DRUG RELEASE PROPERTIES OF CROSS-LINKED

N-CARBOXYMETHYLATED CHITOSAN WAFERS

(MINI DISSERTATION)
by
REFILWE MPHAHLELE
Submitted in partial fulfilment of the requirement for the degree
MASTER TECHNOLOGIAE PHARMACEUTICAL SCIENCES
In the
SCHOOL OF PHARMACY
FACULTY OF HEALTH SCIENCES
TSHWANE UNIVERSITY OF TECHNOLOGY
Supervisor: Prof. J .H. Hamman
Co-supervisor: Mr D. Nazer
2005


DECLARATION BY CANDITATE
I hereby declare that the mini dissertation submitted for the degree M. Tech: Pharmaceutical
Sciences, at Tshwane University of Technology, is my own original work and has not
previously been submitted to any other institution of higher education. I further declare that
all sources cited or quoted are indicated and acknowledged by means of a comprehensive list
of references.
Refilwe Mphahlele
Copyright Tshwane University of Technology






ACKNOWLEDGEMENTS
I would like to acknowledge the invaluable assistance and support that has enabled me to
proceed with this research from the following:
All The School of Pharmacy staff who have supported, helped and guided me throughout
this research. To Des and Sias, thanks a lot for including me in the chitosan team.
The Chemistry Department of Tshwane University of Technology, especially the
Garankuwa and Arcadia campuses; for their support, for availing materials and equipment.
Aventis Pharmaceuticals staff in Pretoriafor their support, time and equipment.
Mr N.F.H Makhubela for his assistance with the NMR spectrophotometer.
My loving familys patience, assistance and support.











DEDICATION



















In loving memory of my late parents, Christine and J oseph Morekhure.
Lord, thank you for the ethos and the strength.


TABLE OF CONTENTS
ABSTRACT ......................................................................................................................... 1
1 CHAPTER 1: Introduction ................................................................................... 3
1.1 Background and justification.....................................................................................3
1.2 Chitosan.....................................................................................................................3
1.3 Derivatives of chitosan..............................................................................................4
1.4 Modified drug release dosage forms.........................................................................5
1.5 Ibuprofen...................................................................................................................6
1.6 Research problem......................................................................................................7
1.7 Hypothesis.................................................................................................................7
1.8 Aims and objectives..................................................................................................8
1.9 Study methods and design.........................................................................................8
2. CHAPTER 2: CHITOSAN AND DERIVATIVES.............................................. 9
2.1 Introduction...............................................................................................................9
2.2 Properties and applications of chitosan.....................................................................10
2.2.1 Physicochemical properties.......................................................................................11
2.2.2 Uses and applications of chitosan..............................................................................13
2.2.2.1 Controlled drug release systems................................................................................13
2.2.2.2 Drug targeting............................................................................................................14

i


2.2.2.3 Other applications......................................................................................................14
2.3 Derivatives of chitosan..............................................................................................15
2.3.1 N-Phthaloyl chitosan.................................................................................................16
2.3.2 N-trimethyl chitosan chloride (TMC)........................................................................17
2.3.2.1 Physicochemical properties.......................................................................................19
2.3.2.2 Absorption enhancing properties...............................................................................19
2.3.3 Carboxymethyl chitosan (glycine glucan).................................................................20
2.3.3.1 Physicochemical properties.......................................................................................21
2.3.2.2 Absorption enhancing properties...............................................................................21
2.4 Conclusion.................................................................................................................23
3. CHAPTER 3: MODIFIED RELEASE DOSAGE FORMS ............................... 25
3.1 Introduction...............................................................................................................25
3.2 Nomenclature............................................................................................................26
3.2.1 Delayed release..........................................................................................................26
3.2.2 Targeted release.........................................................................................................27
3.2.3 Extended release dosage forms.................................................................................27
3.2.4 Repeat action.............................................................................................................27
3.2.5 Prolonged release.......................................................................................................27
3.2.6 Sustained release.......................................................................................................27
3.2.7 Controlled release......................................................................................................27

ii


3.3 Design and function of modified release dosage forms............................................28
3.3.1 Ideal properties of modified release dosage forms....................................................28
3.3.2 Types of modified release dosage forms...................................................................29
3.3.3 Selection of the type of dosage form.........................................................................33
3.3.4 Mechanisms of drug release......................................................................................33
3.3.5 Classification of modified release dosage forms.......................................................34
3.3.5.1 Monolithic matrix delivery systems..........................................................................35
3.3.5.2 Reservoir or membrane-controlled systems..............................................................35
3.4 Chitosan as a drug carrier..........................................................................................36
3.4.1 Cross-linking of polymers to form matrices and/or hydrogels..................................37
3.4.2 Coating of dosage forms............................................................................................39
3.5 Conclusion.................................................................................................................42
4. CHAPTER 4: N-CARBOXYMETHYL CHITOSAN PREPARATION AND
EVALUATION........................................................................................................... 43
4.1 Introduction...............................................................................................................43
4.2 Materials and equipment used...................................................................................43
4.3 Synthesis of N-Carboxymethyl chitosan...................................................................44
4.4 Characterisation of the synthesised N-carboxymethyl chitosan................................45
4.4.1
1
H and
13
C NMR characterisation.............................................................................52
4.4.2 Infrared (IR) characterisation....................................................................................55

iii


4.5 Cross-linking and wafer preparation.........................................................................56
4.6 Evaluation f N-carboxymethyl chitosan wafers........................................................56
4.6.1 Drug content..............................................................................................................56
4.6.2 Swelling properties....................................................................................................58
4.6.3 Ibuprofen release tests at 60 rpm and 37C...............................................................59
4.6.3.1 Dissolution profile at pH 5.8.....................................................................................60
4.6.3.2 Dissolution profile at pH 7.4.....................................................................................62
4.6.3.3 Dissolution profile at pH 8.0.....................................................................................64
4.7 Data analysis..............................................................................................................65
4.7.1 Mean dissolution time...............................................................................................65
4.7.2 Fit factors...................................................................................................................67
4.8 Conclusion.................................................................................................................68
5 Prospective studies....................................................................................................69
BIBLIOGRAPHY 70
APPENDICES 76

iv


ABSTRACT
The delivery of drugs at the right time in a safe and reproducible manner to a specific target
at the required level has spurred the advancement of many innovative drug delivery system
technologies. These technologies for effective drug delivery include nanotechnology,
implants, microencapsulation, chemical modification as well as cell and peptide
encapsulation. The use of absorption enhancing polymers in bioadhesive drug formulations
to promote drug penetration through and between intestinal cells has been a subject of
various investigations.
Biodegradable, bioadhesive and biocompatible chitosan has emerged as a multifunctional
excipient that has been extensively explored for use in several modified release dosage forms
(MRDFs) including hydrogels, microspheres, wafers, beads/microgranules and transdermal
drug delivery systems. The advantage of most MRDFs is the elimination of the typical peak
and valley fluctuations in plasma drug concentration after repeated dosing with
conventional, immediate release dosage forms. Furthermore, protonated chitosan has been
reported to interact with components of the tight junctions, thereby enhancing paracellular
transportation of macromolecules across the gastrointestinal epithelium. An increased
residence time has been attributed to its bioadhesive nature. These extraordinary
characteristics are marred by the limited solubility of chitosan; hence the need to produce
derivatives with increased solubility over a wide pH range.
The primary amino groups in the chitosan polysaccharide impart chemical reactivity that
enables regioselective chemical modification. This results in the formation of various useful
derivatives including N-phthaloylated chitosan, N,N,N-trimethyl chitosan, N-carboxymethyl
chitosan and others. The use of cross-linked N-carboxymethyl chitosan wafers (or matrix
systems) was explored in this study for controlled release of the model drug, ibuprofen.
Theoretically, the added carboxyl (COOH) group should enhance its chelation properties,
which in turn are linked to its hydrogel formation properties. Barium chloride was used as
the cross-linker to ionically bind N-carboxymethyl chitosan chains to each other in order to
produce a hydrogel that was utilised to produce the wafers. The cross-linked N-

1
carboxymethyl chitosan was the only excipient used to make the hydrogel wafers thereby
reducing interference or erroneous deductions in the investigation.
The aim of this study was to investigate the hypothesis that drug release can be controlled by
wafers consisting of cross-linked N-carboxymethyl chitosan in such a way that constant drug
quantities are released over an extended period of time. The drug release behaviour of the
cross-linked N-carboxymethyl chitosan wafers was evaluated in three different phosphate
buffers with pH values of 5.8, 7.4 and 8.0, respectively, to emulate the pH variation along the
gastrointestinal tract.
The results of this study demonstrated that the rate of ibuprofen release from the N-
carboxymethyl chitosan wafers was lower than its release from an immediate release dosage
form in the control group at all the pH values. However, the rate of ibuprofen release from
the wafers was higher in the slightly acidic environment (pH 5.8) as compared to the neutral
and alkaline environments. This higher release rate at a slightly acidic environment was
attributed to the excess protons (H
+
) that compete with the barium cation (Ba
2+
) for
interacting with the N-carboxymethyl chitosan chains resulting in reversed cross-links with a
consequent faster erosion rate of the wafers at this pH value. This was confirmed by the
mass reduction of the wafers in the test for swelling properties.
The cross-linked N-carboxymethyl chitosan wafers showed potential as controlled release
dosage forms with higher mean dissolution time values as compared to the control group, but
different formulation aspects should be investigated in future studies to improve on the
performance of these wafers. Inclusion of binders and plasticisers to improve the mechanical
strength should be studied.





2
CHAPTER 1: INTRODUCTION
1.1 BACKGROUND AND JUSTIFICATION
The concentration of a drug in the blood fluctuates over successive administrations of
conventional single unit dosage forms. The main reason for this is because conventional
dosage forms are designed to release the complete dose of the drug immediately after
administration (i.e. burst release effect). This causes the drug blood concentration to rise to a
high value followed by a subsequent fall to a very low level as a result of elimination.
Forgotten doses or overnight no-dose periods may contribute further to this problem. These
fluctuating drug blood levels can be addressed by means of formulating dosage forms with
pre-determined drug release profiles. The ideal drug delivery system would keep the drug
blood plasma level constant over the treatment period after administration of a single dose
(Colett & Moreton, 2002).
These modified release dosage forms render invaluable therapeutic benefits such as reduced
frequency of drug administration, better patient compliance, improved control over the
maintenance of the plasma drug concentration, reduction in the severity and incidence of
localised gastrointestinal side effects as compared to conventional dosage forms as well as
cost saving because of better disease management (Colett & Moreton, 2002).
In this study, an attempt is made to formulate a controlled release drug delivery system in the
form of a cross-linked N-carboxymethyl chitosan matrix system or wafer.
1.2 CHITOSAN
Chitosan is formed by partial deacetylation (40 - 98%) at the N-position of the linear polymer
of N-acetylglucosamine (i.e. chitin), which is found in crustacean shells and is also present in
some microorganisms and fungi such as yeasts. It is a linear amino polysaccharide made up
of copolymers of N-acetyl-D-glucosamine and D-glucosamine. -1,4-gylcosidic bonds link-
up the sugar backbone to form (1-4)-2-amino-2-deoxy--D-glucan. The chemical structure
of a portion of the polymer chain is given in Figure 1.1. The absence of -glycosidase in the

3
upper part of the gastrointestinal tract implies that the -1,4-gylcosidic bond of chitosan will
only be degraded by bacterial -glycosidases in the lower part of the colon.

Figure 1.1: Chemical structure of a portion of a chitosan polymer chain (Kurita, 2001).
Chitosan is a bioadhesive, biocompatible, biodegradable, water insoluble, weak basic
polymer with a pKa of about 6.2 to 7.0, which means that it is insoluble in neutral and
alkaline media (Thanou, Verhoef & Junginger, 2001 and Hejazi & Amiji, 2003). Its
molecular mass ranges between 50 000 and 2 000 000 Da and it exists in a quasi-globular
conformation, which is stabilised by extensive intra- and intermolecular bonding. This is the
result of the highly reactive amino and hydroxyl groups, which contribute to the high
viscosity of chitosan solutions and also enhance chelation. It exhibits polymeric cationic
characteristics as well as gel and film-forming properties and enhances the rate of drug
absorption (Illum, 1998) with no indication of epithelial damage or cytotoxicity (Thanou,
Verhoef &Junginger, 2001).
Chitosan has various pharmaceutical uses such as a drug carrier, absorption enhancement,
controlled drug release, gene delivery, metal chelating agent, artificial skin, fungicide,
contact lens, weight loss aid, cholesterol lowering agent, burn dressings, clarification of
beverages and as a food supplement (Senel & McClure, 2004).
1.3 DERIVATIVES OF CHITOSAN
Derivatives of chitosan formed by N-substitution with carboxyl bearing groups showed
increased aqueous solubility and zwitterionic character. This allows for the formation of
clear gels at neutral and alkaline pH values but promotes aggregation in an acidic pH
environment (Thanou, Verhoef & Junginger, 2001). The aggregation behaviour led to the

4
need to cross-link the molecules to overcome cationisation of the amino groups and to
stabilise chitosan in acidic solutions.
Carboxymethyl chitosans are potentially multi-dentate ligands. Hejazi and Amiji (2003)
explained that these structural features render them potentially able to chelate metal ions like
Ca
2+
, Ni
2+
and Ba
2+
,

via the nitrogen lone pairs and the negative charge of the carboxylic
group to form stable complexes. Carboxymethyl chitosans aid with absorption by enhancing
paracellular transport through the controlled, transient and reversible opening of intestinal
tight junctions without being absorbed or interacting with charges of the lipid bilayer of the
cell membrane.
N-trimethyl chitosan chloride (TMC) is a soluble cationic methylated derivative of chitosan
(Le Dung et al., 1994), which can widen the paracellular route for the passage of hydrophilic
and macromolecular drugs after mucosal administration (Kotze et al., 1997 and Hamman et
al., 2003).
1.4 MODIFIED DRUG RELEASE DOSAGE FORMS
After oral administration of a conventional, immediate release dosage form, there is an
increase in the plasma drug concentration followed by an exponential decay. To achieve a
predetermined kinetic profile of plasma drug concentration it may be necessary to modify the
dosage form. An ideal modified release dosage form (MRDF) should be capable of releasing
a known amount of drug at a predetermined rate in order to produce an optimum therapeutic
effect. The most common advantage of modified release dosage forms is the elimination of
the typical peak and valley fluctuations in plasma drug concentration after repeated dosing
with conventional dosage forms (Lund, 1994).
There has been considerable interest in developing controlled or sustained drug delivery
systems using polymeric microspheres. Chitosan has attracted attention as a potential matrix
for use in controlled release dosage forms due to its favourable characteristics such as its
biodegradability and non-toxic nature (Thanoo, Sunny & Jayakrishnan, 1992). Cross-links
between polymer functional groups result in decreased hydrophilicity, thus slowing down the
rate of permeation of biological fluids throughout the matrix system. Consequently, the rate

5
of drug diffusion through the hydrated polymer also decreases. The use of complexation
between oppositely charged macromolecules to prepare chitosan beads as a controlled release
formulation for drugs is therefore a very useful way of modifying drug release (Shu & Zhu,
2000)
1.5 IBUPROFEN
The model drug, ibuprofen, is also known as 2-(4-isobutylphenyl)propionic acid and the
chemical structure is shown in Figure 1.2. Ibuprofen is a weakly acidic non-steroidal anti-
inflammatory drug that is slightly insoluble in water. A pK
a
of about 3.6 implies insolubility
in an acidic medium.
CHCOOH
CH
3
CH
2
C
CH
3
CH
3
H

Figure 1.2: Chemical structure of ibuprofen or 2-(4-isobutylphenyl) propionic acid.
The biological half-life of 1.8 to 2 hours means that an oral dosage formulation of this drug
will be rapidly and almost completely absorbed from the gastrointestinal tract with peak
plasma concentrations occurring within 1-2 hours. The main adverse effects of ibuprofen are
gastrointestinal disturbances. In addition, poor solubility in water together with the short
plasma elimination half-life affect its bioavailability hence ibuprofens suitability for
modified release dosage forms. A wide therapeutic range or window of 1200 to 1800 mg/day
up to a maximum of 3200 mg/day allows formulation into controlled release dosage forms
with minimal risk of toxicity. Some of the therapeutic parameters mentioned above are
illustrated in Figure 1.3 (Martindale, 2002 and Ashford, 2002).

6

Figure 1.3: A plasma concentration-time curve illustrating the parameters associated with
the therapeutic or pharmacological response (Ashford, 2002)
1.6 RESEARCH PROBLEM
The drug dose is released immediately after administration of conventional dosage forms
with a quick rise in drug blood level and a subsequent decrease due to elimination. These
fluctuations may lead to toxic effects (at the peaks) or no therapeutic effect at all (at the
valleys). These typical peak and valley drug plasma levels may be overcome by
formulation of a modified release dosage form with predetermined drug release rate.
The question that needs to be answered in this study is: Can drug release be controlled by
wafers consisting of cross-linked N-carboxymethyl chitosan in such a way to ensure constant
drug release over an extended period of time?
1.7 HYPOTHESIS
Drug release will be controlled by wafers consisting of cross-linked carboxymethyl chitosan
in such a way that constant drug quantities are released over an extended period of time.

7
1.8 AIMS AND OBJECTIVES
The aim of this investigation is to determine if the rate of ibuprofen release can be sustained
over an extended period by the formulation of cross-linked N-carboxymethyl chitosan
wafers. This will be supported by the following objectives:
The synthesis, infrared (IR) and nuclear magnetic resonance (NMR) characterisation
of the chitosan derivative, N-carboxymethyl chitosan,
The identification of an appropriate cation or ligand for the preparation of cross-
linked N-carboxymethyl chitosan hydrogel to prepare wafers containing ibuprofen as
a model drug,
The evaluation of the drug release properties of the wafers by means of dissolution
tests at different pH values (representative of an acidic, neutral and alkaline
environment as in the gastrointestinal tract) and
Analysis of the dissolution profiles by calculating the mean dissolution time (MDT),
difference factor (f
1
) and similarity factor (f
2
).
1.9 STUDY METHODS AND DESIGN
This is a quantitative research study with a true experimental design where the dependent
variable (ibuprofen release) was manipulated (formation of cross-linked N-carboxymethyl
chitosan wafers) to measure the effect, but all other conditions were kept constant. Control
groups were included to indicate that the measured effect was indeed caused by the
manipulation and not by other external factors or chance interferences. The dissolution tests
were done in triplicate at different pH values and the results were reported with an analysis of
the dissolution curves in terms of the mean dissolution time (MDT) as well as the difference
(f
1
) and similarity factors (f
2
) for the efficient differentiation between overall release
patterns or the border line release profile differences.



8
CHAPTER 2: CHITOSAN AND CHITOSAN DERIVATIVES
2.1 INTRODUCTION
Chitosan is a cationic amino polysaccharide produced by the incomplete alkaline N-
deacetylation of chitin, a naturally occurring nitrogeneous polysaccharide and supporting
material in the exoskeletons of crustaceans and insects. Chitin is structurally similar to
cellulose. The latter is a commonly used tablet excipient and has hydroxyl groups at the C-2
position in contrast to chitin which has acetamido groups [i.e. NH(C=O)CH
3
] at this position.
Chitosan has in addition to the acetamido groups also amino groups, NH
2
, at a certain
number of the C-2 positions as shown in Figure 2.1 (Kurita, 2001).
Chitosan has remarkable intrinsic properties such as biodegradability, bioadhesiveness
biocompatibility and drug absorption enhancement properties and has been used in drug
controlled release systems. Some of these unique characteristics are obscured by the limited
solubility of chitosan in more basic aqueous media similar to the intestines and the colon.
Consequently, this prompted extensive investigations to modify its structure and to
investigate pharmaceutical properties of various chitosan derivatives (Kurita, 2001).
Muzzarelli et al. (1982) demonstrated that N-carboxymethylation of chitosan could be
regioselectively synthesized using commercial grade reagents. Babak et al. (1999)
demonstrated that the surface activity of carboxymethyl chitin could be improved. A
separate study by Babak & Rinaudo (2002) confirmed the interaction between hydrosoluble
chitosan derivatives with surfactant to form surfacatant-polyelectrolyte complexes via
electrostatic and hydrophobic interactions.
A study by Kotz et al. (1997) and Hamman et al. (2003) revealed that the charge density
and the structural features of chitosan salts and N-trimethyl chitosan chloride (TMC) are
important factors that determine their potential use as absorption enhancers. Other TMC
studies by Kotz et al. (2002) demonstrated that TMC is an absorption enhancer over a wide
pH range and that TMC polymers with high degrees of quarternisation are less mucoadhesive
compared to those with lower degrees of quartenisation and this was attributed to changes in

9
the flexibility of the polymer chain. The same study revealed that TMC had no toxic effects.
According to Hamman & Kotze (2002), TMC reduced the transepithelial electrical resistance
(TEER) of Caco-2 cell monolayers in a slightly acidic environment (pH 6.2). TMC polymers
with higher degrees of quaternisation were found to have a higher number of positive charges
available for more electrostatic interactions between TMC and the cell membranes, resulting
in a greater number of tight junctions that are opened to allow for paracellular movement of
ions and thereby reduction of TEER.
2.2 PROPERTIES AND APPLICATIONS OF CHITOSAN
Chitosan refers to a large number of heteropolymers which differ in their degrees of N-
deacetylation (40 - 98%), viscosity grades and their molecular weights which vary between 2
000 Da for oligomers and 50 000 2 000 000 Da for polymers (Hejazi & Amiji, 2003). The
chemical structures of cellulose, chitin and chitosan including the synthetic step of partial
basic N-deacetylation of chitin to form chitosan are presented in Figure 2.1.


Figure 2.1: Comparison of the chemical structures of cellulose, chitin and chitosan
including N-deacetylation of chitin to form chitosan (Kurita, 2001).
Figure 2.1 shows that chitosan is a polysaccharide made up of copolymers of N-acetyl-D-
glucosamine and D-glucosamine with a sugar backbone linked by -1,4 gylcosidic bonds to
form -(14) 2-amino-2-deoxy--D-glucan (Kurita, 2001). The glycosidic linkages of
chitosan are relatively stable against alkali but cleaved with acid and undergo total hydrolysis

10
with warm hydrochloric acid (HCl) to give the constituting monosaccharide, D-glucosamine
whilst mild acidic hydrolysis yields N-acetyl-D-glucosamine. In addition, the two hydroxyl
groups in the repeating hexosamine residue give rise to various novel biofunctional
hydrophobic macromolecular products with longer side chains. Enzymatic hydrolysis of
chitosan with lysozyme results in oligomer production whilst chitosanase from Bacillus sp.
gives dimer to pentamer products without producing the monomer. Enzymatic reactions that
occur in the lower part of the colon result in non-toxic degradation products (Kurita, 2001).
2.2.1 Physicochemical properties
Most naturally occurring polysaccharides like cellulose, dextran, pectin, alginic acid, agar,
agarose and carragenans are neutral or acidic in nature whereas chitosan is a weak base with
a pK
a
value of about 6.2 - 7.0 and is therefore insoluble in neutral/aqueous and alkaline pH
media.
The presence of the amino group has a significant contribution to the characteristics of
chitosan as illustrated below:
The amine groups of chitosan are protonated in an acidic medium resulting in a
soluble polysaccharide with a relatively high charge density; one positive charge for
each D-glucosamine unit (Hejazi & Amiji, 2003).
Ravi Kumar, (2000) pointed out that due to the presence of the primary amino
groups, which are absent in cellulose, chitosan undergoes reactions typical of amines
with the most important ones being the Schiff reactions and N-acylation illustrated in
Figures 2.2 and 2.3, respectively.

Figure 2.2: Reaction of the free amino group of chitosan with an aldehyde to give the
corresponding Schiff base (Kurita, 2001).

11

Figure 2.3: Fully acylated chitosan at the amine and hydroxyl groups (Kurita, 2001).
Hejazi & Amiji (2003) stated that chitosan exists in a quasiglobular conformation
stabilised by extensive intra- and intermolecular hydrogen bonding as a result of the
highly reactive amino and hydroxyl groups, which also accounts for the high
viscosity of chitosan solutions. The amino groups also impart secondary amine
reactivity for modification reactions, which are not possible cellulose.
Chitosan forms hydrogels of which the swelling kinetics is affected by the extent of the
dissociation of the hydrogen bonds. They exhibit polymeric ionic characteristics and film
forming properties due to intermolecular hydrogen bonding (Illum, 1998; Thanou, Verhoef &
Junginger, 2001). Kurita, (2001) pointed out that chitosan is soluble in dilute solutions of
hydrochloric acid and aqueous organic acids such as formic acid, acetic acid, oxalic and
lactic acids and forms water-soluble salts with organic and inorganic acids such as glutamic
acid, acetic acid and hydrochloric acid. Chitosan with a low degree of deacetylation (i.e.
40%) is soluble up to a pH of 9, whereas highly deacetylated chitosan (i.e. 85%) is soluble
only up to a pH of 6.5. The latter has increased viscosity and an extended conformation with
a more flexible chain because of the charge repulsion in the molecule (Hejazi & Amiji,
2003). According to Senel & McClure, (2004) the high molecular weight of chitosan and a
linear unbranched structure make chitosan an excellent viscosity-enhancing agent in acidic
environments. It behaves as a pseudoplastic material exhibiting a decrease in viscosity with
increasing rates of shear. Kurita, (2001) explains that the degree of acetyl [NH(C=O)CH
3
]
substitution affects the adsorption of Cu(II). The adsorption capacities increase markedly in
the region of low substitution with increased hydrophilicity and decreases with further acetyl
substitution, which results in enhanced hydrophobicity.

12
2.2.2 Uses and applications of chitosan
Chitosans unique characteristics, low toxicity and FDA approval as a pharmaceutical
excipient (Muzzarelli, 1996) have increased its pharmaceutical applications and other uses as
indicated below.
2.2.2.1 Controlled drug release systems
Controlled drug release systems are designed to produce an optimum therapeutic response,
less side effects, prolonged efficacy, enhanced safety and reliability of drug therapy,
regulated drug release rate and reduced frequency of drug administration to encourage patient
compliance (Collet & Moreton, 2000). Chitosan has been utilised in several modified release
dosage forms as explained in the following examples.
pH-dependent chitosan hydrogels (Chen, Tian & Du, 2003), which are highly
swollen, hydrophilic polymer networks that can absorb large amounts of water and
drastically increase in volume depending on the molecular structure, gel structure,
degree of cross-linking, content and state of the water in the hydrogel (Yao et al.,
1998).
Chitosan tablets for controlled release as observed in anionic-cationic inter-polymer
networks (Mi et al., 1997). They reported alginate as an anionic polyelectrolyte to
control the swelling and erosion rates of chitosan tablets in acidic media.
Cross-linked chitosan microspheres for the controlled release of ibuprofen,
griseofulvin and aspirin (Arica et al., 2002 and Thanoo, Sunny & Jayakrishnan,
1992).
Controlled release studies by Gupta & Kumar, (1999) on diclofenac sodium release
from chitosan beads/microgranules and Arica et al. (2002) on sustained release of
ibuprofen from chitosan microspheres.

13
Transdermal drug delivery systems were designed using cross-linked chitosan
membranes to regulate propranolol release rate, which was found to depend on the
cross-link density within the membranes (Thacharodi & Rao, 1995).
2.2.2.2 Drug targeting
Chitosan can be used for drug release at specific sites, which include oral gene delivery
involving inhibition of DNA degradation facilitated by chitosan-DNA complexes, mucosal
vaccination, stomach-specific drug delivery, intestinal delivery and colon-specific delivery of
drugs (Hejazi et al., 2003).
2.2.2.3 Other applications
These are attributed to chitosans unique properties that promote its extensive use in industry
as outlined by Ravi Kumar, (2000) for various purposes such as
Removal of harmful metals for the detoxification of hazardous waste due to chitosans
versatility to adsorb metal ions and surfactants as well as chemical modification to
attract dyes and other moieties (Ravi Kumar, 2000),
Antacid and anti-ulcer characteristics which prevent drug irritation in the stomach and
thereby making chitosan a potentially suitable carrier in sustained drug delivery
systems (Gupta & Kumar, 2000 and Hou et al., 1985),
Antibacterial activity was demonstrated on the growth of E. coli and Fusarium
Alternaria attributed to the interaction of the chitosan C-2 cationic amino groups with
the anionic groups of the microorganisms (Hirano, 1995),
Fat trapping involves the positively charged chitosan on the N-position binding free
fatty acids and bile components thereby retarding their absorption (Muzzarelli, 1999),
Photography involves the use of chitosans optical characteristics, film forming
ability, resistance to abrasion and fungicidal properties (Muzzarelli, 1997),

14
Opthalmology exploits chitosans optical clarity and correction, mechanical stability,
immunological compatibility to make contact lenses (Ravi Kumar, 2000),
Chromatography utilises the chiral field of chemically modified chitosan stationary
phase for asymmetric recognition and optical resolution of racemic mixtures (Senso,
Oliveros & Minguilln, 1999).
2.3 DERIVATIVES OF CHITOSAN
The pH-dependent solubility of chitosan presents a potential problem in the design and
formulation of sustained release dosage forms because of the variation in pH along the
gastrointestinal tract as presented in Figure 2.4. In addition, poor mechanical strength, the
quest for efficacy, safety and non-toxicity of chitosan controlled-release drug delivery
systems substantially influenced the course of chitosan research. This resulted in chemical
modifications by substitution at the N and/or O-position(s) to improve dissolution in various
media (Le Dung et al., 1994; Kurita, 1997; Thanou, Verhoef & Junginger, 2001; and Van der
Lubben et al., 2001).

Figure 2.4: pH variation along the gastrointestinal tract (Ashford, 2002).

15
The characteristic crystalline structure with intermolecular forces, limited solubility and
multifunctional nature of chitosan make chemical modifications difficult and necessitate
careful manipulation and proper control of the reactions such as the use of appropriate
regioselective reagents and reaction conditions which are mainly heterogeneous (Ravi
Kumar, 2000). Reactions with ketoacids followed by sodium borohydride reduction produce
glucans with proteic (e.g N-carboxymethyl chitosan) or non-proteic (e.g N-carboxybenzyl
chitosan) amino groups (Kurita, 2001 and Ravi Kumar, 2000). Different derivatives have
been explored together with trimethyl chitosan as absorption enhancers and for controlled
drug delivery by various researchers including Chen, Tian & Du, (2003), Thanou, Verhoef &
Junginger, (2001) and Babak et al, (1999) and Hamman & Kotze (2002).
2.3.1 N-Phthaloyl chitosan
N-Phthaloyl chitosan is formed by treating chitosan with excess phthalic anhydride at 120-
130
o
C to form both partial O- and fully N-phthaloylated chitosan that is soluble in dimethyl
sulfoxide to protect the amino functionalities of the chitosan. Hydrazine can be used to
regenerate the free amino group(s) thereby providing a key intermediate to prepare
derivatives with well-defined structures under mild conditions and perfect discrimination of
functional groups for regioselectivity and quantitative viewpoints as illustrated in Figure 2.5.

Figure 2.5: Typical reactions to illustrate that the phthaloyl group is suitable for protection
of the amino group (Kurita, 2001 and Nishimura et al., 1991).

16
This derivative is also useful for solubilisation in organic solvents since the bulky phthaloyl
group prevents hydrogen bonding by eliminating hydrogens from the amino group (Kurita,
2001 and Nishimura et al., 1991). Other applications include the synthesis of non-natural
branched polysaccharides with potential biological functions such as immuno-adjuvant
activities and those with amino sugar branches, as shown in Figure 2.6, which have been
found to exhibit antimicrobial activity that is better than chitosan (Kurita et al., 2000).

Figure 2.6: Synthesis of polysaccharides with amino sugar branches by using an oxazoline
derived from N-acetylglucosamine to C-6 silylated OH acceptor in 1,2-
dichloroethane/camphorsulfonic acid at 80
o
C (Kurita, 2001 and Kurita et al, 1998).
2.3.2 N-trimethyl chitosan chloride (TMC)
Amines are Brnstead bases and hence are also nucleophiles that undergo bimolecular
nucleophilic substitution, S
N2
, reactions with alkyl halides to form alkylammonium ions.
Further alkylation may proceed if more N-H bonds are present as in amine quaternisation
(Loudon, 1995).
Exhaustive methylation of the primary amine of chitosan yields quaternary N-trimethyl
chitosan iodide as illustrated in Figure 2.7.

17

Figure 2.7: Preparation of a N,N,N-trimethyl chitosan iodide (monomer shown) by
methylation of chitosan (Thanou, Verhoef & Junginger, 2001).
According to Loudon (1995), alkyl substitution tends to stabilise the basicity of amines. This
can be explained by the fact that the alkyl groups exert a polarisation effect on the nitrogen
atom wherein the electron clouds of the alkyl group distort so as to create a net attraction
between them and the positive charge of the ammonium ion as illustrated in Figure 2.8.

Figure 2.8: Reinforcement of the basicity of an amine by quaternisation (Loudon, 1995).
Other known quaternary salts applied in the medical field are triton B and benzalkonium
chloride, a common antiseptic and a surfactant in water and is also soluble in organic
solvents (Loudon, 1995).



18
2.3.2.1 Physicochemical properties
N,N,N-trimethyl chitosan chloride is a fully ionic compound and has higher aqueous
solubility than chitosan in a much broader pH range, acidic to neutral media. This is
attributed to the fact that the alkyl groups tend to prevent hydrogen bond formation between
hydoxyl and amine groups of the chitosan backbone (Thanou, Verhoef and Junginger, 2001).
A study by Snyman et al. (2002) revealed that reductive alkylation results in an increase in
the molecular weight of the polymer due to the addition of methyl groups to the amino group
of the repeating monomers. The same study demonstrated that the intrinsic viscosity, as an
indication of the molecular weight, decreases with an increase in the degree of quaternisation
of the TMC polymers.
2.3.2.2 Absorption enhancing properties
The charge, charge density and the structural features of chitosan salts and N-trimethyl
chitosan chloride (TMC) are important factors which determine their potential use as
absorption enhancers for peptide drugs by reversibly interacting with components of the tight
junctions between epithelial cells and thereby widening paracellular routes for the passage of
hydrophilic and macromolecular drugs after mucosal administration (Kotze et al.,1997;
Hamman et al., 2001 and Van der Lubben et al., 2001).
A study by Thanou, Verhoef and Junginger, (2001) indicated that a threshold value of the
charge density of TMC is necessary to trigger the opening of the tight junctions at neutral pH
values. In addition, some studies revealed that the TMC polymer does not provoke cell
membrane damage on Caco-2 intestinal monolayers during enhancement of the transport of
hydrophilic macromolecules. This suggests that the mechanism of opening the tight
junctions is similar to that of protonated chitosan, that is, a specific interaction of the cationic
polymer with components of the tight junctions.
Figure 2.9 is an illustration of the junctional complexes between adjacent epithelial cells
including the tight junctions.

19

Figure 2.9: Tight junctions between adjacent epithelial cells.
Van der Lubben et al., (2001) indicated that the positive charge on TMC is only suitable for
improved delivery and absorption of hydrophilic, macromolecular drugs with neutral or basic
properties and that N-substitution of chitosan with moieties bearing carboxyl groups, yielding
polyampholytic (zwitterionic) polymers is a promising approach, hence the investigation of
carboxymethyl chitosan as a drug absorption enhancer and a potential candidate for novel
drug delivery systems.
2.3.3 Carboxymethyl chitosan (glycine glucan)
Muzzarelli et al. (1982) pointed out that this N-carboxymethyl chitosan is a novel
polyampholyte, which may be prepared from a variety of chitosans differing in molecular
sizes, molecular-weight distributions, and degrees of deacetylation by treating them with
various amounts of glyoxylic acid. In addition, the N- and O-derivatives are chemically
different because the new group [NHCH
2
COOH] at the N-position is chemically similar to
glycine and hence raises interest in pharmaceutical chemistry. The lone pairs from the N-C-
C-O sequence of glycine contribute towards chelation properties superior to those exhibited
by tertiary amines of ethylene diammine tetraacetic acid (EDTA).

20
Carboxymethyl chitosan is synthesised by the reaction shown in Figure 2.10 and has amino,
glycine or acetamido groups at the C-2 position interspaced by glucose rings.

Figure 2.10: Formation of N-carboxymethyl chitosan from the reaction of chitosan with
glyoxylic acid followed by reduction with sodium borohydride (Thanou, Verhoef &
Junginger, 2001). MCC = monocarboxymethyl chitosan
2.3.3.1 Physicochemical properties
Thanou, (2001) observed that carboxymethyl chitosan with 87 - 90% degree of substitution
has polyampholytic (zwitterionic) character, which allows the formation of clear gels or
solutions depending on the polymer concentration at neutral and alkaline pH values but
aggregates under acidic conditions. Another significant characteristic is the complete
solubility of N-carboxymethyl chitosan at all pH values.
2.3.3.2 Absorption enhancing properties
Robinson & Lee, (1997) noted that bioadhesive agents allow close contact of peptides to the
mucous lining, while at the same time minimising transit so that a high concentration
gradient across the membrane can be maintained for extended periods of time. This may
permit penetration enhancers and enzyme inhibitors to be used at lower concentrations
thereby lessening toxicity and irritation. This is achieved by the interaction of a number of
hydrophilic groups such as carboxyl (COOH), hydroxyl (OH), amide and sulfate (SO
4
)
groups. In addition, some bioadhesive agents have inherent penetration-enhancing effects
because they are effective ion chelators. These bioadhesive compounds can chelate calcium
ions (Ca
2+
) in physiological buffers to affect the opening of tight junctions that is calcium
dependent. In view of the description of a bioadhesive and the structure and nature of

21
carboxymethyl chitosan, it seems to be a potential candidate for absorption enhancing
bioadhesives as it contains the carboxyl, hydroxyl and amide groups. They further highlight
the limitations associated with bioadhesive agents as:
Fouling of the bioadhesive sites of the polymer before reaching the desired target,
Rapid rate of mucus (or mucin) turnover making long-term adhesion impossible or too
slow to allow further passage of drug delivery system.
Thanou, Verhoef and Junginger, (2001) demonstrated that the polyanionic
monocarboxymethyl chitosan (MCC) concentration necessary to induce a 50% decrease in
transepithelial electrical resistance (TEER), an indication of opening of the tight junctions,
was several times higher than that of polycationic N,N,N-trimethyl chitosan chloride (TMC)
at a neutral pH. In the same study, intra-duodenally administered MCC-bound Anti-Xa
serum and onset of absorption was delayed compared to when TMC was used. The delay
was attributed to the fact that MCC is unstable in acidic pH at the beginning of the duodenum
suggesting better absorption enhancing effect in intestinal medium.
As indicated under 2.2.2.1, a recent study by Di Colo et al., (2004) confirmed that
polyanionic N-carboxymethyl chitosan failed to enhance intraocular (pH 2) drug penetration
but increased precorneal ofloxacin retention due to its viscosity-increasing effect and
mucoadhesive binding to ofloxacin. The polyampholytic character favoured the formation of
clear gels or solutions through metal chelation and this depended on the concentration of the
polymer. The chitosan residue retained the protonated structure, which is essential for
enhancing paracellular drug absorption across the epithelial tight junctions even as
monocarboxymethyl chitosan.
Robinson & Lee, (1997) further advanced a concept of intelligent polymers, which refer to
soluble, surface-coated or cross-linked polymer systems that exhibit relatively large, sharp
physical or chemical changes in response to small physical or chemical stimuli such as
temperature, pH, solvent or electric field. They can change their properties rapidly according
to the environment. These polymers may be useful in the development of the next generation
of bioadhesive polymers that can target a drug to the desired site of absorption.

22
The results from studies by Thanou et al., (2001) and Di Colo et al., (2004) present a
problem for carboxymethyl chitosan use in the formulation of controlled drug release
delivery systems if linked to pH variations along the gastrointestinal tract. Muzzarelli et al.,
(1982) indicate that the chelation of metals by the carboxylate ion, -COO

, of carboxymethyl
chitosan causes cross-linking of the polymer chains to yield swellable material or hydrogel in
pH dependent processes. The resultant reversible ionically-linked hydrogel is thought to be
significantly less cytotoxic than the hydrogel formed by glutaraldehyde (covalently-linked)
and is prepared in a simple and mild method for hydrogel formation. In addition, the
ionically cross-linked hydrogels seem to have more potential in the medical and
pharmaceutical fileds, since they are often biocompatible and have been investigated for
controlled drug delivery. Their disadvantages are the possible lack of mechanical stability
and the risk of dissolution of the system due to a highly pH-sensitive swelling (Berger et al.,
2004).
2.4 CONCLUSION
The production of chitosan from an abundant natural product, chitin, is done under moderate
conditions using simplified methods. Chitosan is a nitrogeneous polysaccharide with
favourable characteristics, but is insoluble at neutral and alkaline pH values. The remarkable
physicochemical properties of N-carboxymentyl chitosan, a chitosan derivative, including
complete aqueous solubility and the fact that it may be synthesised from commercial grade
reagents (Muzzarelli et al, 1982) make the exploration of cross-linked N-carboxymentyl
chitosan in various controlled drug release dosage forms an economically viable venture.
Moreover, the uses of chitosan and N-carboxymentyl chitosan in drug delivery have been
shown in several studies. Cross-linked chitosan has been shown to be a suitable matrix for
peroral microspheres for the controlled release of griseofulvin (Chithambara Thanoo, 1992;
Sunny & Jakakrishnan, 1992). Gupta & Kumar, (2000) demonstrated that the pH-dependent
pulsed release behaviour of glutaraldehyde and spacer group glycine cross-linked peroral
chitosan beads and microgranules could be altered by modifying the formulations to obtain
the desired controlled drug delivery systems. N-carboxymentyl chitosan has been found to
elicit negligible toxicity (Muzzarelli et al., 1982; Le Dung et al., 1994).

23
Previous studies to investigate nonparenteral routes of administration to overcome problems
like patient non-compliance with the parenteral route while maintaining therapeutic plasma
drug concentrations, have confirmed that N-carboxymentyl chitosan can be safely
administered via various routes including the ocular (Di Colo et al., 2004) and peroral route
(Thanou et al., 2001; Chen, Tian & Du, 2004).
The unique physicochemical properties of N-carboxymethyl chitosan suggest that this
chitosan derivative is a potentially viable candidate for various dosage forms including
controlled release dosage forms.















24
CHAPTER 3: MODIFIED RELEASE DOSAGE FORMS
3.1 INTRODUCTION
Various innovative technologies for effective drug delivery have been developed, including
implants, nanotechnology, microencapsulation, chemical modification and others. This
development in technology was spurred by the quest to deliver drugs at the right time in a
safe and reproducible manner and to a specific target at the required concentrations.
Modified release drug formulations attempt to obtain zero-order release or a slow first-order
system to maintain required blood drug levels over extended periods. The drug delivery rate
is intended to balance the drug elimination rate as represented mathematically in Equation
3.1 (Jantzen & Robinson, 2002; Orive et al., 2003).
d d e
xV xC k
lim
out Rate in Rate = = (3.1)
C
d
is the desired drug level, V
d
is the volume of distribution and k
elim
is the rate constant for
drug elimination. Figure 3.1 shows the pharmacokinetic patterns of a drug after the
administration of a conventional, controlled and sustained release dosage form, respectively.

Figure 3.1: Drug plasma-time profile of a zero-order controlled release, a slow-first-order
sustained release and a conventional dosage form (Jantzen & Robinson, 2002).

25
The advancement of drug delivery system development is influenced by physiological factors
such as the acidic conditions of the stomach, the first-pass effect of the liver and the intestinal
reduction of drug bioavailability. Physicochemical factors including aqueous solubility,
ionisation, pKa, dose size, partition coefficient and stability also had a significant role in the
course of modified release dosage form development. Moreover, over the years researchers
have managed to address several issues such as the need for suitable approved scientific
research, the impact of altered scientific policy on specific financial support, government
regulations and market forces present challenging barriers (Orive et al., 2003 and Jantzen &
Robinson, 2002).
3.2 NOMENCLATURE
Several literature sources such as Shargel and Yu, (1999) and Collet and Moreton, (2002)
acknowledge different terms to describe modified release dosage forms (MRDFs), which
include delayed release, extended release, repeat action, prolonged action, sustained release
and controlled release.
3.2.1 Delayed release
There is a release of discrete portion(s) of drug at a time(s) other than promptly after
administration although one portion may be released immediately after administration as a
loading dose. Common examples in this category are enteric-coated products such as
microspheres/beads in capsules (Shargel & Yu, 1999).
3.2.2 Targeted release
Drug release occurs at or near the intended physiologic site of action or site of absorption.
This may have either immediate or extended release characteristics as in nitroglycerine
sublingual tablets and transdermal patches (Transderm-Nitro

), respectively (Shargel & Yu,

1999).



26
3.2.3 Extended release dosage forms
There is a slow drug release and plasma concentrations are maintained at a therapeutic level
for a prolonged period, usually between 8 and 12 hours (Collet & Moreton, 2002).
3.2.4 Repeat action
The first dose is released fairly soon after administration, the second or third doses are
subsequently released at intermittent intervals after administration of a single dosage form
(Collet & Moreton, 2002)
3.2.5 Prolonged release
The drug is provided for absorption over a longer period of time than that for a conventional
dosage form. Onset of action is thought to be delayed because of an overall slower release
rate from the dosage form (Collet & Moreton, 2002).
3.2.6 Sustained release
There is an initial release of drug sufficient to provide a therapeutic dose soon after
administration and then a gradual release over an extended period of time usually to try and
mimic zero-order release by providing a drug in a slow first-order manner (Jantzen &
Robinson, Collet & Moreton, 2002).
3.2.7 Controlled release
The drug is released at a constant rate and provides plasma concentrations that remain
invariant with time (Collet & Moreton, 2002). This type of drug delivery system attempts to
control drug concentrations in the target tissue. The aim is to maintain therapeutic blood or
tissue drug levels constant over an extended period. This is usually accomplished by zero-
order drug release from the dosage form (Jantzen & Robinson, 2002).



27
3.3 DESIGN AND FUNCTION OF MODIFIED RELEASE DOSAGE FORMS
Several control mechanisms have been developed to achieve slow, controlled release of a
drug from tablets. These include drug transport by diffusion, dissolution, erosion, convective
flow accomplished by osmotic pumping and ion exchange control. Diffusion controlled and
dissolution controlled systems are the oldest, successful and most commonly used since they
are relatively easy and less expensive to manufacture (Alderborn, 2002 and Jantzen &
Robinson, 2002).
3.3.1 Ideal properties of a modified release dosage form
The ideal properties of a MRDF include the following (Shargel & Yu, 1999):
Efficacy and reduced or no toxicity, i.e. there should be no dose dumping or abrupt
release of a large amount of the drug. A single dose should show steady-state levels
within the therapeutic plasma levels comparable to those reached by using multiple
doses of a conventional dosage form as illustrated in Figure 3.2.

Figure 3.2: Plasma levels of a drug from a conventional tablet containing 50 mg of drug
administered at 0, 4 and 8 h in curve A compared to a single 150 mg drug dose given in an
extended release dosage form in curve B (Shargel & Yu, 1999).

28
Controlled drug release to control the therapeutic effect,
Consistent pharmacokinetic performance between individual dosage units to allow
for the maximum amount of drug to be absorbed,
Minimum patient-to-patient variation and better patient compliance,
Cost saving for treatment of patients, especially chronic conditions, even though the
cost of manufacture of a MRDF is generally higher than the cost of a conventional
dosage form,
3.3.2 Types of modified release dosage forms
Drug delivery system development has advanced extensively up to a point where there are
several simple and complex structured MRDFs already on the market. Most of the
characteristics outlined in 3.3.1 are satisfied, albeit the cost of manufacture is a hindrance for
most types of MRDFs. Shargel & Yu, (1999) describe various types of extended release
dosage form products as follows:
Pellet or bead type sustained release refer to beads prepared by coating drug powder
onto preformed cores called nonpareil seeds which are made from slurry of starch,
sucrose and lactose. The drug beads, which may act as rapid-release carriers for the
drug, may be further coated with protective coating to allow a sustained or prolonged
release of the drug. Dissolution may also be controlled by variation in bead blending
or coating with different materials. The use of pH sensitive enteric coating materials
plus blending aid in providing two doses of a drug in one formulation which is ideal
for delivering the drug to the gastrointestinal tract regions with different pH values.
Prolonged-action tablets where drug release is controlled by altering the solubility so
that the tablet dissolves over a period of several hours. This is not easily
reproducible and therefore this dosage form is usually not reliable.
Ion-exchange applies in systems where the insoluble drug-resin complex dissociates
in the gastrointestinal tract in the presence of the appropriate counter ions. The

29
released drug then dissolves in the fluids of the gastrointestinal tract and is rapidly
absorbed. This dosage form is, however, not reliable because of uncontrollable
levels of counter ions and variability among individuals and resins may provide a
potential means of interaction with nutrients and other drugs.
A core tablet is a tablet within a tablet and is used as a slow drug-release component.
The outside shell contains a rapid-release dose of drug. The core material may be
surrounded by hydrophobic excipients so that the drug leaches out over a prolonged
period of time. This type of preparation is sometimes called slow erosion core tablet
because the core generally contains either no or insufficient disintegrant to fragment
the tablet. The composition of the core may range from waxy to gummy or
polymeric material.
Gum-type matrix tablet have a remarkable ability to swell in the presence of water to
form a gel-like consistency which provides a natural barrier to drug diffusion from
the tablet. The gel-like material is quite viscous and may not disperse for hours
thereby providing a means for sustaining the drug release over an extended period
until all the drug has been completely dissolved and has diffused into the intestinal
fluid. A common gel-forming material is gelatine. Modification of the release rates
of the product may further be achieved with various amounts of talc or other
lipophilic lubricant.
Micro encapsulation is a process of encapsulating microscopic drug particles with a
special coating material, thereby making the drug particles more desirable in terms of
physicochemical characteristics such as aspirin covered with ethylcellulose.
A polymeric matrix tablet involves the use of polymeric materials in prolonging the
release rate of a drug. Prolonged release may last for days and weeks rather than for
a shorter duration as with other techniques. The first example of an oral polymeric
tablet is Gradumet

, which is marketed as an iron preparation. The plastic matrix

provides a rigid geometric surface for drug diffusion so that a relatively constant
drug release rate is obtained. In the case of the iron preparation, the matrix reduces

30
the exposure of the irritating drug to the gastrointestinal mucosal tissues. The matrix
is usually expelled unchanged in the faeces after all the drug has been leached out.
The matrix tablets for oral use are generally quite safe, however, for certain patients
with reduced gastrointestinal motility caused by disease, the polymeric matrix tablet
should be avoided. This is because obstruction of the gastrointestinal tract by the
matrix tablets has been reported. The use of the matrix tablet in implantation has
been more popular than orally administered products.
Osmotic controlled release is a fairly new concept in controlled-release preparations
where drug delivery is precisely controlled by the use of an osmotic controlled
device. It pumps a constant amount of water through the system dissolving and
releasing a constant amount of drug per unit time through the orifice. This device
consists of an outside layer of semi-permeable membrane filled with a mixture of
drug and osmotic agent. When the device is placed in water, osmotic pressure
generated by the osmotic agent within the core causes water to move into the device
through the membrane thereby forcing the dissolved drug to move out of the delivery
orifice. The process continues until all the drug is released. The rate of drug
delivery is relatively unaffected by the pH of the environment. The osmotic delivery
system has become a popular drug vehicle for many products that require extended
period of drug delivery for 12 to 24 hours some examples being Adalat CR

and
Efidac 24

. The osmotic preparation available for implantation is known as the

osmotic mini-pump.
Transdermal therapeutic systems (TTS) are intended for delivering a dose of
medication across the skin for systemic drug absorption. It can deliver the drug dose
through the skin in a controlled rate over an extended period of time. A semi-
permeable membrane next to the reservoir layer controls drug diffusion.
A monolith or matrix TTS has an occlusive backing layer to protect the drug matrix
which comprises a suspension of drug in equilibrium with its saturated solution for
maximum thermodynamic activity. The adhesive layer contains dissolved drug in
equilibrium with the matrix and attaches the patch to the skin (Barry, 2002).

31
Physicochemical and pharmacodynamic properties are the crucial criteria for the
choice of drug(s) suitable for TTS. Physicochemical properties of the drug include a
small molecular size (<500 Daltons) and high lipid-solubility. The elimination half-
life should not be too short to avoid too frequent patch application per day. . The
drug potency should be fairly high so that only a small systemic drug dose is needed
and the size of the patch (dose is also related to surface area) need not be
exceptionally large, not greater than 50 cm
2
(Guy, 1996). After the application of a
transdermal patch, there is generally a lag time for the onset of drug action, due to
the drug's slow diffusion into the dermal layer (Shargel & Yu, 1999)
Implants and inserts: polymeric drug implants can deliver and sustain drug levels in
the body for an extended period of time. Both biodegradable and non-biodegradable
polymers can be impregnated with drugs in a controlled drug delivery system. Levo-
norgestrel implants (Norplant

system) is a set of six flexible closed capsules made

of silastic (dimethylsiloxane/methylvinylsiloxane copolymer), each containing 36 mg
of the progestin, levonorgestrel. The capsules are sealed with silastic adhesive and
sterilized. The Norplant

system is available in an insertion kit to facilitate

subdermal insertion of all six capsules in the mid-portion of the upperarm. The
levonorgestrel implants are effective up to 5 years for contraception and only needs
to be replaced after this period. An intrauterine progesterone contraceptive system
(Progestasert

is a T-shaped unit which contains a reservoir of 38 mg progesterone.

Contraceptive effectiveness for Progestasert

is enhanced by continuous release of

progesterone into the uterine cavity at an average rate of 65 g/day for 1 year
(Shargel & Yu, 1999).
The number of polymers available for use in drug formulations is increasing and includes
polyacrylate, methacrylate, polyester, ethylene-vinyl acetate copolymer (EVA),
polyglycolide, polylactide, and silicone. Of these, the hydrophilic polymers, such as
polylactic acid and polyglycolic acid, erode in water and release the drug gradually over
time. A hydrophobic polymer such as EVA will release the drug over a longer period of
weeks or months. The rate of release may be controlled by: mixing two polymers and

32
increasing the proportion of the more hydrophilic polymer (e.g low molecular weight
polymer) thus increasing the rate of drug release. For example, the addition of polylactide to
a polylactide-polymer formulation increased the release rate of the drug and enabled the
preparation of a controlled-release system (Bodmeier et al., 1989).
Hydrophobic polymers with linkages susceptible to water hydrolysis are prepared so that
partial breakdown of the polymers allows for desired drug release without deforming the
matrix during erosion. For oral drug delivery, the problem of incomplete drug release from
the matrix is a major hurdle that must be overcome with polymeric matrix dosage forms.
Another problem is that the drug release rates may be affected by the amount of drug loaded.
For implantation and other uses, the environment is more stable, so that a stable drug release
from the polymer matrix may be attained for days or weeks.
3.3.3 Selection of the type of dosage form
The choice of the dosage form is an important factor in the delivery of the drug. It is
especially important to decide whether to formulate the active ingredient in:
A single unit system which includes tablets, coated tablets, matrix tablets or capsules,
A multiple unit system such as granules, beads, capsules or microcapsules (Collet &
Moreton, 2002).
The selection of the appropriate dosage form has to take in account an acceptable level of
variability of performance, the influence of gastrointestinal tract structure and function on the
delivery system, the release mechanism and release profile of the dosage form.
3.3.4 Mechanisms of drug release
Two basic drug release mechanisms are involved namely, dissolution of the active drug
component and the diffusion of dissolved or solubilised species out of the system into the
surrounding fluid. These mechanisms may operate independently, together or consecutively
and there are four processes operating within the context of these mechanisms, which include
(Collet & Moreton, 2002):

33
Hydration of the device (swelling of the hydrocolloid or dissolution of the
channelling agent),
Diffusion of water into the device,
Dissolution of the drug,
Diffusion of the dissolved (or solubilised) drug out of the device.
Drug release may be constant, declining or bimodal: In a constant release system the
modified release dosage form should provide and maintain constant drug plasma
concentrations, in other words, the system should exhibit zero-order kinetics. This led to
considerable effort being put into developing systems that release drugs at a constant rate.
A declining release system is commonly a function of the square root of time or follows first-
order kinetics. These systems cannot maintain a constant plasma drug concentration but can
provide sustained release. Bimodal release is based on the notion that the release rate must
always be slower than the absorption rate even if a constant drug release rate is achievable so
as to regulate drug absorption.
3.3.5 Classification of modified release dosage forms
The composition of MRDFs serves as a basis for their classification. Some components may
include active drug, release-controlling agent(s), matrix/membrane modifier, solubiliser, pH
modifier and/or density/modifier, lubricant and supplementary coatings in various
proportions (Collet & Moreton, 2002).
Alexandridis et al. (2000) point out that the composition of MRDF is channelled by certain
critical physicochemical considerations that include:
Efficient drug loading of the polymer-based delivery system,
Maintenance of the integrity of the drug during the loading process,

34
Proper surface properties of the drug delivery system, which promote the
biocompatibility, bioavailability and stability of the drug delivery system.
The kinetics and the mechanism of drug release from the carrier to the biological
system to achieve optimal drug release profiles.
3.3.5.1 Monolithic matrix delivery systems
Hydrophilic colloid matrices are systems where drug particles are dispersed in a soluble
matrix, in which the drug becomes available as the matrix dissolves or swells. Drugs
dispersed in a soluble matrix rely on a slow dissolution of the matrix to provide a sustained
release system. Alternatively, slowly dissolving fats and waxes undergo surface erosion with
little or no bulk erosion. If the matrix is presented with a conventional tablet geometry the
surface area of the matrix decreases with time with a concomitant decrease of drug release on
contact with the dissolution media (Collett & Moreton, 2002).
Hydrophobic lipid matrices and insoluble polymer matrices have drug particles dispersed in
an insoluble matrix and become available as the solvent enters the matrix and dissolves the
particles. Drug release from these matrices proceeds by way of penetration of fluid, followed
by dissolution of the drug particles and then diffusion through fluid-filled pores. This type of
delivery system would not be suitable for the release of compounds that are insoluble or
which have a low aqueous solubility. Excipients used in the preparation of insoluble
matrices include hydrophobic polymers, such as polyvinyl acetate, ethyl cellulose and some
waxes (Collett & Moreton, 2002).
3.3.5.2 Reservoir or membrane-controlled systems
A drug reservoir such as a tablet or multi-particulate pellet system is coated with a membrane
on the surface and not throughout the system as in the case of the matrix systems. The rate
controlling part of the system is a membrane polymer through which the drug must diffuse
hence the membrane has to be permeable. The membrane neither swells nor erodes during
hydration. Membrane-controlled systems may occur as one of the following types (Collett &
Moreton, 2002):

35
Single unit systems: A film-coated tablet formulation where the core is not
allowed to disintegrate but to dissolve and water is allowed to penetrate the
membrane so that diffusion can occur.
Multiple unit systems: The system is made up of coated spheroids (pellets
approximately 1 mm in diameter) filled into a hard gelatin capsule or rarely
compressed into a tablet. The membrane coating is the critical part of the
formulation as it controls drug release.
Osmotic pump system: Drug is included in a tablet core which is water soluble
which will solubilise or suspend the drug in the presence of water. The tablet core
is coated with a semi-permeable membrane which allows the passage of water in
only, unlike in membrane systems where there is water in and drug out
movement. A hydrostatic pressure builds up and forces/pumps the drug
solution/suspension after the dissolution of the core through a hole (or orifice)
drilled in the coating after the dissolution of the core.
3.4 CHITOSAN AS A DRUG CARRIER
Chitosan is currently receiving a great deal of interest for medical and pharmaceutical
applications due to its favourable intrinsic properties such as biodegradability, bio-
adhesiveness and biocompatibility. The fact that derivatives can be formed to adapt to the
various physiological environments sparked interest in various studies such as the use of
chitosan nanoparticles and beads for controlled drug release (Thanoo, Sunny & Jayakrishnan,
1992; Gupta & Kumar, 2000).
The hydrophilic nature of chitosan and the varying pH regions of the gastrointestinal tract led
to chemical modifications of chitosan in an attempt to make it functional and effective in all
the regions. Examples include mono- and di-carboxymethylated chitosan for heparin
absorption (Thanou, Verhoef & Junginger, 2001) and mucoadhesive and absorption
enhancing properties of trimethyl chitosan chloride (Kotze et al., 2002).


36
3.4.1 Cross-linking of polymers to form matrices and/or hydrogels
Bodmeier, Chen & Paeratakul (1989) refer to cross-linking as the interaction of water-soluble
polymers bearing positively or negatively charged groups to form three-dimensional
networks with molecules of opposite charges, which may be used to entrap water-insoluble
drugs to facilitate their transport. The ionically cross-linkable polymers may be anionic or
cationic in nature and include but are not limited to carboxylic, sulfate, hydroxy and amine
functionalised polymers, normally referred to as hydrogels after being crosslinked. The term
"hydrogel" indicates a cross-linked, water insoluble, water-containing and swellable material
(Ronan & Thompson, 2001).
The cross-linking ions may be anions or cations depending on whether the polymer is
anionically or cationically cross-linkable. Appropriate cross-linking ions include calcium,
magnesium, barium, strontium, boron, beryllium, aluminum, iron, copper, cobalt, lead and
silver ions. Anions may be phosphate, citrate, borate, succinate, maleate, adipate and oxalate
ions. More broadly, the anions are derived from polybasic organic or inorganic acids. The
most preferred cross-linking cations are calcium and barium ions. The most preferred cross-
linking anion is phosphate. Cross-linking may be carried out by exposing the polymers to an
aqueous solution of the appropriate ions (Ronan & Thompson, 2001).
Suitable cross-linkable polymers include but are not limited to, one or a mixture of
polyethylene amine, alginic acid, carboxy methyl cellulose, hyaluronic acid, heparin sulfate,
chitosan, carboxymethyl chitosan, chitin, carboxymethyl starch, carboxymethyl dextra and
chondroitin sulfate. Polymers which are not ionically cross-linkable are used in blends with
polymers which are ionically cross-linkable (Ronan & Thompson, 2001).
Cross-linking may occur via irreversible covalent bonding to form a permanent network with
good mechanical properties, coordinate covalent bonding or ionic bonding to form hydrogels
that are more labile. Most of the cross-linkers used in covalent bonding tend to induce
toxicity if found in free traces before administration such as glutaraldehyde. This problem
may be overcome by reversible ionic cross-linking, which is a viable option for chitosan
because it can chelate with negatively charged ions via the protonated amine groups to form

37
a network through ionic bridges between polymeric chains. These can be characterised by
turbidimetric titration, viscometry or IR spectra (Berger et al., 2004).
Non-ionic cross-linking mechanisms have a higher cross-link density, improved mechanical
properties, i.e. improved stiffness, modulus, yield stress and strength. This may be
accomplished for the ionically cross-linkable polymer by additionally subjecting these to
non-ionic cross-linking mechanisms such as high-energy radiation (gamma rays) or treatment
with a chemical cross-linking agent reactive with groups present in the polymer such that
covalent bonds are formed connecting the polymer network. Another non-ionic cross-linking
mechanism useful with respect to some classes of hydrogel polymers is physical cross-
linking, which is typically accomplished by crystal formation or similar association of
polymer blocks such that the polymer molecules are physically tied together and prevented
from complete dissolution. Non-ionic cross-linking may be carried out prior to, subsequent
to or concurrently with ionic cross-linking (Ronan & Thompson, 2001).
One of the unique properties of polymer hydrogels is that the ionic cross-links can be easily
and selectively displaced in-vivo resulting in swelling and softening, which enhances patient
comfort. Since the non-ionic cross-links are not significantly displaced, the device will retain
its original non-ionically cross-linked shape configuration to a large degree and will not
disintegrate (Ronan & Thompson, 2001).
Berger et al., (2004) divided ionically cross-linked chitosan networks into two groups
depending on the type of cross-linker used, anions or anionic molecules, even though their
characteristics and properties are identical. They also emphasise that ionic cross-linking
requires multivalent counter-ions as cross-linkers to form bridges between polymeric chains.
According to this requirement, monovalent carboxymethylated chitosan is a potential
candidate for cross-linking with multivalent metals. Hejazi & Amiji, (2003) explained that
these structural features also render them potentially able to chelate metal ions with vacant d-
orbitals, like Co
2+

and Zn
2+
,

via the nitrogen lone pairs and the negative charge of the
carboxylic group to form stable complexes. The mechanical strength of such neutral
complexes could aid with the controlled release of drugs.

38
3.4.2 Coating of dosage forms
Coating serves various purposes such as:
Masking of unpleasant taste,
Protection of components from atmospheric degradation,
Separation of reactive ingredients,
The control of the site for drug release as in enteric coating and
Delayed/prolonged absorption of the drug component by retarding drug release
from the dosage form (Hogan, 2002).
There are three major techniques for applying coatings to pharmaceutical solid dosage forms,
namely, sugar coating, compression coating and film coating. The latter comprises tablet
film coating and multi-particulate (micro-encapsulation) coating and performs the
pharmaceutical function of modifying drug release rate of some oral dosage forms (Hogan,
2002) and will therefore be described further.
Film coating is a more recently used technology in tablet coating and almost all newly
launched coated products are film coated (Hogan, 2002). This process involves the
deposition, usually by a spray method, of a thin, polymeric but uniform film onto the surface.
Initially highly volatile organic solvents were used but created many potential problems
including flammability, toxicity, concerns over environmental pollution, costs related to
minimising these problems plus the cost of the solvents themselves. The current film coating
methods rely more and more on water as the prime solvent. Film coating has proven to be a
popular alternative to sugar coating, because it allows additional substrates to be coated other
than just compressed tablets, for example, powders, granules, nonpareils and capsules
(Hogan, 2002).
Continuous spray techniques and manual application procedures have been used to coat a
moving bed of material. Film coating has proved successful as a result of the many

39
advantages offered which include minimal weight increase (typically only 2 to 3% of the
tablet core weight), significant reduction in processing times, increased process efficiency
and output because of improved in-process technology and equipment design, increased
flexibility in formulations and improved resistance to chipping of the coating (Hogan, 2002).
Raw materials used in film coating mainly consist of a polymer, plasticiser, colourant, and
solvent (or vehicle). Good interaction between solvent and polymer is necessary to ensure
that optimal film properties are obtained when the coating dries. This initial interaction
between solvent and polymer yields maximum polymer-chain extension, producing films
having the greatest cohesive strength and, thus, the best mechanical properties (Hogan,
2002).
The major solvents used in film coating typically belong to one of the following classes:
alcohols, ketones, esters, chlorinated hydrocarbons and water. An important function of the
solvent system is to ensure a controlled deposition of the coating material onto the surface of
the substrate so that a coherent and adherent film coat is obtained. Ideal properties for the
polymer include solubility in a wide range of solvent systems to promote flexibility in
formulation, an ability to produce coatings that have suitable mechanical properties and
appropriate solubility in gastrointestinal fluids such that drug bioavailability is not
compromised (Hogan, 2002).
Cellulose ethers are often the preferred polymers in film coating, particularly hydroxypropyl
methylcellulose. Suitable substitutes are hydroxypropyl cellulose, which may produce
slightly tackier coatings and methylcellulose, which is known to retard drug dissolution.
Alternatives to the cellulose ethers are certain acrylics, such as methacrylate and methyl
methacrylate copolymers, and vinyls such as polyvinyl alcohol (Hogan, 2002).
The incorporation of a plasticiser into the formulation improves the flexibility of the coating,
reduces the risk of the film cracking and possibly improves adhesion of the film to the
substrate. To ensure that these benefits are achieved, the platiciser must show a high degree
of compatibility with the polymer and be retained permanently in the film, if the properties of
the coating are to remain consistent on storage. Examples of typical plasticisers include

40
glycerin, propylene glycol, polyethylene glycol, triacetin, acetylated monoglyceride, citrate
esters (e.g. triethyl citrate) or phthalate esters (e.g. diethyl phthalate) (Hogan, 2002).
Functional coating, a modified form of film coating, confers controlled or enteric drug
release properties on dosage forms which such as tablets or granules. This type of coating is
based on mechanical methods such as:
Pan coating,
Air-suspension techniques,
Multi-orifice centrifugal techniques,
Modified spray-drying techniques (Hogan, 2002).
Polymers with restricted water solubility or permeability, such as ethylcellulose and modified
acrylate derivatives, are used in film coating. Particles which have been film coated for
controlled release are filled into hard gelatine capsules, or occasionally compressed directly
into tablets by a process which permits minimal rupture of the applied film (Hogan, 2002).
Multi-particulates, pellets or beads, have a small size, typically 0.7-2.0 mm that allows them
to pass through the constricted pyloric sphincter to distribute along the gastrointestinal tract,
hence their preference over conventional non-disintegrating tablets for controlled release use.
This results in the prevention of multi-lodging in constrictions and consequently in reduced
possible ulceration as smaller drug doses are loaded and any accidental complete release
would not pose a high risk. These systems may be extruded/spheronised granulates which
are produced in modified granulating equipment, with the drug granulation extruded through
a mesh or other device under pressure to form small granulates which are subsequently
spheronised. The non-pareils are sucrose spheres coated with the drug plus an adhesive
water-soluble polymer and may be coated with the controlled-release coating (Hogan, 2002).
Subsequent to the release of the coated pellets from the hard gelatine capsule or tablet, the
drug is released in a predetermined fashion with respect to time via a number of mechanisms.

41
These include diffusion, erosion and osmosis, which involve pressure built-up and the
pushing out of the drug (Hogan, 2002).
Enteric coating techniques are used to protect the tablet core from disintegration in the acid
environment of the stomach, for protection from the irritant effect of certain drugs and/or
facilitated absorption of a drug that is preferentially absorbed distal to the stomach.
Polymers commonly used for the purposes of enteric coating are cellulose acetate phthalate,
polyvinyl acetate phthalate plus suitable acrylic derivatives, which exhibit a differential pH
solubility profile because of the free carboxylic acid groups on the polymer backbone. They
are sparingly soluble in aqueous media at low pH, but as the pH rises they experience a
sharp, well-defined increase in solubility at a specific pH value (Hogan, 2002).
3.5 CONCLUSION
Modified release dosage forms have several advantages over conventional immediate release
dosage forms such as the maintenance of therapeutic drug plasma levels over an extended
period of time. Monolithic matrix systems and coated reservoir systems are different types
of MRDFs with different drug release mechanisms. Hydrophilic polymers have been cross-
linked into three-dimensional networks or hydrogels that may be used to entrap a drug to
form a MRDF. The aim is to release the drug at a constant rate (i.e. by means of zero order
release kinetics).
The relative safety of ionically cross-linked chitosan hydrogels, their ease of preparation as
well as the already indicated remarkable characteristics and reactivity of chitosan offer the
potential to be used in modified release dosage forms, specifically in monolithic matrix
systems. Cross-linked derivatives of chitosan can be formulated into micro or nanoparticles
and matrix systems, which can be used for controlled release in acidic and also in basic
media if the problem of mechanical strength can be overcome due to pH dependent swelling.
Cross-linking with opposite charged molecules form reversible bridges between the polymer
molecules. Ionic cross-linking has the additional advantage of non-toxicity as opposed to the
covalent cross-linking process with toxic substances such as glutaraldehyde.

42
CHAPTER 4: EVALUATION OF A CROSS-LINKED
N-CARBOXYMETHYL CHITOSAN DRUG DELIVERY SYSTEM
4.1 INTRODUCTION
In this study a derivative of chitosan, N-carboxymethyl chitosan, was synthesised and
investigated for its use in the development of a controlled release dosage form (i.e. a wafer or
matrix system). Ibuprofen was selected as the model drug and N-carboxymethyl chitosan
was ionically cross-linked with barium chloride to form a hydrogel. This hydrogel was
mixed with the model drug and cast into a mould. The wafers were removed after drying and
evaluated in terms of drug content, swelling properties and in vitro drug release.
The methods used to synthesise and characterise N-carboxymethyl chitosan as well as the
cross-linking procedure and the method used to manufacture matrix delivery systems or
wafers are described in this chapter. The in vitro release behaviour of these wafers was
determined at pH 5.8, 7.4 and 8.0. The results are reported with an analysis of the dissolution
curves in terms of the mean dissolution time, MDT, as well as the difference and similarity
factors, f
1
and f
2
.
4.2 MATERIALS AND EQUIPMENT USED
The synthetic materials, glyoxylic acid and sodium borohydride, were purchased from Merck
chemicals. Deuterated water, D
2
O, was obtained through Merck chemicals from Germany.
The equipment listed in Table 4.1 were used throughout the study for various purposes
ranging from weighing to characterisation. The manufacturer and the place were they were
made are also indicated.




43
Table 4.1: Equipment used in the synthesis and evaluation of N-carboxymethyl chitosan.
Equipment Manufacturer City and/or country
Hanson SR8 Plus and
Hanson SR2 Plus
(Six station dissolution
apparatus)
Hanson Research Chatsworth, Carlifornia,
USA
Mettler Toledo Balance AB
204/S capacity max 220 g
min 10 mg
Mettler Toledo Switzerland.
Microprocessor pH meter
Hanna pH 211
Hanna Instruments Portugal
Shimadzu UV/VIS
spectrophotometer 1601
Shimadzu Tokyo, Japan
Thermo Nicolet Erweka 360
FTIR ESP Spectrometer
Thermo Nicolet Corporation Madison, USA
Varian Mercury 300 MHz
NMR Spectrometer
Varian Incorporation Darmstadt, Germany.

4.3 SYNTHESIS OF N-CARBOXYMETHYL CHITOSAN
The N-carboxymethyl chitosan synthesis method was adapted from the methods described by
Thanou, Verhoef and Junginger, (2001) and Muzzarelli et al. (1982). Briefly, the method
involved preparation of a chitosan solution, 1 g in 100 ml of 0.15 M aqueous acetic acid
overnight. This solution was treated with 4 g of synthetic grade glyoxylic acid at 25
o
C while
stirring with a magnetic stirrer. This mixture was allowed to stand for one hour and was then
reduced with addition of 3.6 g synthetic grade sodium borohydride (NaBH
4
), which was
dissolved in 5 ml of water.
Figure 4.1 depicts the reaction between chitosan and glyoxylic acid during the synthesis
process. The product was precipitated with alcohol, isolated by centrifugation and then
freeze-dried with a system equipped with cold trapping.

44

Figure 4.1: The reaction between chitosan and glyoxylic acid followed by reductive
alkylation to form N-carboxymethyl chitosan (Thanou et al., 2001). MCC = mono-
carboxymethyl chitosan.
4.4 CHARACTERISATION OF THE SYNTHESISED N-CARBOXYMETHYL
CHITOSAN
Spectroscopy, the study of the interaction of matter and light or electromagnetic radiation,
can be used to determine unknown molecular structure. The most common type being
absorption spectroscopy and involves certain wavelengths of radiation. Absorption is
determined as a function of wavelength, frequency or energy in an instrument called a
spectrometer or spectrophotometer. The basic components include a radiation source, the
sample or material to be examined and a detector to measure the intensity of the radiation
that is finally recorded as a spectrum of the sample. The spectrum of a compound is
determined by its chemical structure or constituent functional groups. Various types of
spectroscopy are used including nuclear magnetic resonance (NMR), infrared (IR) and
ultraviolet-visible (UV/VIS) spectroscopy.
Nuclear magnetic resonance (NMR) utilises the fact that protons have nuclear spins that can
have quantum numbers of +
1
/
2
or -
1
/
2
as in the case of the electron. The nuclear spin acts like
a tiny magnet and therefore responds to a magnetic field. The two spin states have identical
energies when a magnetic field is not applied. Energy absorption by nuclei in a magnetic
field is termed nuclear magnetic resonance and is illustrated in Figure 4.2. Magnetic
resonance energy is low (approximately 0.02 J mol
-1
) and corresponds to the frequency
modulation (FM) radio waves of the electromagnetic spectrum.

45

Figure 4.2: Nuclear magnetic resonance or absorption of energy (E) by nuclei with spin
+
1
/
2
causing these nuclei to invert their spins and assume a more energetic state with spin
1
/
2
. This energy absorbance by nuclei is detected in an NMR spectrometer (Loudon, 1995).
A schematic diagram of the functional components of the NMR spectrometer is illustrated in
Figure 4.3.

Figure 4.3: NMR spectrometer showing radio-frequency (rf) energy absorbed from a coil
connected to the source. The magnetic field changes with the current. The receiver coil
detects E which is amplified and presented as a spectrum (Loudon, 1995).

46
It is essential for the sample to be free-flowing for an NMR test, therefore solvents which do
not interfere with the desired nuclear spin are necessary. Although deuterium has a nuclear
spin, proton NMR and deuterium NMR require different frequencies (deuterium absorptions
are therefore not detected under conditions used for proton NMR). Solvents devoid of
protons such as carbon tetrachloride (CCl
4
) or carbon disulfide (CS
2
) may be used if the
sample is soluble in them.
Typical frequencies used in NMR experiments range between 60 and 500 megahertz (MHz).
A chemical shift of a test functional group (hydrogen) is defined as the difference between its
absorption position and that of a reference (TMS for hydrogen) i.e. -
TMS
in Hz, divided by
the operating frequency in MHz as shown in Equation 4.1. This equation converts chemical
shifts to a form that is independent of the operating frequency.
MHz in frequency Operating
Hz in

shift Chemical
o
TMS
=

(Loudon, 1992) (4.1)
The units for the chemical shift are parts per million or ppm. The ppm scale increases
downfield or in a de-shielded direction from the reference toward a lower magnetic field.
Proton NMR or
1
H NMR is used to deduce stereochemically non-equivalent protons present
with absorptions within a chemical shift range of 0-10 ppm. The number of signals indicates
how many kinds of protons a molecule contains. The intensities of the signals vary with the
number of each kind of nuclei present and the splitting of a signal into several peaks gives an
indication of the environment of a proton with respect to other nearby protons.
Characteristic proton chemical shifts for some functional groups are illustrated in Table 4.2.
Theory and experiment have shown that a proton with a particular environment has about the
same chemical shift irrespective of the frequency of the NMR used nor the molecule that it
forms part of.

47
Table 4.2:Characteristic proton chemical shifts of some functional groups (Morrison &
Boyd, 1992).
Class of compound Functional group Proton chemical shift,
, (ppm)
Alcohols
C OH H

3.4-4
Ethers
C O H R

3.3-4
Esters
C O H RCO

3.7-4.1
Esters type 2
C H OR CO

2.0-2.2
Acids
COOH C H

2.0-2.6
Carbonyl compounds
C H C O

2.0-2.7
Aldehydic
C O R
H

9.0-10.0
Hydroxylic
H O R

1.0-5.5
Phenolic
O H Ar

4.0-12.0
Enolic
C
H
O C

15.0-17.0
Carboxylic
RCOO H

10.5-12.0
Amino
N R
H
H

1.0-5.0

Carbon-13 NMR, CMR or
13
C NMR spectroscopy applies the same principles as proton
NMR concerning the nuclear spin of
1
/
2
,.even though the CMR runs are more difficult to
obtain. The reason being that the only isotope of carbon that has a nuclear spin is the carbon-

48
13 isotope (or
13
C), hence CMR does not measure the resonance of the natural carbon isotope
(
12
C). The
13
C isotope has a very low natural abundance of 1.10% as illustrated in Table 4.3.
This results in a weaker
13
C resonance moreover that no coupling occurs between the natural
carbon isotope (
12
C) atoms.
Table 4.3: Properties of some nuclei with
1
/
2
spin
Isotope Relative sensitivity Natural abundance, % Magnetogyric ratio
1
H 1.00 99.98 26.753
13
C

0.0159 1.10 6.728
19
F 0.834 100 25.179
31
P 0.0665 100 10.840

Information about the carbon skeleton is obtained from the CMR spectrum. Once again, the
CMR runs last for a longer time with a chemical shift range of about 0-200 ppm and are more
number of signals varies with the number of different types of carbons or sets of
stereochemically equivalent carbons in a molecule. The splitting of a signal indicates the
number of hydrogens attached or coupled to each carbon and this may be suppressed by
decoupling the hydrogens so that there is no splitting of peaks, i.e. single peaks are obtained.
The chemical shift provides information about the hybridisation (sp
3
, sp
2
or sp) of each
carbon and also about the electronic environment of each carbon with respect to other nearby
carbons or functional groups (Morrison & Boyd, 1992).
sensitive to small changes in chemical environment. Some CMR characteristic chemical
shifts are depicted in Table 4.4.

49
Table 4.4: Typical CMR chemical shift ranges for some common functional groups.
Functional group
13
C chemical shift,
, (ppm)
Functional group
13
C chemical shift,
, (ppm)
CH
3
,
CH
2
,
CH
,
C

0-55
C C

100-145
C C

65-85
C N

25-70
C O

40-80
C
O

190-220
C

110-160
C Cl

20-60
C
O
O

160-190
C Br

10-50
C N
O

160-180

Infrared (IR) absorptions can be expected only for vibrations that cause a change in the
molecular dipole moment i.e. for IR active molecules. A given type of functional group
absorbs in the same general region of the IR spectrum. An IR spectrum is a plot of the
infrared radiation transmitted through a sample as a function of the wavenumber or
wavelength of the radiation. Schematic diagram of the functional components of an IR
spectrometer is illustrated in Figure 4.4.

50

Figure 4.4: Schematic diagram of an infrared spectrometer showing splitting of IR radiation
by a mirror into sample (S) and reference (R) beams (Loudon, 1995).
Characteristic regions and functional groups for IR absorption are depicted in Table 4.5.
Table 4.5: Regions of the infrared spectrum (Loudon, 1995)
Wavenumber range
(cm
-1
)
Type of absorptions Name of region
3400-2800
2250-2100
1850-1600
O-H, NO, C-H stretching
CN, CC stretching
C=O, C=N, C=C stretching
FUNCTIONAL GROUP
1600-1000 C-C, C-O, C-N stretching;
various bending absorptions.
FINGERPRINT
1000-600 C-H C-H BENDING




51
Although Loudon (1995) gives the major IR spectrum regions shown in Table 4.5 he
concedes that:
The IR spectra of most compounds contain many more absorptions than can be
readily interpreted,
Certain absorptions are diagnostic, that is, they indicate with reasonable certainty
that a particular functional group is present. For example, an intense peak in the
17001750 cm
-1
region implies the presence of a carbonyl, C=O group,
Other peaks are confirmatory, i.e. the peaks can be found in other types of
molecules but their presence confirms a structural diagnosis made in other ways
as in the absorption of the CO bond in the 12001050 cm
-1
region, which
could indicate an ester, ether, alcohol or carboxylic acid and
Subtle differences in structure generally give discernible differences in the IR
spectrum particularly in the fingerprint, 1000 cm
-1
and 1600 cm
-1
.
4.4.1
1
H and
13
C NMR characterisation
Formation of N-carboxymethyl chitosan as the product was confirmed with both
1
H and
13
C
NMR spectrometry on the Varian Mercury 300 MHz NMR Spectrometer made by Varian
Incorporation of Darnstadt, Germany. About 35 mg of CMC was dissolved in about 0.2 ml
of deuterated water (D
2
O from Merck chemicals) in a nuclear magnetic resonance (NMR)
tube. Tetramethylsilane (TMS) was used as a reference for mono-carboxymethyl chitosan
proton resonance. Chitosan did not dissolve in deuterated water, D
2
O, hence an attempt to
obtain an NMR spectrum in D
2
O was futile.
Salient features of N-carboxymethyl chitosan are the C-2 carbon bearing either the acetamido
group, NH(C=O)CH
3
or the amine group, NH
2
, as shown in Figure 4.1. The additional
functional group absent in chitosan is the glyoxylate residue at the N position and contains
the carboxylic group, COOH. Therefore an enlarged CO stretch and a prominent COO
near the C=O stretch may confirm the presence of a carboxylic group.

52
The
1
H NMR spectrum obtained for the synthesised N-carboxymethyl chitosan is shown in
Figure 4.3, while the proton-decoupled
13
C spectrum is illustrated in Figure 4.4.

Figure 4.3:
1
H NMR spectrum (Varian Mercury 300 MHz NMR Spectrometer) of the
synthesised N-carboxymethyl chitosan.
The intensities of the signals varied with the number of each kind of nuclei present and the
splitting of a signal into several peaks is indicative of the environment of a proton with
respect to surrounding protons (Loudon, 1994). The
1
H NMR peak at 2.879 ppm is attributed
to the -H of the -CH
2
COOH from glyoxylic acid, which was added to the chitosan molecule
during the synthesis process and this concurs with the results obtained by Le Dung (1994) for
N-carboxymethyl chitosan. The H of COOH is expected further downfield due to the
deshielding effect of the CO
-
2
group.


53

Figure 4.4: The
13
C NMR proton-decoupled spectrum (Varian Mercury 300 MHz NMR
Spectrometer) of the synthesised N-carboxymethyl chitosan.
The
13
C is decoupled, i.e. the carbon-proton splitting is removed because of the low natural
abundance of
13
C. Therefore each chemically non-equivalent set of carbons appears as a
single line. A larger range (about 200 ppm) of chemical shifts is observed than that of
1
H
NMR. The signals are more sensitive to small changes in chemical shift and hence give rise
to more distinct resonance for each chemically non-equivalent set of carbons compared to
proton signals in
1
H NMR.
The
13
C NMR spectrum confirms the presence of the COOH at a chemical shift of 176.784
ppm, which is fairly close to that of Le Dung (1994) at 180 ppm and complies with
characteristic CMR chemical shifts by Loudon (1995) depicted in Table 4.3. The acetamido
group signal could probably be alongside the CO
2
-carbon signal.

54
4.4.2 Infrared (IR) characterisation
Infrared (IR) characterisation was done using potassium bromide (KBr) powder as a
dispersion prism medium. A spectrum was recorded between 1800 to 400 cm
-1
to establish
the presence or absence of functional groups at their characteristic band positions. The IR
spectra of the starting polymer (i.e. chitosan) and the product (i.e. N-carboxymethyl chitosan)
are shown in Figures 4.5 and 4.6, respectively.

Figure 4.5: IR spectrum of chitosan (Thermo Nicolet; Erweka 360 FTIR, ESP)

Figure 4.6: IR spectrum of N-carboxymethyl chitosan (Nicolet; Erweka 360 FTIR, ESP).

55
The IR spectrum for the synthesised carboxymethyl chitosan compares well with the IR
spectrum obtained by Muzzarelli et al. (1982) for N-carboxymethyl chitosan of various
degrees of substitution. The CO
2
H band at 1736.65 cm
-1
is in agreement with the discussion
in Loudon (1995) and is distinctly absent in the un-substituted chitosan IR spectrum shown in
figure 4.3. The symmetrical CO
2
stretching vibration at 1396.30 cm
-1
compares to that of the
literature 1400 cm
-1
and confirms the presence of the CO
-
2
from the CO
2
H group.
4.5 CROSS-LINKING AND WAFER PREPARATION
The wafers were prepared with only cross-linked N-carboxymethyl chitosan as an excipient.
Neither diluents nor any other excipient were added. A mass of 4 g of synthesised N-
carboxymethyl chitosan was weighed and then dissolved by stirring rigorously with a
magnetic stirrer in 100 ml of distilled water. A mass of 4 g of ibuprofen, the model drug,
was added while stirring the above solution at room temperature. The pH of this mixture was
adjusted to a range between 6.0 and 7.0. A volume of 12 to 15 ml of this solution was
transferred drop-wise into 100 ml of 20% w/v aqueous barium chloride (BaCl
2
) cross-linking
solution and stirred until an insoluble material or hydrogel was observed. The resulting
hydrogel was centrifuged at 1500 rpm periodically until the solid mass was reasonably free
of the supernatant liquid.
Hydrogel units of a mass of 2.5 g each were transferred with moulding using two spatulas
into round shaped wells on a plastic tray. These moulded gels were then dried at 40
o
C for
about 48 hours with continual monitoring, further moulding, compressing and turning over
where necessary. These dried matrix systems or wafers were then stored in a desiccator until
further use.
4.6 EVALUATION OF N-CARBOXYMETHYL CHITOSAN WAFERS
4.6.1 Drug content
An accurately weighed mass of 100 mg of a cross-linked wafer was transferred into a 100 ml
volumetric flask and 60 ml of 0.1 M NaOH was added to the flask. The mixture was stirred
for 24 hours and then made up to volume with the sodium hydroxide solution. A portion of

56
the supernatant liquid was transferred into a UV cuvette. The absorbance was determined at
264 nm, the wavelength of maximum absorbance, against a blank. This test was done in
triplicate.
An ibuprofen calibration curve was obtained by measuring the absorbance of samples on the
from a range of ibuprofen dilutions in 0.1 M NaOH including 0.16; 0.20; 0.25; 0.32; 0.40 and
0.48 mg/ml. The ibuprofen calibration curve with the equation describing the linear
relationship as well as the regression (R
2
) is shown in Figure 4.5 and this equation was used
to calculate the drug content.
y =1.8681x +0.008
R
2
=0.9973
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0 0.1 0.2 0.3 0.4 0.5 0.6
Concentration (mg/ml)
A
b
s
o
r
b
a
n
c
e

a
t

2
6
4

n
m

Figure 4.6: Ibuprofen calibration curve
The drug content is expressed as a percentage calculated by means of Equation 4.2.
100
) 100 (

% x
mg wafer of mass
drug of mass
content Drug = (4.2)
The ibuprofen concentrations as well as the percent drug content are reported in Table 4.6.

57
Table 4.6: Ibuprofen determination at 264 nm for drug content of CMC wafers.
Sample number Abs at 264 nm Conc. (mg/ml) % drug content
1 0.6320 0.334029 33.4
2 0.7260 0.384348 38.4
3 0.6520 0.344735 34.5
Average drug content/wafer 0.354371 35.4

4.6.2 Swelling properties
The N-carboxymethyl chitosan wafers (or matrix systems) were accurately weighed (W
0
) and
immersed in buffer solutions of pH 5.8, 7.4 and 8.0, respectively. At 15 min time intervals
the wet wafers were weighed after they were wiped with soft paper tissue (W
t
). The degree
of swelling (or hydration ratio) at time t may be calculated using Equation 4.3
o
o t
W
W W
ratio Hydration

= (4.3)
W
t
and W
0
are the weights of the wet wafers at time t and in the dry state respectively.
The mass of the wafers, before and after dispersion into the buffer solutions, is given in Table
4.7. It is clear from the results that the swelling properties of the N-carboxymethyl chitosan
wafers could not be measured due to erosion and dissolution of part of the wafers. The mass
of the wafers decreased after dispersion in the liquid due to the loss of part of the wafer
because of the reversal of the cross-links and dissolution of the surface molecules. The
decrease in mass of the wafers happened at a faster rate in the slightly acidic environment
(pH 5.8) as compared to the neutral (pH 7.4) and alkaline (pH 8.0) environments.


58
Table 4.7: Mass of the N-carboxymethyl chitosan wafers at different time intervals at three
different pH values
Mass in grams of the wafers at 15 minute intervals in buffer media; Time
(minutes)
pH 5.8 pH 7.4 pH 8.0
0
0.4456 0.4692 0.4586
15
0.3060 0.3556 0.3531
30
0.2983 0.3321 0.3178
45
0.2871 0.3056 0.2927
60
0.2758 0.3043 0.2912
75
0.2747 0.3026 0.2848
90
0.2593 0.2937 0.2813
105
0.2552 0.2915 0.2749
120
0.2402 0.2756 0.2676

4.6.3 Ibuprofen release tests at 60 rpm and 370.5
o
C.
Dissolution tests were done in different buffered media to measure the rate and extent of
ibuprofen release from the wafers. The drug release behaviour of the cross-linked N-
carboxymethyl chitosan wafers was evaluated in three different phosphate buffers with pH
values of 5.8; 7.4 and 8.0, respectively. These buffer solutions were prepared by mixing
different volumes of 0.2 M KH
2
PO
4
and 0.2 M NaOH aqueous solutions as described in the
USP XXI. The pH was adjusted to the correct value as measured with a pH meter and
sufficient quantities of each buffer were prepared for all the dissolution tests. A control was
prepared by accurately loading an empty capsule with accurately weighed 35 mg of
ibuprofen raw material.

59
The paddle method (USP XXI) was used to conduct the dissolution tests and each experiment
was done in triplicate. The paddle position was set at 2.5 cm from the bottom of the flask
and the speed was adjusted to 60 rpm. A wafer (test group) or ibuprofen capsule (control
group) was transferred into a round-bottom dissolution vessel containing 900 ml of the
buffered dissolution medium at 37 0,5
o
C. Paper clips were used as sinkers to prevent the
capsules from floating.
Aliquots of 5 ml were withdrawn and immediately replaced with 5 ml of the dissolution
medium to maintain a constant volume of 900 ml. The samples were taken at the following
time intervals: 15, 30, 45, 60, 90, 120, 150, 180, 240, 300, 360, 420 and 480 minutes.
Stirring with the paddles was continued for up to 24 hours and the speed was increased to
200 rpm for the last 15 minutes in order to estimate the 100% release point. The samples
were filtered through 0.45 m membranes and the absorbance was determined at 264 nm in
the UV-VIS spectrophotometer against an appropriate buffer as a blank.
4.6.3.1 Dissolution profile at pH 5.8
The cumulative percentage values of ibuprofen released from the N-carboxymethyl chitosan
wafers at pH 5.8 are given in Table 4.8 using values from appendix A. These cumulative
percentage release values were plotted as a function of time and are shown in Figure 4.6.
The relatively fast release of the ibuprofen from the wafers can possibly be explained by the
relatively fast erosion of the wafers in this slightly acidic environment. This is in agreement
with the mass reduction of the wafers obtained from the test conducted for measurement of
the swelling properties (paragraph 4.5.2). The N-carboxymethyl chitosan was cross-linked
by using an ionic cross-linker (BaCl
2
) to form reversible bridges between the polymer
molecules. Moreover, Muzzarelli et al (1982) demonstrated the pH dependency of
insolubilization of metal ions by chelation with soluble N-carboxymethyl chitosan to form
hydrogels that is favourable in basic medium. These links between the polymer molecules in
the wafers are reversible and are degraded during the dissolution test with subsequent release
of the entrapped ibuprofen molecules.


60
Table 4.8: Cumulative % ibuprofen released from carboxymethyl chitosan wafers at pH 5.8.
Time Control Wafer 1 Wafer 2 Wafer 3
15 62.7 48.6 43.6 59.0
30 71.4 50.5 48.0 67.4
45 76.8 53.3 51.2 74.3
60 85.1 54.8 50.7 73.8
90 83.9 56.5 58.1 74.8
120 85.4 57.7 55.4 74.3
150 87.2 60.3 57.8 75.7
180 88.2 62.1 60.2 78.2
240 90.0 68.2 65.2 83.4
300 92.0 70.8 67.5 83.4
360 93.8 76.1 73.8 86.6
420 93.8 77.2 75.4 87.7
480 95.2 78.4 74.2 88.2

0
10
20
30
40
50
60
70
80
90
100
0 15 30 45 60 90 120 150 180 240 300 360 420 480
Time (min)
%

i
b
u
p
r
o
f
e
n

r
e
l
e
a
s
e
d
Control (pH 5.8) N-carboxymethyl chitosan wafer

Figure 4.6: % ibuprofen released as a function of time in phosphate buffer (pH 5.8).

61
4.6.3.2 Dissolution profile at pH 7.4
The cumulative percentage values of ibuprofen released from the N-carboxymethyl chitosan
wafers at pH 7.4 are given in Table 4.9. These cumulative percentage release values were
plotted as a function of time and are shown in Figure 4.7.
Table 4.9: Cumulative percentage ibuprofen released from N-carboxymethyl chitosan
wafers at pH 7.4.
Time Control Wafer 1 Wafer 2 Wafer 3
15 93.0 3.5 - 1.0
30 101.5 5.3 2.3 4.8
45 101.8 5.9 2.3 4.4
60 101.8 4.8 0.9 3.5
90 101.3 5.9 0.2 4.9
120 101.4 9.5 1.3 7.7
150 101.0 14.0 2.0 11.3
180 100.6 18.1 4.0 14.3
240 101.1 26.0 8.1 20.9
300 99.9 32.9 20.7 30.9
360 100.0 38.0 29.5 38.5
420 99.7 42.4 34.0 48.9
480 98.5 45.7 39.8 52.6




62
0
10
20
30
40
50
60
70
80
90
100
110
0 15 30 45 60 90 120 150 180 240 300 360 420 480
Ti me (mi n)
%

i
b
u
p
r
o
f
e
n

r
e
l
e
a
s
e
d
Control (pH 7.4) N-carboxymethyl chitosan wafer

Figure 4.7: % ibuprofen released as a function of time in phosphate buffer (pH 7.4).
The ibuprofen was relatively slowly released from the N-carboxymethyl chitosan wafers in
the neutral environment (pH 7.4) in which ibuprofen is better soluble as compared to an
acidic environment. Furthermore, the total cumulative quantity of ibuprofen released at the
end of the 8 h dissolution test was below 50%. This may be due to the relatively slow
erosion of the wafers at this pH value with a subsequent slower release of the entrapped
ibuprofen molecules in the matrix system. The reversible ionic links between the polymer
molecules were apparently not degraded at the same rate at this neutral pH value as compared
to a slightly acidic environment.
It is recommended that the formulation of N-carboxymethyl chitosan wafers be investigated
in future studies to enhance swelling to increase the total quantity of drug released over a
period of 8 h. Disintegrating excipients with a swelling mechanism or channelling agents
can be incorporated in different ratios to optimise the rate of drug release.



63
4.6.3.3 Dissolution profile at pH 8.0
The cumulative percentage values of ibuprofen released from the N-carboxymethyl chitosan
wafers at pH 8.0 are given in Table 4.10. These cumulative percentage release values were
plotted as a function of time and are shown in Figure 4.8.
The release of the ibuprofen from the N-carboxymethyl chitosan wafers at an alkaline pH
value (pH 8.0) is similar to that of the release at a neutral pH value. However, the total
cumulative quantity of ibuprofen released at the end of the 8 h dissolution test was higher as
compared to that released at the neutral pH value. This can possibly be explained by the
relatively better solubility of the ibuprofen at an alkaline pH value.
Table 4.10: Cumulative percentage ibuprofen released from N-carboxymethyl chitosan
wafers at pH 8.0.
Time Control Wafer 1 Wafer 2 Wafer 3
15 89.2 4.7 15.1 3.9
30 102.6 9.7 8.0 5.3
45 102.8 5.0 0.9 6.3
60 101.3 8.4 0.1 8.0
90 100.6 14.1 0.5 14.7
120 99.7 22.3 0.7 19.8
150 99.5 31.2 2.3 31.9
180 99.0 38.1 3.2 41.8
240 98.6 50.7 7.1 59.0
300 97.6 69.1 14.4 73.5
360 97.2 79.8 28.9 80.4
420 96.4 86.6 40.0 85.8
480 96.2 90.2 55.9 88.2


64
0
20
40
60
80
100
120
0 15 30 45 60 90 120 150 180 240 300 360 420 480
Ti me (mi n)
%

i
b
u
p
r
o
f
e
n

r
e
l
e
a
s
e
d
Control (pH 8.0) N-carboxymethyl chitosan wafer

Figure 4.8: % ibuprofen released as a function of time in phosphate buffer (pH 8.0).
The results from the dissolution tests were analysed for a quantitative comparison between
the dissolution of test groups and that of the control and between the dissolutions in the
different dissolution media.
4.7 DATA ANALYSIS
4.7.1 Mean dissolution time
The dissolution profiles were quantified by calculation of the mean dissolution time (MDT)
that is defined in equation 4.4 (Pillay & Fassihi, 1998):

=
n
i
M
M
t
i t MDT
1

(4.4)
M
t
is the fraction of dose released in time, = (t i t

i
+ t
i-1
)/2 where t
i
is the sampling time and
M corresponds to the loading dose.

65
MDT reflects the time for the drug to dissolve and is the first statistical moment for the
cumulative dissolution process that provides an accurate drug release rate (Reppas and
Nicolaides, 2002:232; Sousa et al., 2002:111). It is a more accurate expression for drug
release rate than the time of 50% dissolution (T
50%
). A higher MDT value indicates greater
drug retarding ability (Vueba et al., 2004). MDT values calculated for the N-carboxymethyl
chitosan wafers as well as control groups at each of the three buffer solutions are presented in
Table 4.11.
Table 4.11: MDT values for ibuprofen control groups and N-carboxymentyl chitosan
(CMC) wafer groups.
pH 5.8
Control
pH 5.8
CMC
pH 7.4
Control
pH 7.4
CMC
pH 8.0
Control
pH 8.0
CMC
MDT value 10.64 12.04 6.93 22.26 6.67 24.18

The MDT calculated values in each buffer solution and for each control-CMC pair indicate
higher drug retarding ability of the CMC formulation, i.e. higher values indicate the ability to
retard drug release (Vueba et al., 2004). The largest value was obtained for the basic
medium, followed by the neutral medium and then the acidic medium (with fastest release).
In addition, the MDT values for the control and the CMC matrix are fairly close suggesting a
fairly similar extent of drug retarding effect. These results are in agreement with the
cumulative percentage release-time profiles shown in Figures 4.6, 4.7 and 4.8.
4.7.2 Fit factors
The difference factor (f
1
) is defined in Equation 4.5 (Moore and Flanner, 1996).
% 100
1
1
1
x f
n
t
t
n
t
t t
R
T R

=
=
=

(4.5)

66
The difference factor describes the relative error between two dissolution profiles (reference
and test), n is the number of withdrawal points, R
t
is the reference assay at time point t, w
t
is
an optional weight factor and T
t
is the test assay at time point t for Equations 4.4 and 4.5.
The percent error expressed by f
1
is zero when the test and reference are identical and
increases proportionally with the dissimilarity between profiles, in other words, a lesser value
is desirable for f
1
.
The similarity factor f
2
is a measure of similarity in the percentage dissolution between two
dissolution curves and is defined by Equation 4.6 (Moore and Flanner, 1996):

+ =

100 ) ( 1 log 50
5 . 0
1
2
1
2
x T R w f
n
t
t t t n (4.6)
Where n is the number of withdrawal points, R
t
is the percentage dissolved of reference at the
time point t and T
t
is the percentage dissolved of test at the time point t.
A value of 100% for the similarity factor (f
2
) suggests that the test and reference profiles are
identical. Values between 50 and 100 indicate that the dissolution profiles are similar whilst
smaller values imply an increase in dissimilarity between release profiles (Flanner & Moore,
1996; Pillay & Fassihi, 1998).
Detailed calculations of the fit factors are shown in appendix A.
Table 4.12: The difference factor (f
1
) and similarity factor (f
2
) values calculated for the
dissolution profiles of the reference (control group) and test (N-carboxymethyl chitosan
wafers) groups at each pH value
.
pH 5.8 pH 7.4 pH 8.0
F
1
5.23 1.16 1.27
F
2
36.37 3.58 6.87


67
The similarity factor (f
2
) is higher at pH 5.8 than at pH 7.4 and 8.0, respectively. This
represent a higher similarity between the wafer and control in the slightly acidic environment
as compared to the higher pH values, which can be observed in the dissolution curves.
However, none of the ibuprofen release profiles from the wafers was similar to that of the
control (i.e. not statistically significantly similar as indicated by a value between 50 and 100).
4. CONCLUSION
The rate of ibuprofen release from the N-carboxymethyl chitosan wafers was lower than the
release from the immediate release dosage form in the control group at all the pH values.
However, the rate of ibuprofen release from the wafers was higher in the slightly acidic
environment (pH 5.8) as compared to the neutral and alkaline environments. This can be
attributed to a faster erosion rate of the wafers at this pH value as the Ba
2+
-CMC cross-links
are reversed due to the competition by the increased H
+
concentration in the acidic buffer.
This is confirmed by the erosion of the wafers in the test for swelling properties.
The cross-linked N-carboxymethyl chitosan wafers showed potential as controlled release
dosage forms with higher MDT values as compared to the control group, but different
formulation aspects should be investigated in future studies to improve on the performance of
these wafers.








68
5 PROSPECTIVE STUDIES
Improvement and refining of the synthesis process so as to optimise the yield, to
shift from synthetic to commercial grade reagents as indicated by Muzzarelli
(1982) and towards production scale.
An exhaustive study of the effect of temperature and pH on the formation of the
imine, an intermediate during N-carboxymethyl chitosan sysnthesis and also on
gel formation
In-depth study of adsorption properties to establish a link between the results and
the unique characteristics of chitosan and the derivatives.
Different formulation aspects to be investigated in future studies to improve on
the performance of these wafers.












69
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Influence of cellulose ether polymers on ketoprofen release from hydrophilic matrix tablets.
European jourmal of pharmaceutics and biopharmaceutics. 58: 51-59.
Yang, L. & Alexandidis, P. 2000. Physicochemical aspects of drug delivery and release
from polymer- science. 5: 132-143














75
APPENDIX A: IBUPROFEN RELEASE PROFILES
APPENDIX A.1: Fit factors for control and cmc wafer in pH 5.8 buffer
A value of 100% for the similarity factor (f
2
) suggests that the test and reference profiles are
identical. Smaller values imply an increase in dissimilarity between release profiles (Flanner
& Moore, 1996; Pillay & Fassihi, 1998). A zero value for f
1
means the test and reference are
identical and f
1
increases proportionally with the dissimilarity between profiles.
# pnt. 13
Dissolution profiles at 60 rpm and 37 0.5
o
C
f1 = 5.2328
% Ibuprofen released f2 = 36.37123385
Time Control 5.8 CMC 5.8 ABS(R-T) (R-T)
2
Criterion: f1<15 and f2>50
0 0 0 0.0000 0
15 62.7 50.4087 12.2913 151.07606
30 71.37 55.2795 16.0905 258.90419
45 76.78 59.5599 17.2201 296.53184
60 85.07 59.7689 25.3011 640.14566
90 83.86 63.1085 20.7515 430.62475
120 85.4 62.4563 22.9437 526.41337
150 87.17 64.6108 22.5592 508.9175
180 88.24 66.825 21.4150 458.60223
240 90.05 72.2521 17.7979 316.76524
300 92.01 73.9157 18.0943 327.40369
360 93.76 78.8507 14.9093 222.28723
420 93.78 80.0944 13.6856 187.29565
480 95.19 80.2438 14.9462 223.38889


76
APPENDIX A.2: Fit factors for control and cmc wafer in pH 7.4 buffer
# pnt. 13
Dissolution profiles at 60 rpm and 37 0.5
o
C
f1 = 1.159248
%Ibuprofen release
f2 = 3.582373
Time Control 7.4 CMC 7.4 ABS(R-T) (R-T)
2
Criterion: f1<15 and
f2>50
0 0 0 0 0
15 93 6.388047 86.604893 7500.407
30 101.48 4.125695 97.354305 9477.861
45 101.78 4.187168 97.592832 9524.361
60 101.8 3.046412 98.753588 9752.271
90 101.3298 3.674758 97.655042 9536.507
120 101.4301 6.147616 95.282484 9078.752
150 101.0381 9.079435 91.958665 8456.396
180 100.5996 12.15256 88.44704 7822.879
240 101.0888 18.32869 82.76011 6849.236
300 99.94834 28.16289 71.78545 5153.151
360 100.0304 35.34302 64.68738 4184.457
420 99.73678 41.75271 57.98407 3362.152
480 98.49432 46.02105 52.47327 2753.444






77
APPENDIX A.3: Fit factors for control and cmc wafer in pH 8.0 buffer
# pnt. 13
Dissolution profiles at 60 rpm and 37 0.5
o
C
f1 = 1.273588
%Ibuprofen release f2 = 6.871017
Time
Control 8.0 CMC 8.0
ABS(R-T) (R-T)
2
Criterion: f1<15 and f2>50
0 0 0 0 0
15 89.23517 7.886578 81.34859 6617.593
30 102.6484 7.646573 95.00183 9025.347
45 102.8243 4.059655 98.76465 9754.455
60 101.3252 5.50448 95.82072 9181.61
90 100.6015 9.749489 90.85201 8254.088
120 99.73406 14.22917 85.50489 7311.086
150 99.47845 21.76781 77.71064 6038.944
180 99.01334 27.7138 71.29954 5083.624
240 98.58563 38.9069 59.67873 3561.551
300 97.56634 52.31588 45.25046 2047.604
360 97.241 63.03761 34.20339 1169.872
420 96.35637 70.82384 25.53253 651.9101
480 96.24004 78.08469 18.15535 329.6167






78
APPENDIX A.4: Fit factors for controls in pHs 7.4 and 5.8 buffers
# pnt. 13
Dissolution profiles at 60 rpm and 37 0.5
o
C
f1 = 15.0850243
%Ibuprofen release f2 = 38.0164289
Time Control 7.4 Control 5.8 ABS(R-T) (R-T)
2
Criteriuon: f1<15 and
f2>50
0 0 0 0 0 Criterium:
15 93 62.7 30.29294 917.66221 f1<15
30 101.48 71.37 30.11 906.6121 f2>50
45 101.78 76.78 25 625
60 101.8 85.07 16.73 279.8929
90 101.3298 83.86 17.4698 305.19391
120 101.4301 85.4 16.0301 256.96411
150 101.0381 87.17 13.8681 192.3242
180 100.5996 88.24 12.3596 152.75971
240 101.0888 90.05 11.0388 121.85511
300 99.94834 92.01 7.93834 63.017242
360 100.0304 93.76 6.2704 39.317916
420 99.73678 93.78 5.95678 35.483228
480 98.49432 95.19 3.30432 10.918531






79
APPENDIX A.5: Fit factors for controls in pHs 8.0 and 5.8 buffers
# pnt. 13
Dissolution profiles at 60 rpm and 37 0.5
o
C
f1 = 13.69948
Time Control 8.0 Control 5.8 ABS(R-T) (R-T)
2
f2 = 39.20081
0 0 0 0 0 Criterium:
15 89.23517 62.7 26.53517 704.11525 f1<15
30 102.6484 71.37 31.2784 978.33831 f2>50
45 102.8243 76.78 26.0443 678.30556
60 101.3252 85.07 16.2552 264.23153
90 100.6015 83.86 16.7415 280.27782
120 99.73406 85.4 14.33406 205.46528
150 99.47845 87.17 12.30845 151.49794
180 99.01334 88.24 10.77334 116.06485
240 98.58563 90.05 8.53563 72.856979
300 97.56634 92.01 5.55634 30.872914
360 97.241 93.76 3.481 12.117361
420 96.35637 93.78 2.57637 6.6376824
480 96.24004 95.19 1.05004 1.102584







80
APPENDIX A.6: Fit factors for controls in pHs 7.4 and 8.0 buffers
# pnt. 13
Dissolution profiles at 60 rpm at 37 0.5
o
C
f1 = 1.945442
Time Control 7.4 Control 8.0 ABS(R-T) (R-T)
2
f2 = 81.06113
0 0 0 0 0 Criterium:
15 93 89.23517 3.75777 14.12084 f1<15
30 101.48 102.6484 1.1684 1.365159 f2>50
45 101.78 102.8243 1.0443 1.090562
60 101.8 101.3252 0.4748 0.225435
90 101.3298 100.6015 0.7283 0.530421
120 101.4301 99.73406 1.69604 2.876552
150 101.0381 99.47845 1.55965 2.432508
180 100.5996 99.01334 1.58626 2.516221
240 101.0888 98.58563 2.50317 6.26586
300 99.94834 97.56634 2.382 5.673924
360 100.0304 97.241 2.7894 7.780752
420 99.73678 96.35637 3.38041 11.42717
480 98.49432 96.24004 2.25428 5.081778











81