4.
__0 0—
OH

Figure 28. Transesterfication reaction with maleated starch

H20

PCL, marketed under the trade name TONE polymers, was obtained from Dow

Chemicals. TONE polymers are homopolymers of s-caprolactone, a seven-member ring

compound. The chemical structure of the PCL polyester is shown in Figure 29.

- -

R0, /

/

O

 

 

Figure 29. Chemical structure of polycaprolactone

For these experiments, lower molecular weight was used; TONE P-737 and P-757

polymers of Mn 32,000 g/mol and 43,000 g/mol respectively.

56

6.2 Results

An FTIR confirmed the formation of the maleated thermoplastic starch (MTPS) in Figure

30. The strong double bond of the maleic acid group can be seen at thel 703 cm'I peak.

Control Starch

0.9

0.8

0.7

 

0.6 MTPS

 

 

 

 

0.5

 

0.4

0.3

 

"I""l""l""l""l""I""l""I""l""l""l""l""l'"'l""l""
4000 3750 3500 3250 3000 2750 2500 2250 2000 1750 1500 1250 IMO 750 500

Figure 30. FTIR of Starch and MTPS

The PCL/MTPS was able to be oxidized slightly and form a water-soluble fraction.
However the yield of that product was less then 0.1% and is was not able to be
characterized. While there is potential with this reaction, improvements in the reaction
setup would have to be tailored to this reaction, such as a more efficient way to mill the

material before reacting.

57

Chapter 7. Drug Release Profiles

 

Figure 31. Structure of ofloxacin

Ofloxacin is an antibacterial agent belonging to the fluoroquinolone family with a
molecular weight of 361.37. Of the available fluoroquinolones, ofloxacin is one of only
usually given as a single agent and has been shown to have the best aqueous humor
penetration. As an ophthalmic formulation, ofloxacin is formulated as a 0.3% w/v
solution and goes by the trade name OCUFLOX ®. According to Allergan’s prescribing
information packet, OCUFLOX solution is unbuffered and formulated with a pH of 6.4
(range - 6.0 to 6.8). It has an osmolality of 300 mOsm/kg. Ofloxacin is a fluorinated 4-
quinolone which differs from other fluorinated 4-quinolones in that there is a six member

pyridobenzoxazine ring from positions 1 to 8 of the basic ring structure.

7. 1 Drug Release Results
The drug release profiles were studied using a Perkin Elmer’s Lambda 900

ultraviolet-visible Spectrophotomer. The absorbance spectrum for the drug ofloxacin can

be seen in Figure 32.

58

 

 

 

 

 

 

 

 

 

 

 

 

 

 

a- .. _____1_-. _ -

 

 

 

Absorbance
/
I

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

4...”-..... -_... W... -...e ........

\_
mmmmmmmwwmmmmmmmo
Vlbiele’ghrrn

Figure 32. UV-VIS Curve for Ofloxacin

The strongest peak at 290 nm was used to determine concentration of ofloxacin as
compared to a calibration curve. The absorbance at concentrations of 0.0036% w/v to
0.00075%w/v was found to linearly dependent and measurable using the parameters
described in the analytical technique chapter. Some of the samples had to be diluted to 1
part sample to 2 parts plain tear solution to have samples in a measurable range. A
concentration method was developed to read the output only the absorbance at 290 nm.
This eliminated the need of developing full spectra for all of the samples. The calibration
confirmed that absorbance was linear with concentration from a range of absorbance

from 0-2.5

59

 

Calibration Curve of Ofloxacin in Simulated Tear

 

 

 

Solution
2 3
8 2.5 2: 69101x
N 2 R = 0.9998
15
3 1.5
5 1
E
,, 0.5 ——-————
0 l
< o I
0.0000% 0.0005% 0.0010% 0.0015% 0.0020% 0.0025% 0.0030% 0.0035% 0.0040%;
1

 

Conc. (va°/o) I

 

Figure 33. Calibration Curve for Ofloxacin
The drug release profiles were conducted using two different set-ups a more

equipment became available. The setup are explained in Chapter 4 and here will be

referred to as the stir plate method and the USP transdermal method.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Drug release 010.3% ofloxacin in simulated tear
solution
40% _, ----- no matrix I
8 35% — -+—1%,Gelrite(TM)
§ 323’ " +1%,dicarboxystarch
o o ‘_,.-A'
320% _..---"'
g 15% ,,,,,
«5 10% .......... 6"" .. _______________ 4
°\° 5% "—i—WV ‘4
0% , T
0 60
Time (mins)

 

 

 

Figure 34. Comparison of dicarboxy starch to gelrite

60

 

 

 

 

 

 

 

 

 

 

 

Drug release of 0.3% ofloxacin
Stirplate Method I
100% ““‘T’ ”T” ' o no matrix I
90% ‘ r + 1% Gelrite I
80% * A 1% Dicarbcxy starch (rxn 36a)
3 702/0“ . ° -O--1%advantagel
g 6004* . +1%DimrboxyStarch(rxn11)
g 50 /o ‘ —o—1%advantagel and dextrose
\° 40% " A
° 30% . A _____ —o
20% - * e ——————
10% W
0% . t T r r
0 100 200 300 400 500 600
Time (wins)

 

 

Figure 35. Drug release of various materials

Stir plate method results

Initially the release of the dicarboxy starch was compared to that of Gelrite to see
if it exhibited similar release properties. As seen in Figure 34, over a period of one hour
the dicarboxy starch and the Gelrite release the ofloxacin in a similar manner.

Results fi'om the USP method
Figure 25 shows the release profiles from three different dicarboxy starches (20%
dicarboxy, 50% dicarboxy and 80% dicarboxy) compared to the release profile of Gelrite.
All were formulated using 1wt% of the matrix in a phosphate buffer, pH =7.4. There is
no statistical different between the release profiles of the materials with different carboxy
concentrations. This is important because the optimization of the material in the previous

chapter assumed that all the materials would have the same release profile.

61

 

Release of 0.3% Ofloxacin in Simulated Tear Solution

 

60%

50% ~

 

 

 

40%

30%

    

 

% release

. .av‘"

   

. o' '
' I
'.
.' ".J
o

 

 

 

, —o—50% dicarboxy starch. 1%;
L?“ ' --I- ~ - 80% dicarboxy starch, 1%;

 

 

20%

10%-

 

 

 

------a--- 20% dicarboxy starch, 1%;
- - -l~ - - no matrix; 0.3% drug
- - at - -— Gelrite 1%. 0.3% drug

 

 

 

 

J
I I I I 1

20 30 40 50 60 70

Time (mins)

 

Figure 36. Release profiles using USP dissolution system

 

80%
70%
60%
50%
40%
30%
20%
10%

0%

% release

 

L

Release of Ofloxacin from 1% dicarboxy starch matrix

 

 

0

its

at A

 

 

 

D 0(1-
b

A 0.3% ofloxacin
e 0.1% ofloxacin
x 0.15% ofloxacin
O 0.09% ofloxacin

bl.
>x

D.

 

i T l 0.2% ofloxacin

 

 

 

50 100 1 50
Time (mins)

Figure 37. Release with varying drug concentration

62

 

7.2 Modeling the Diffusion Method
The data obtained from the USP dissolution method was used compared to the models

predicted by the Higuchi Model presented in Chapter 2. As indicated by the model the
release of the drug from a swollen hydrogel is diffusion controlled and should follow a
square-root time dependence when the percentage released is less than 60%. As seen in
Figure 38, the drug release is consistent with that model because all experimental data for
each of the matrices can be fitted with a linear fit with a R2 value greater than 0.97. The
varying slopes of the lines indicates that there are different apparent diffusion coefficients
for each of the different materials. This confirms the fact that material can be engineered
to change the release profiles. The release profile with no matrix appears linear when
plotted against the square root of time, however there is a higher linear correlation when

it is plotted against time which is consistent with standard diffusion through a membrane

 

 

      
 
   

 

 

 

[Saltzman].
Square root time dependence of the drug release
0.3% ofloxacln
60%
050% dicarboxy starch. 1%; R2 = 0 9676 R2 _._ 0.9723
50% - I 80% dicarboxy starch. 1%;
x20% dicarboxy starch. 1%;
3 40% * Ano matrix; 0.3% drug
0
0‘ 30% 2
g R = 0.9835
n 20% 2
a! R = 0.9904
10%
0% r . r
0 2 4 6 8 10

Square root of time (mlns‘0.5)

 

 

 

Figure 38. Square root time dependence of the oi'loxacin

Following the Higuchi model presented in Chapter 2, for the same material, surface area
and volume, the diffusion coefficient should be able to be calculated by changing the

concentration of the drug. To calculate this, a formula with 1% of the 20% dicarboxy

63

starch was made up with varying concentrations ofloxacin. These runs were conducted
once each. The percent released was plotted against the square root of time and the
$10pes of the lines were found. According to Higuchi model when rearranged for the
dimensionless percent released, the slope of the line, y, should be equal to:
=2A(Dm/1c)°'5 (8)
Following this equation the slope of the lines showed be equal for the same matrix and
release area. As seen if Figure 39, the slopes vary for each of the release profiles. Since
each of these runs were conducted only once, the difference could be due to the number
of runs. The 0.3% formulation may becoming close to the solubility limit of the drug in
which case the Hi guchi model presented would deviate. If the values for the 0.3% value
are removed, the average of the slope of the lines becomes 0.0665 :1: 9% which is
considered a reasonable deviation. Using that number an apparent diffusion coefficient

can be calculated with the answer being 0.000225 cmz/s.

 

Calculation of diffusion coefficient
Release of Ofloxacin from 1% dicarboxy starch matrix

 

   
 
 
    
 

 

 

 

120%
A 0.3% ofloxacin y = 0.0727x y = 0.0702x
100% — I 0.1% ofloxacin R2 = 0.9849 R2 = 0.9859
0 0.15% ofloxacin
q, 80% - o 0.09% ofloxacin y = 0.0624x
8 I 0.2% ofloxacin R2 .-. 03905
2 60% ~ _
g y - 0.0606x
o 2 =
.\ 40% y R 0.9809
20% + y = 0.0422x
R2 = 0.9796
0% . . .
0 5 10 15

 

square root of time (mins)

 

 

Figure 39. Calculation of diffusion coefficient

64

In conclusion, it appears that the release profiles can be modeled using the Higuchi
equation. Monitoring the drug concentration with the UV-visible spectrophometer

provides a good means to analyze the release profile.

65

Chapter 8. Conclusions and Recommendations

8. 1 Conclusions
I This engineered dicarboxylate material can be characterized by FTIR and titration

methods to analyze the amount of added carboxylate groups.

I The dicarboxy starch is water dispersible and shows drug release profiles
consistant of hydrogel matrices. This would make it suitable for an ocular drug
delivery system.

I Using the diffusion model presented, the material could be engineered to have
exhibit desired diffusion properties.

I This material has the potential to be used in a variety of drug delivery applications
including topical and depot ocular, medicated wound dressing.

I Carboxylated polysacchrides with grafied biodegradable hydrophobic branches
can be produced, however those reactions are not currently optimized.

I From observation, this material appears to have a good ability to adhere to
biological surfaces. The mucoadhesive properties need to be evaluated further,
but there are is the potential for muccoadhesive applications such as nasal drug
delivery.

I This material is worth pursuing for commercial applications.

66

8.2 Kinetic considerations and scale up

Below in Figure 40, a large-scale batch process for the production of the dicarboxy starch
is proposed. For the situation were this would be used to to produce enough material to
be formulated at a lwt.% for a major product such as a glaucoma treatment the following

would be suggested.

Table 8. Reaction Specifications

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Formulated product required/annually 200000 kg
Assume 10,000,000 bottles & 20 ml each
Amount of dicarboxy starch needed/annually 2000 kg
# of batch runs annually 50
Amount/batch needed for 42% dialdehyde 40 kg
Yield = 63.47%
eactants/ batch
Starch 60.9 kg
Sodium m-periodate 33.8 kg
Water 6282.4 kg
Acetic Acid 13.6 kg
Hydrogen Peroxide 45.1 kg
Na—EDTA 0.8 kg
Sodium Chlorite 42.5 kg

 

Reactor 1 would be a 500 gallon stainless steel jacketed reactor. Water would be used to
heat the reactor to 40° C. The second reactor would be a stainless steel 1250 gallon
reactor. Chlorine byproduct will have to be controlled and quenched accordingly. The

ethanol/water washwater could be recycled using a basic distillation column.

Special considerations that would have to be addressed include:

I A suitable filter that would ionically repel the material or atleast, not attract it

67

I The iodate can be reoxidized electrolytically to periodate or can be oxidized to
paraperiodate using sodium hypochlorite which then will release the

metaperiodate ion.

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69

Chapter 9. Examination of business potential

9. 1 Overview of market

Market for Drug Delivery S 2stems

Pharmaceutical companies are facing intense pressure to develop new and better drugs.
R&D expenditures grew from $18 billion in 1990 to $43.8 billion in 2000, reflecting this
competitive pressure. Drug delivery technologies are increasingly recognized as a critical
strategic tool, allowing companies to make their drugs easier to administer and more
effective. Currently, within the $337 billion global pharmaceutical market, products
incorporating drug delivery systems constitutes 13%. The overall market for advanced
drug delivery systems is expected to jump from $16.28 billion in 2000 to $27.35 billion
in 2005. The figure below depicts the trends for increased use of drug delivery for both

new and existing drugsl.

 

Pharmaceutical Companies Projects Incorporating

 

 

 

    

Drug Delivery Systems
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Figure 41. Drug delivery growth

 

I Findlay G. Kennani F. Making Drug Delivery Alliances Successful. CMR lntemational. 2000. www.gmr.9_rg(dggrpt.html

7O

Mrket [or Ocular T herageutics
There is strong agreement among industry analysts that ocular therapeutics represents
substantial business opportunity. Major medical insurance programs cover the expense

of topical ocular prescription drugs, with appr0priate co-pays applied.

Glaucoma, Antibiotics and Anti-A llergy T herapeutics._ Projected growth in the
ophthalmic market for topical products for glaucoma, antibiotics and anti-allergy
therapeutic areas — our initial focus - is for these markets to approximately double by
2010 to over $8 billion (from $3.6 billion in 2000). For example, according to estimates
from Aciont, the current market size for anti-inflammatories to treat intermediate and
posterior uveitis is $600million2. The glaucoma market is the only one in which gelling
ocular products have been introduced. Merck’s Timoptic XE® was introduced first,
followed by Alcon (Falcon). The market positioning for both has been that the gel
increases efficacy and decreases systemic exposure as a major advantage. After the
introduction of Xalatan® and other similar prostaglandin glaucoma drugs with superior
intraocular pressure (IOP) lowering, market share for timolol and other glaucoma
medications declined. However, Timoptic XE still sells well and had an 18% increase in

sales 3Q 2003 relative to 3Q 2002.2

Retinal Degenerative Disease Therapeutics. Biopolymer Innovations’ (BI) secondary
targets include treatments of such retinal degenerative diseases as age- related macular

degeneration (AMD) and diabetic retinopathy (DR). The incidence of these diseases is

 

2 Merck & Company, Inc. , financial disclosure, third quarter, 2003.

71

expected to increase substantially with the aging of the US. and world population. While
there are currently few effective treatments on the market, the pharmaceutical companies
are directing large amounts of research and development funds to these areas; many more
drugs are expected in the relative near-term. The growth in age-related macular
degeneration (AMD) and diabetic retinopathy (DR) drugs is expected to grow
exponentially to about $10 billion in 2010 from $800 million) in 2000. According to
estimates from Aciont, the current market size for chronic macular edema associated with
diabetes is approximately $1.2 billion, for anti-angiogenic activity to treat "wet form" age
related macular degeneration is $1.2 billion, and anti-angiogenic activity to treat diabetic

retinopathy is $1 billion3'

International market growth

Growth trends in economically advanced countries are similar to those in the US. Our
pharmaceutical partners will vary on their relative market size and distribution reach in
international locations; yet international market growth in general will be robust. Third
world countries, in contrast, are still focused largely on the prevention of blindness (the
most common cause of blindness is Vitamin A deficiency). Some opportunity may exist
in third world markets for the development of an inexpensive glaucoma agent (timolol

reformulation, for example).

 

3 2http://www.aciont.cor'n/products.htrn

72

COME TI T ION PROFILE
There are three companies that currently offer an in situ gelling drug delivery system:
Merck, Falcon, and DelSite. Their products along with other products in development are

compared in the chart below.

0 Merck. Merck currently has the only true in situ forming ocular gelling product
on the market. Gelrite®, registed to Merck, is a naturally occurring

polysaccharide and is used in their product Timoptic.

- Falcon (Alcon). Falcon’s “gel forming solution” is not a true in situ forming gel

and therefore does not increase bioavailability

- DelSite. DelSite markets an in situ forming gel for multiple applications and
routes of administration. The polymer is a naturally occurring acidic polymer

derived from aloe vera. No topical ocular applications are described.

Listed below are companies that are working on ocular drug delivery technologies across
the spectrum of eye conditions and diseases, and that could possibly have programs
similar to the ocular gelling technology of B1. None of these companies is publicly
stating that in situ forming gels for ocular applications are under development. Also,

none has announced that it is working with modified biopolymers in the area of ocular

73

topical delivery (or depot delivery). The competitors were compared on the following

three criteria, described below:

Synthetic or Natural. In general, “natural” means that the products are based on

 

materials that are common to the body, with known metabolism pathways. This
suggests high biocompatibility with the body, and the material would not be seen
as a foreign substance. A naturally occurring substance at low dosages will have

no harmfirl effects, leading to quicker FDA approvals.

Easy to Use. Will the product potentially increase patient compliance, or at least
not decrease it? Some new technologies are difficult to use and potentially
decrease compliance — they must be administered by a doctor or are very
disconnected to the current way people treat eye diseases. The ability to reduce

dosing frequency was also considered.

Broad Application. Broad application is defined as: modifiable to conform to
characteristics of the drug to create the optimum formulation; the drug

formulation/ reformulation process is quick and simple; it is easy to manufacture.

OcularGelTM performs very well on all three criteria:

74

0 Natural Product
0 It is based on materials the body can recognize and metabolize.
0 Easy to Use
0 It canbe formulated in current drugs without changing the way drugs are
administered.
0 Can decrease the frequency of dosing
o Broad application
0 Can be engineered to conform to the drugs properties
0 Can be used in other routes of administration besides ocular

o Is simple to manufacture, ship and formulate

75

Table 9. Competitors in the area of ocular drug delivery

 

 

Technology (A)lliances/
Company Name Name (P)arent Co.
Currently in the market:

CP Kelco GelriteTM A: Merck
Falcon “Gel forming
, solution”
Delsite GelSiteTM P: Carrington
Biotechnologies Laboratories
(TX) Inc.
Under development or veg little market share:
InSite Vision DuraSite A:
(CA) Bausch&Lomb;
Japan's SSP
Co.
Oculex A: Allergan
Pharmaceuticals
Inc. (CA)
Oxigene CA4P A: Arizona
State; U of
Lund; Baylor,
U of Florida
Escalon Medical Ocufit A: West
(PA) Pharmaceutical
Services
BioSante CAP
Pharmaceuticals, Nanoparticles
Inc. (IL)
Lipocore Galacticles
Ophthalmic
Retinapharma PhotoTarget A: John
(PA) 1'“ System Hopkins

Synthetic
or
Natural

Synthetic
Natural

Natural

Synthetic

Synthetic

Synthetic

Synthetic

Synthetic

Natural

Synthetic

Easy
to
Use

Yes
Yes

Yes

Yes

No

No

No

No

Yes

Broad
application

No

No

Yes but not
for ocular

applications

Yes

No

No

 

76

 

C US T Oil/ER PROFILE

Pharmaceutical companies are relying increasingly on novel drug delivery technologies
to differentiate their products,to extend product life cycles, and to sustain their high
growth and profitability. Trends are for large pharmaceutical companies to develop

strong alliances with drug delivery partners, including acquisitions, as drug delivery

represents an area outside their core research competence. Some of the specialized firms

work in partnership with the pharmaceuticals,and others independently develop their
platforms with the goal of selling the platform or licensing the technology to major

pharmaceutical companies once development is complete.

6 Largest Pharma with Ocular Drugs
$Mi|lions (Year 2000)

 

El Pfizer

 
    
 

$320 $2 $700

“:2; 11mph: ._‘. ..

i. Allergan é

      

Alcon

 

Figure 42. Largest Pharmaceutical Companies with Ocular Drugs

Our customers are pharmaceutical companies with treatments for glaucoma, eye
infections, allergies, dry eye and inflammation. The customer base consists of major

manufacturers - Pfizer, Merck, Alcon, Allergan, Novartis, and Bausch & Lomb - and a

77

number of smaller manufacturers and generic drug companies. The current market shares

for ocular drugs for all major companies are illustrated below.

BI will pursue discussions and negotiations with the major pharmaceutical companies
with treatments in its initial target therapeutic areas — glaucoma, antibiotics, and anti-
allergy therapeutics — once the initial proof of concept is complete. Our initial partnership
will involve joint development to ensure we maximize our learning about the platform’s
characteristic and performance, and that it is performing well in its initial application.

We also anticipate that this initial project will be for an ocular drug that is currently on
the market, or one that was previously marketed that could benefit greatly by the
OcularGelTM drug delivery system. The advantage of starting with a known product is
that the formulation issues related to the incorporation of OcularGelTM will be simpler
and more straightforward. Subsequent partnership relationships will remain in the initial

target areas, yet could include projects involving new drugs.

The following chart shows major pharmaceuticals companies with ocular treatments in

BPS initial target therapeutic areas, as representative of our potential customer base.

78

Si

 

Ht .-

Table 10. Customer Profile

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Predicted
Com an Dru Chemical EA __Patent _U_s_e 1:32:32
——2—l _g —— Approval Expiration /Mechanism ($MM)
FY 2003
Open Angle
Glaucoma - $1,051
Pharmacia Prostaglandin $623
/ Pfizer Xalatan Latanoprost 1996 2006-201 1 F 2A1pha (2003)4
Macular
Novartis Visudyne Verteporfin 2000 2007-2016 Degeneration
Novartis Zaditor Anti-allergy
Open Angle
Glaucoma -
Alpha 2 $249
Allergan Alphagan Brimonidine 200 l 201 2-201 5 Stimulator $286. 85
Open Angle
Glaucoma -
Prostaglandin
Allergan Lumigan Bimatoprost 2001 2012 F 2Alpha $181 .35
Open Angle
Glaucoma -
lsopropyl Prostaglandin
F ujisawa Rescula Unoprostone 2000 2008-201 1 F 2Alpha $7
Alcon Patanol Anti-allergy $2526
Ciloxan 2004 2004
Vi gamox ligamox (CiloxanL Anti-infection
Alcon Travatan Glaucoma $135T
Glaucoma -
Carbonic
Trusopt/ Anhydrasc
Merck Cosopt Dorzolamide 1994 2003—2008 Inhibitor $460
Timoptic/ Glaucoma -
Timoptic 1982- Non selective
Merck XE Timolol 1993 2006 beta blocker $159
Chibroxin Anti-infection
Bausch & OTC &
Lomb prescript. $467.97
Senju OTC 180408

 

 

 

 

4

http://www.pfizer.com/are/investors reports/annual 2003/financi312003pdf. after the purchase of
Pharmacia

’ht_t;p://www.shareholder.com/AGN/EdgarDetail.cfm‘?Companyl D=AGN&C1K=850693&FlD=892569-04-
307&SlD=04-00#’ 102', page 24

 

6 hpp://media.co;p_orate-imet/media files/NYS/ACUrgports/ZOO3ar.pdf, page 29
7 http://www.ba_u_sch.com/uglvision/gbout/investor/pdfs/2003annual.pdf, page 23

79

 

 

9.2 Path to commercialization

Product Development: T imetrames and Milestones

The phases of product development are depicted in Table l and described below.

Phase 1 -Proof of Concept

A modified starch gel has been completed that can increase the residence time on the
corneal epithelium. As described earlier, it is known that modified polysaccharides,
when applied topically to the eye, are not significantly irritating. Gellan gum is a good
example, since this polymer is similar in structure to OcularGelW. Current activity

involves (see Table 1 below):

0 Characterization (final) of the reaction conditions to produce OcularGelTM

o Demonstration of increased ocular bioavailability with the compound timolol

0 Characterization of reactions conditions continues, as does evaluation of new
reactions

0 Demonstration of safety in rabbit and dog, again while further characterization of

reactions conditions continues

We are confident that we will not experience any significant technology development

obstacles. The key possible obstacles, and the reasons for our confidence, are as follows:

 

8 hgp://www.senju.co.jp/english/part/e-stgtisticshtml 2001 revenue in Japanese currency

80

I Inability to produce OcularGelTM consistently. This is very unlikely as the
chemistry is well understood, and the gelling properties demonstrated.

0 Lack of an increase in ocular bioavailability. This is very unlikely as this effect is
a property of all gels.

0 Significant irritation in rabbit and/or dog GLP safety studies. This is very
unlikely as polysaccharides with very similar structure have been demonstrated to

not be irritating in similar tests.

Phase 2 — Applications Development

After the concept has been proved to be completely safe and effective, six to eight drugs
will be tested extensively with OcularGelTM to determine the best potential
commercialization partners. The tested needed for FDA approval to test in humans will
be conducted at this time.

I Extensive in vitro studies to determine the best partners

I Extensive formulation studies

I Bioavailability testing in dogs and primates

Phase 3- Commercialization

BI will pursue three methods of commercialization of OcularGelTM for prescription

drugs. These methods and their time frames are described below:

81

1. Licensing of OcularGelTM for u_se with Current and/or Previou_slv Mar‘keted Products.
BI will target drugs that will realize important benefits through incorporation of
OcularGelTM, particularly related to their safety and efficacy. The process of
reformulation by integration of OcularGelTM into existing products is identical for
aqueous based products (solutions) or suspensions, with the exception of specific pH and
solubility requirements for a given drug. While each case might be slightly different, for
the most part the reformulation will simple involve the addition of OcularGelTM at 0.5 —

5% by weight.

Integration of OcularGelTM into an existing product with patent protection on its active
pharmaceutical ingredient (API) will be done in partnership with the original
manufacturer. In the case of off patent drugs, which are readily available through
commercial sources, the integration of OcularGelTM can be done solely by B1, or in
partnership with the original manufacturer or a generic manufacturer. There are a large
number of such products, from antibiotics to glaucoma agents. Selection of these targets

will be made based on market size and share, and potential profitability.

In the cases of B1 pursuing sole development/formulation of a product, it will seek a

partnership for marketing and distribution.

2. Licensing of OculprGelTM for 1g with Productgunde; Develonmyj

82

Integration of OcularGelTM into products under development will be done in
partnership with the original manufacturer. A key variable in these projects is the time
needed for FDA approval, which varies significantly by application. For example,
products to treat age-related macular degeneration (AMD) or diabetic retinopathy would
receive very rapid approval and the inclusion of OcularGelTM would not be an issue
because of the immediate need for a product on the market where none exists currently.
Products to treat serious infections may also be on a “fast track” to approval because of
the severity of condition. On the other hand, treatments for ocular inflammation or
glaucoma would require more extensive safety data because there are currently adequate

drugs on the market to treat these conditions.

3. Direct Sgle of OcularGelTM PM

The entire OcularGelTM platform could be sold directly. A major manufacturer would
pursue this strategy to have proprietary use of OcularGelTM in all of its products, and to
prevent OcularGel’sTM use in competitive products. Merck is an example of such a
manufacturer. In this relationship, BI would work directly with the partner to continue to
improve the platfomi, as the partner requires. Pursuit of this commercialization route
would likely end BI’s involvement in starch based gels, as defined by the patent. BI

would pursue development of depot and other platforms.

Table 11 below shows the timeframes and milestones for the Product Development Plan.

83

Table 11. Path to commercialization

FIRST

1: 1st polymerselected

. safety and

bioavailability testing with

 

84

Product Development Plan

We envision our product evolution pathway in the following stages:

Short-term goals (one to three years)

Reformulate existing IP protected drugs for glaucoma, infection, inflammation,
and allergy. Described earlier under “Product development milestones and
timeframes” reformulation of existing drugs represents the quickest
commercialization route.

Reformulate oflpatent drugs. Also described earlier under “Product development
milestones and timeframes”.

Formulate with new products under development

Reformulate over the counter (OTC) preparations. This will not generate large of
amounts of revenue for the company, but it could represent a way to generate a

larger market recognition for our company.

Long-term goals (three to six years)

Develop depot IP for retinal diseases — age-related macular degeneration (AMD)
and diabetic retinopathy (DR). This is at the concept stage of development. The
theoretical properties of esters of OcularGelTM may be ideal for local depot

injections.
Develop Non-Ocular Drug Delivery. This is at the concept stage of development

Gene Targeting. This is at the concept stage of development

85

We anticipate the following time frames for accomplishing the stages above:

Table 12. Projected Product Development Timeline

2004 2005 2006 2007 2008 2009 2010
Existing drugs
Off
New drugs
OTC

Non-ocular drug
de '

Retinal diseases
Gene targeting

 

Proprietagz rights

The materials under development are proprietary and will be protected under patents. The
proof of concept research is occurring at Michigan State University (MSU), and the
patents will be filed through the university. A composition of matter patent will protect
the technology platform, and that is currently patent pending. A licensing arrangement
has been negotiated with MSU in which Biopolymer Innovations will have exclusive
rights to all of the patents around this particular drug delivery platform, including future
inventions.

Technology for aspects of modifying the hydrophobic/hydrophilic balance of the material
that will be licensed to BI for drug delivery applications are covered in eight patents by

R. Narayan.

86

Governmental approvals

As OcularGelTM is not an active ingredient, i.e., it is an excipient, the FDA approval
process is rapid compared to having a new active agent approved. The FDA gives only

guidelines for approval of excipients in topical ocular formulations.

Permission will be needed from the FDA for evaluation of the OcularGelTM product in
humans. The preclinical studies conducted by Biopolymer Innovations are designed to
meet FDA standards for this approval process. This requires a Good Laboratory Practice
(GLP) study in a dog and rabbit, for duration of one month, at higher dosing frequency
and gel concentration levels compared to anticipated levels in humans. This study will be

completed at an appropriate contract research organization (CRO).

As described earlier in Table l 1, we anticipate that OcularGel'l'M will be ready for human
trials starting in early 2005. The duration of that testing should be completed by will be
dependent on the partnering drug but could take as little as six months if the drug has
already been approved by the FDA.

Production
There are two production processes involved - first to produce OcularGelW, and second
to produce the drug with the gel incorporated.
It is assumed that larger pharmaceutical companies will want to oversee the production of
OcularGel 1'” once is licensed to them. However, if desired, BI will commercially

produce OcularGelTM at a Good Manufacturing Practice (GMP) approved manufacturing

87

site and commercial production of the pharmaceutical product incorporating OcularGelW

will be conducted by the licensee using their own GMP facilities.

88

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