REVIEW OF LITERATURE…
2.1.
ANATOMY OF COLON
The digestive tract is the system of organs within multicellular animals
that takes in food, digests it to extract energy and nutrients, and expels the
remaining waste. The major functions of the GI tract are ingestion, absorption and
defecation.
In a normal human adult male, GIT is approximately 6.5 metres (20 feet
long) and consists of upper and lower GI tracts. The upper GI tract consists of the
mouth, pharynx, oesophagus and stomach. The lower GI tract comprises the small
intestine, large intestine and anus.A Large intestine is wider and shorter than the
small intestine (approximately 1.5 metres in length as compared to 6.7 to 7.6
metres in length for the small intestine). The colon is 1.5 cm long and it itself is
made up of caecum, the ascending colon, hepatic flexure, transverse colon,
splenic flexure, descending colon and the sigmoid colon. The structural features
are depicted in Figure 2.1 and anatomical features of small intestine & large
intestine are summarised in Table 2.1.
Figure 2.1 Structural features of large intestine
5
REVIEW OF LITERATURE…
Table 2.1 Anatomical features of small intestine and large intestine
Characteristics
Organ
Small Intestine
Duodenum
Jejunum
Ileum
It is 25-30 cm long section. It serves as a receiving area for
chemicals and partially digested food from stomach.
It is about 40% of the small gut in man. It comes in contact with
large number of intestinal cells containing thousands of tiny finger
like projections called villi which increases surface area to absorb
most of the nutrients into blood
It is about 60% of the intestine in man. It contains goblet cells and
peyer’s patches. Here remaining nutrients are absorbed before
moving into the large intestine.
Large Intestine
Caecum
Ascending
colon.
Hepatic
flexure
Transverse
colon
Descending
colon
Sigmoid
colon
Rectum
It is about 6-7 cm in length. It is the pouch where the large
intestine begins. It is where ileum opens from one side and
continues with the colon.
It is about 20 cm long. It is the part of the large intestine that goes
from the bend on the right side below the liver and the caecum.
It is on the right side of the body near the liver. It is the right angle
bend in the colon that marks the connection of the ascending colon
and transverse colon.
It is about 45 cm long. It is the largest and most mobile part of the
colon (Meschan, 1975). It attaches the ascending colon to the
descending colon by crossing the abdominal cavity. Its diameter
varies from 9 cm in caecum to 2 cm in sigmoid colon; its average
diameter is about 6.5 cm (Mrsny, 1992).
It is about 30 cm long. It traverses inferiorly along the left
abdominal wall to the pelvic region.
It is about 40 cm long. It is the part of the colon that forms an angle
medially from the pelvis to form an S-shaped curve.
It is about 12 cm in length. It is a short, muscular tube that forms
the lowest portion of the large intestine and connects it to the anus.
Faeces collects here until pressure on the rectal walls cause nerve
impulses to pass to the brain, which then sends messages to the
voluntary muscles in the anus to relax, permitting expulsion.
Source:-David, 1992
6
REVIEW OF LITERATURE…
The colon serves following important functions:
1.
Creation
of
suitable
environment
for
the
growth
of
colonic
microorganisms.
2.
Absorption of potassium and water from the lumen concentrating the
faecal
contents, secretion and excretion of potassium and
bicarbonate ions (Watts and Illum, 1997).
3.
Through the muscular movements of colon faecal matter is pushed along
until finally, the walls of the sigmoid colon contracts, causing the faeces to
move into the rectum.
4.
Synthesis of vitamin K by colonic bacteria promotes a valuable
supplement to dietary sources and makes clinical vitamin K deficiency
rare.
The colon is mainly situated in the abdomen; the rectum is primarily a
pelvic organ. Further, the histological and microscopic structural evaluation of
colon shows four layers: serosa, the mascularis externa, the submucosa and the
mucosa (Figure 2.2 - 2.3).The serosa is the external coat of the large intestine and
consists of aerolar tissue that is covered by single layer of squamous mesothelial
cells. The major muscularis coat of the large intestine is the muscularis externa.
This is composed of an inner circular layer of fibers that surrounds the bowel. The
submucosa is the layer of connective tissue that lies immediately beneath the
mucosa. The colonic mucosa is further divided into three layers: the muscularis
mucosae, the lamina propria and the epithelium.
Figure 2.2 Histological features of Colon (Source:- Harshmohan, 2003)
7
REVIEW OF LITERATURE…
Figure 2.3 Microscopic structure of colon (Source:- Kaye et al.,
1973)
2.2
CHALLENGES IN THE DESIGN OF COLONIC DELIVERY
DOSAGE FORMS
Formulations for colonic delivery are, in general, delayed-release dosage
forms which may be designed either to provide a ‘burst release’ (Pinhasi et. al.,
2004) or a sustained/prolonged release once they reach the colon. The proper
selection of a formulation approach is dependent upon several important factors,
which are listed below:
a)
Pathology and pattern of the disease, especially the affected parts of the
lower GI tract or physiology and physiological composition of the healthy
colon if the formulation is not intended for localized treatment.
b)
Physicochemical and biopharmaceutical properties of the drug such as
solubility, stability and permeability at the intended site of delivery, and
c)
The desired release profile of the active ingredient.
Formulation of drugs for colonic delivery also requires carefull
consideration of drug dissolution and/or release rate in the colonic fluids.
Generally, the dissolution and release rate from colonic formulations is thought to
be decreased in the colon, which is attributed to the fact that less fluid is present in
the colon than in the small intestine (Takaya et. al., 1998). The poor dissolution
and release rate may in turn lead to lower systemic availability of drugs. These
issues could be more problematic when the drug candidate is poorly water-soluble
and/or requires higher dose for therapy. Consequently, such drugs need to be
delivered in a presolubilized form, or formulation should be targeted for proximal
colon, which has more fluid than in the distal colon (Basit and Bloor, 2003).
8
REVIEW OF LITERATURE…
Likewise, colonic formulations for polar drugs (Log Poct/pH 7.4 buffer < 1.0)
including proteins and peptides require use of absorption enhancing agents (also
known as absorption promoters). Examples of suitable absorption enhancers
include fatty acids (Watts and Illum, 2001; Takada and Murakami, 2005), bile
salts (New, 1998) and chelating agents (Miyauchi, 1990). Few factors usually
considered to be important during colonic drug delivery are summarized in Table
2.2.
2.2.1 Barriers in Colonic Absorption
Drug absorption from colon can be limited by a number of barriers (Figure
2.4).
a)
Mucosal and Physical Barriers
Besides providing a stable pH environment, the mucus layer adjacent to
the colonic mucosa also acts as a diffusion barrier (Smith et. al., 1986). The
mucus layer at the epithelial surface, due to it’s highly charged and sieve like
nature presents a formidable thermodynamic barrier to the transit of large
negatively charged drug molecules. Cephalosporins, penicillins
and aminoglycosides are few examples of small molecule drugs that can bind to
negatively charged mucus (Niibuchi et. al., 1986). This might facilitate longer
colonic residence time and hence environmental or enzymatic degradation.
Alteration of this layer using mucolytic agents has been implicated in a variety of
disease processes and pathological conditions.
At the mucosal surface there is a layer of relatively unstirred water layer
which also acts as a barrier to colonic transport. All molecules must pass through
this area by diffusion, and thus molecular size and other determinants of
diffusibility, such as polarity, affects themovement of drug molecules towards the
mucosa. Some viscous soluble dietary fibres may increase the thickness of this
layer by reducing intraluminal mixing (Johnson and Gee, 1981).
However, physical barrier to drug absorption, at the level of epithelium, is
the lipid bilayer of the individual colonocytes and the occluded junctional
complex between the cells (Powell, 1981). Hence, the passage of drugs is via
transcellular route or the paracellular route. As the cell cytoplasm has a number of
enzymes, transit through cell cytoplasm may result in extensive enzymatic
degradation that makes uptake of drug molecules into blood capillaries or
lymphatic sinuses problematic. Therefore, successful passive transcellular
9
REVIEW OF LITERATURE…
transport requires a drug moiety that is stable to the multiple environments
encountered in its transit through the colonic epithelial cells.
b)
Chemical Barriers
Some dietary fibres, such as pectin and chitosan (Vahouney et. al., 1981)
have cation-exchange properties, which may bind charged molecules such as bile
acids. This binding is increased at the low pH encountered in the colon (Eastwood
and Hamilton, 1968) and may be
Table 2.2 Factors affecting colonic drug absorption
Route and rate of delivery to
colon
Oral, rectal
Dosage form
Does it need release or activation by bacteria?
Lipid solubility
May be affected by pH
Colonic residence
Site of colon (proximal or distal) important-may be affected by
diet, drugs, disease, exercise, etc.
pH microclimate
pka of drug
Mixing rate and resistance of
contents
Mucus barrier, unstirred
layer
Solidity or viscosity of content may be affected by diet
Thickness may be increased by dietary components
Mucosal pore size
Permeability, absorption characteristics
Concentration
Dilution by luminal contents especially with high water-holding
capacity dietary fibre
Bacterial metabolism
May be affected by diet or antibiotics
(Edwards et. al., 1997)
10
REVIEW OF LITERATURE…
Figure 2.4 Barriers to colon drug delivery (Source: - Randall, 1992)
a factor in the immobilisation of some drugs. In addition, drug molecules could be
trapped within the solid matrix of the concentrated dietary residue or within the
entangled chains of a soluble dietary fibre.
c)
Gastrointestinal Transit
The transit through the GI tract is highly variable and depends on many
factors (Hunter et. al., 1982; Davis et. al., 1984; Devereux et. al., 1990; Price et.
al., 1993; Meier et. al., 1995; Brown et. al., 1998). Gastric transit of single-unit
non-disintegrating dosage forms has been reported to vary from 15 min to more
than 3 h (Kaus et. al., 1984). Table 2.3 gives overview of transit time of dosage
forms in GIT.
d)
Gastrointestinal pH
The progressive change in pH along the human GI tract has been well
characterized (Dressman et. al., 1993; Evans et. al., 1988). pH in different regions
of GIT is shown in table 2.4.
e)
Microbiological Barriers
The human GIT, the major port for the entry of drugs into humans,
consists of a complex ecosystem incorporating aerobic and anaerobic
microorganisms (Simon and Gorbach, 1984; Rubinstein, 1990; Watts and Illum,
1997). The flora becomes diverse and luxuriant in the colon (Figure 2.5). The
microflora of the stomach is normally sparse and bacterial concentration is less
than 103 CFU/ml. The microflora in stomach is predominately gram positive and
11
REVIEW OF LITERATURE…
aerobic. The most commonly isolated species are streptococci, staphylococci,
lactobacilli and fungi (Simon and Gorbach, 1984). The
Table 2.3 Transit time of dosage forms in GIT
Organ
Stomach
Transit time (h)
Reference
< 1 (Fasting), > 3 (Fed)
Hardy et al., 1987
3-4
Hinton et al., 1969
33 (Men)
Hinton et al., 1969
47 (Women)
Hinton et al., 1969
Small intestine
Colon
Table 2.4 pH in different regions of GIT
pH
Region of GIT
1.0-2.5
Stomach
Reference
Evans et al., 1988
Evans et al., 1998;
Duodenum
5.1-6.6
Jejunum
5.2-6.2
Ileum
6.8-7.8
Caecum
6.4
Bussemer et al., 2001
Large
Ascending colon
5.7
Bussemer et al., 2001
intestine
Transverse colon
6.6
Bussemer et al., 2001
Descending colon
7.0
Bussemer et al., 2001
Small
intestine
Ashford, 2002
Ashford, 2002
Evans et al., 1988;
Ashford, 2002
Figure 2.5 Concentration of bacterial flora in different regions of the
gastrointestinal tract
12
REVIEW OF LITERATURE…
bacterial concentration in the duodenum is of the order of 103 –104 CFU/ ml and
the predominant species are aerobic and gram positive. These include
Streptococci, Staphylococci, Lactobacilli and anaerobic Veillonella. In the
jejunum and the upper ileum very few microorganisms are present which include
Lactobacilli and Enterococci. In the distal ileum, gram-negative bacteria begin to
outnumber the gram-positive organism. The bacterial concentration becomes high.
Coliforms are consistently present and anaerobic bacteria such as Bacteroides,
Bifidobacterium, Fusobacterium, and Clostridium are found in high concentration
(Gorbach et. al., 1967a; Gorbach and Levitan, 1970; Drasar and Shiner, 1969;
Gorbach, 1971; Thadepalli et. al., 1979). The human colon is a dynamic and
ecologically diverse environment, containing over 400 distinct species of bacteria
with a population of 1011 to 1012 CFU/mL (Cummings and Macfarlane, 1991;
Gorbach, 1971). The most important anaerobic bacteria found in colon are
Bacteroides,
Bifidobacterium,
Eubacterium,
Lactobacillus,
etc.
greatly
outnumbering other species.
The colonic microflora produce a variety of enzymes that are not present in the
stomach or the small intestine and could therefore be used to deliver drugs to the
colon after enzymatic cleavage of degradable formulation components or drug
carrier bonds (enzyme-controlled drug delivery).
2.2.2 Inflammatory Bowel Disease
Inflammatory bowel disease (IBD) is a chronic inflammatory condition of
gastrointestinal tract. Ulcerative colitis (UC) and Crohn’s disease (CD) are its two
major types. UC is limited to large intestine, whereas in CD inflammation of
almost every part of gastrointestinal tract occurs (Gramlich et. al., 2007).
Although IBD represented mainly by ulcerative colitis and Crohn’s disease but
also include noninfectious inflammations of the bowel (Strober et. al., 2007).
Inflammatory diseases in the gastro-intestinal tract have been known for
thousands of years. The incidences of IBD are found mainly in areas such as
Northern Europe and North America, but continue to rise in low –incidence areas
such as Southern Europe, Asia and other parts of developing world. As many as
1.4 million persons in the United States and 2.2 million persons in Europe suffer
from this disease (Loftus et. al., 2004). IBD and various other diseases related to
colon are depicted in Figure 2.6, Table 2.6.
13
REVIEW OF LITERATURE…

Etiopathogenesis
Although the exact etiology of ulcerative colitis and Crohn’s disease is
unknown, similar factors are believed to be responsible for both conditions (Table
2.5).
Figure 2.6 Diagrammatical presentation of Colon associated diseases
Table 2.5 Proposed etiologies for inflammatory bowel disease
Factors
Causes
Viruses (eg. measles)
Infectious Agents
Commensal bacteria
Mycobacteria
Chlamydia
Metabolic defects
Genetics
Environmental factors
Connective tissue disorders
Geographical and socioeconomic factors
Diet
Smoking
Altered host susceptibility
Immune defects
Immune-mediated mucosal damage
Stress
Psychological factors
Emotional or physical trauma
Occupation
Barrier functions
Impaired intestinal permeability
(Source:- Modified from Dipro, J.T and Schade, R.R, 2005)
14
REVIEW OF LITERATURE…
Table 2.6 Colon targeting diseases, drugs and target sites
Target Disease Conditions
Sites
Topical /
Local
action
Symptoms
Drugs and active agents
Inflammatory Bowel Disease
Crohn’s
disease
Diarrhea, Abdominal pain and cramping,
Mesalamine, Hydrocortisone,
blood in stool, ulcers, reduced appetite andBudenoside, Prednisolone,
weight loss
Sulfasalazine, Olsalazine,
Balsalazide, Infliximab
Ulcerative Inflammation in the rectum, rectal bleeding, Mesalamine, Sulfasalazine,
colitis
Balsalazide, Infliximab,
Azathioprine and Mercaptopurine
rectal pain
Irritable bowelAbdominal pain or cramping, a bloated
Dicyclomine, Hyoscine,
syndrome feeling, flatulence, diarrhea or constipation —Propantheline, Cimetropium,
people with IBS may also experience
Mebeverine, Trimebutine,
alternating bouts of constipation and
Scopolamine, Alosetron,
diarrhea, mucus in the stool
Tegaserod
Colorectal A change in bowel habits, narrow stools,
5-Flourouracil, Leucovorin,
cancer
rectal bleeding or blood in stool, persistent Oxaliplatin, Irinotecan,
abdominal discomfort, such as cramps,
Bevacizumab and Cetuxim B
abdominal pain with bowel movement,
unexplained weight loss
Diverticulitis Formation of pouches (diverticula) on the Bactrim, Flagyl, Sulfatrim
outside of the colon due to bacterial infectionMetronidazole
Antibiotic Overgrowth of Clostridium difficile and its
associated subsequent toxin production
colitis
Hirschsprung'sSevere form of constipation in which bowel
disease movement occurs only once or twice a week
To prevent
---Systemic
action
first pass
metabolism of
orally ingested
drugs
Oral delivery
---of peptides
Ulcerative Ulcerative proctitis, pancolitis, fulminant
colitis
colitis
15
Clindamycin, Broad-Spectrum
Penicillins (Eg, Ampicillin,
Amoxicillin) and Cephalosporins
Metronidazole, vancomycin,
loperamide, botulinum toxin
Steroids
Insulin
Prednisolone Metasulfobenzoate,
Tixocortol Pivalate, Fluticasone
Propionate, Beclomethasone
Dipropionate
And
Budesonide, Azathiorpine/6Mercaptopurine.
REVIEW OF LITERATURE…
2.2.3
Animal Models for Inflammatory Bowel Disease
The animal models used to investigate inflammatory bowel disease are
shown in Table 2.7. However, models used by the induction of chemicals i.e.
inducible colitis models are discussed here.
Inducible Colitis Models
1)
Trinitrobenzene sulfonic acid (TNBS)-induced colitis
It has been reported that colitis would occur by treatment with a TNBS
enema after destruction of the mucosal barrier with an ethanol enema (Morris et.
al., 1989; Wirtz et. al., 2007). Ethanol is required to break the mucosa; barrier,
whereas TNBS is believed to haptenize colonic autologous or microbiota proteins
rendering them immunogenic to the host immune system. As CD4+ T cells have
been shown to play a central role in chronic TNBS colitis, this model is useful to
study T helper cell-dependent mucosal immune responses (Neurath et. al., 19995).
The TNBS colitis model has been very useful in studying many important aspects
of gut inflammation, including cytokine secretion patterns, mechanism of oral
tolerance, cell adhesion and immunotherapy. Acute colitis in rats is associated
with mucosal permeability as a consequence of epithelial necrosis and elevations
in colonic myeloperoxidase activity. A high damage score is observed, which is
apparently related to increase in the number of macrophages and granulocytes.
Murine TNBS colitis was initially describe in SJL/J mice, a mouse strain with
high susceptibility, that develops chronic TNBS colitis was characterized by a
predominant TH1-mediated immune response with dense infiltrations of
lymphocytes/macrophages and thickening of the colon wall. TNBS/ethanolinduced colitis in SJL/J mice is characterized by a transmural granulomatous
inflammation with severe diarrhea, weight loss and thickening of the bowel wall.
The chronic stage is associated with an activation of the mucosal immune system
and an increase in the number of infiltrating lymphocytes, especially CD4+ T
cells in the lamina propria (Neurath et. al., 1995). However, studies with IFN-γ-/mice on a Balb/c background showed that in these mice. TNBS colitis may be
associated with TH2-mediated colonic patch hypertrophy (Dohi et. al., 1999 and
2000).
2)
Oxazolone colitis
16
REVIEW OF LITERATURE…
A number of animal models of colitis have been reperted, but all are Thelper type-1 colitis, except for TCRα-/- mice. In 1998 it was reported that an
enema of oxazolone with ethanol would induced colitis (Boirivant et. al., 1998).
Rectal administration of the hapten reagent oxazolone dissolved in ethanol
induces severe colitis in rats or mice, characterized by weight reduction, diarrhea
and marked loss of goblet cells, leading to high death rates
Table2.7 Animal models of chronic intestinal inflammation
Name of the Category
Models
Geneticallyengineered/Transgenic
animal models




Gene knockout mouse models


Mouse and rat models with gene
overexpression
IL-2 knockout/IL-2 Rα knockout
mice
IL-10/CRF2-4 knockout mice
STAT-3 knockout mice
T-cell receptor-α chain knockout
mice
TNF-3 UTR knockout mice
Trefoil factor-deficient mice
 IL-7 transgenic mice
 STAT-4 transgenic mice
 HLA B27 transgenic rat


Models of spontaneous colitis
Inducible colitis models
Dextran sulfate sodium colitis Adoptive
transfer models
17
C3H/HeJBir mice
SAMP/Yit mice
 Trinitrobenzene sulfonic acidinduced colitis
 Oxazolone colitis
 Dextran sulfate sodium induced
colitis
 Acetic acid induced colitis
 Indomethacin induced colitis
 Carrageenan colitis
 Peptidoglycan-polysaccharide
colitis
 CD4+/CD45RB high T-cell
transfer colitis
 Colitis induced by transfer of
hsp60-specific CD8 T-cells
REVIEW OF LITERATURE…
IL-interleukin, TNF-tumor necrosis factor, UTR- untranslated region, hsp-heat
shock protein. (Modified source:- Hibi et. al., 2002).
(Ekstrom et. al., 1998; Boirivant et. al., 1998). The peak body weight loss and
diarrhoea is seen on the second day after enema and symptoms diminish after 1012 days. Colitis, accompanied by ulcers, is localized in the distal colon.
Histopathological studies show that the number of epithelial cells, goblet cells and
glands are decreased compared with controls. In contrast to TNBS colitis, these
findings closely resemble those to UC. The stimulated lamina propria
lymphocytes in this model produce increased amounts of IL-4 and IL-5, but not
IFNγ, which suggests that the colitis in this model is induced by a T-helper type-2
response. In contrast to several other murine colitis models, treatment with
neutralizing anti-IL-4 antibodies or a decoy IL-13Ra2-Fc protein ameliorates
disease (Boirivant et. al., 1998; Heller et. al., 2002). Therefore, this model has
been used to study the contribution of TH2-dependent immune response to
intestinal inflammation. In comparison with TNBS, this agent caused colitis
earlier.
3)
Dextran sulfate sodium (DSS) colitis
Feeding mice for several days with DSS polymers in the drinking water
induces a very reproducible acute colitis characterized by bloody diarrhoea,
ulcerations and
infiltrations with granulocytes (Okayasu et. al., 1990;
Mahler et. al., 1998; Wirtz et. al., 2007). It is generally believed that DSS is
directly toxic to gut epithelial cells of the basal crypts and affects the integrity of
the mucosal barrier. As T and B-cells-deficient C.B-17scid or Rag1-/- mice also
develop severe intestinal inflammation, the adaptive immune system obviously
does not play a major part (at least in the acute phase) in this model (Dieleman et.
al., 1994). Hence, the acute DSS colitis model is particularly useful to study the
contribution of innate immune mechanisms of colitis. In addition, the DSS model
has been shown to be study epithelial repair mechanisms (Wiliams et. al., 2001).
In susceptible strains, the administration of DSS for several cycles (e.g., 7 days
DSS, 14 days water) results in chronic colitis and if combined with single dose of
the genotoxic colon carcinogen azoxymethane (AOM). Patients with UC have
increased risk for the development of colon cancer (Jess et. al., 2006). As colonic
inflammation is suggested to play a key role in IBD-related colorectal cancer, the
18
REVIEW OF LITERATURE…
AOM/DSS model is a very useful tool to study mechanisms linking inflammation
to colon carcinogenesis.
4)
Acetic acid induced colitis
The intrarectal administration of diluted acetic acid in to rodents or rabbits
leads to epithelial injury and increased permeability followed by an acute
mucosal/transmural inflammation in a dose-dependent manner (MacPherson et.
al., 1978). In the original description of the model, 0.5 ml of 10-15% acetic acid
diluted with water was instilled in to the rectum of Sprague-Dawley rats. After
10s of surface contact, the acdic solution was withdrawn, and the lumen was
flushed three times with0.5 ml saline. In a later modification (Yamada et. al.,
1991), 1 ml 4% acetic acid (pH 2.3) was slowly infused 5 cm into the rectal lumen
of a anesthetized rat. After a 30s exposure, excess fluid was withdrawn, and the
colon was flushed with 105 ml physiological buffer solution. Many further
modifications have been introduced throughout the years, and most subsequent
studies used 15 to 30sexposures to 4% or 5% acetic acid in both enema and
ascending colon models because higher concentrations induced frequent
perforations. This reproducible model is easy to use and valuable for studying
early events of inflammation after mucosal injury and wound healing. Mucosa and
submucosal inflammation followed initial injury and was associated with
activation of arachidonic acid pathways (Elson et. al., 1995).
5)
Indomethacin induced colitis
In rats subcutaneous injection or oral administration of indomethacin
causes chronic ulcerations and transmural inflammation in the small bowel
(Banerjee and Peters, 1990; Yamada et. al., 1993). It was shown that
indomethacin induces small intestinal and colonic ulceration in a dose-dependent
fashion in rodents (Elson et. al., 1995). Initial epithelial damage is mediated partly
by synthesis inhibition of the protective prostaglandins PGE1, PGE2 and
prostacyclin. Germ-free rats and rats treated with antibiotics do not show chronic
inflammation, indicating a pivotal role of the enteric microflora for disease
development. This model has the advantage of being easily induced, acute or
chronic phases.
6)
Carrageenan colitis
Degraded carrageenan polymers in the drinking water of guinea pigs and
mice lead to mucosal inflammation of the caecum within a week, which extends
19
REVIEW OF LITERATURE…
to the left side of the colon within 3-6 weeks after treatment (Marcus et. al., 1989;
Kitsukawa et. al., 1992). Removing the polymers from the drinking water
prolongs the colitis for 1-2 weeks, while
Prolonged treatment is lethal after 7-8 weeks (due to sepsis). Carragenan affects
epithelial cells and severely impairs the mucosal barrier. Several studies have
shown that presence of anaerobic bacteria (in particular Bacteroides species) is
important for the development of mucosal lesions and ulcerations (Breeling et. al.,
1998), although the exact pathophysiological mechanisms remain unclear.
7)
Peptidoglycan-polysaccharide colitis
Intramunal injection of the bacterial cell wall component peptidoglycan-
polysaccharide (PG-PS) into the distal colon of rats induces transmural
enterocolitis (Sartor et. al., 1988). In genetically susceptible animals chronic
granulomatous colitis with thickening of the colon wall and infiltrating
lymphocytes, macrophages and neutrophils develops after 3-4 weeks. PG-PS
increases mucosal permeability, myeloperoxidase activity, enhances NO
production and collagen synthesis. Treatment with recombinant IL-1 receptor
antagonist (Carter et. al., 2001) or IL-10 (Herfarth et a;., 1996) attenuates disease,
particularly the chronic stages of nonpathogenic resident enteric bacteria are
sufficient to induce acute and chronic colitis resident enteric bacteria are sufficient
to induce acute and chronic colitis in a susceptible host, when they penetrate into
the colon wall.
2.2.4 Permeation of drugs across colon
Drug permeation in the colon takes place by two routes: paracellular route
and transcellular route. Transcellular absorption involves passage of the drugs
through cells and this is the route lipophilic drugs takes, whereas paracellular
absorption involves the transport of drug through the tight junctions between cells
and is the route most hydrophilic drugs takes. The poor paracellular absorption of
many drugs is due to the fact that the epithelial cell junctions are very tight
(Powell, 1981).
Colon is a more selective site for the drug absorption than small intestine
for many drugs. Drugs shown to be well absorbed include diclofenac (Gleiter et.
al., 1985), ibuprofen (Wilson et. al., 1989), and theophylline (Staib et. al., 1986).
Drugs shown to be less absorbed include atenolol, cimetidine, hydrochlorthiazide,
lithium (Ehrlich and Diamond, 1983).
20
REVIEW OF LITERATURE…
The rate and extent of drug absorption from the colon is thought to be
slower than small intestine due to lock of villi, microvilli and circular folds etc.
The single most significant barrier to epithelial transport of drugs in the colon is at
the level of epithelium. The drugs intending to pass from the epical to basolateral
surface of the epithelial barrier must do by passing through either colonocytes (the
transcellular route) or between adjacent colonocytes (the paracellular route).
Transit through the cell cytoplasm may result in extensive enzymatic degradation.
Therefore, successful passive transcellular transport requires a drug moiety that is
stable to the multiple environments encountered in its transit through the colonic
epithelial cells.
The drug molecules that successfully transverse the physical and
enzymatic barrier of the colonic mucosa are taken up by the venous and lymphatic
drainage systems. In general, the hydrophilic drugs not degraded by local
enzymes in the submucosa, are taken up via the venous drainage and transported
to the liver through the hepatic portal system. A significant amount of metabolism
of drugs can occur in the liver prior to reaching the systemic circulation.
Alternatively the hydrophobic drugs are transported via the lymphatic sinuses and
enter the systemic circulation without undergoing metabolic breakdown (Watts
and Illum, 1997).
Small amphipathic drugs (i.e. molecules having both hydrophilic and
hydrophobic properties) have a reasonable probability of transcellular transport by
sequential partitioning from an aqueous to a lipid and then back to an aqueous
environment. Such drug movements begin in extracellular aqueous environment,
move into lipid bilayer of the plasma membrane, then to the cytoplasmic
environment, and so on, with multiple possible routes through the cell. Exit from
the cell at the basolateral side could follow the same or similar events. Transit
through the cytoplasm may result in extensive enzymatic degradation. Therefore
successful passive transcellular transport requires a drug moiety that is stable to
the multiple environments encountered on its transit through the colonic epithelial
cell (Mrsny, 1992).
Passive and active transport processes in the colon result in the net
secretion of potassium and bicarbonate and the net absorption of sodium and
chloride. Water passively follows the uptake of sodium and chloride, resulting
into dehydration of colon chyme as it is processed into faeces. Although this net
21
REVIEW OF LITERATURE…
flux of water can also act as a driving force in the uptake of water-soluble drug
molecules but mainly is transported by carrier-mediated mechanisms (Fig.2.6).
Carrier-mediated uptake can occur through two basic mechanisms: passive,
facilitated diffusion and active transport. Similar to amphipathic partitioning,
passive or facilitated diffusion is driven by the differences in the chemical
potential of the drug. Facilitated drug uptake however, requires a trans-membrane
carrier at the apical plasma membrane (composed of protein or a glycoprotein
molecule or complex) while amphipathic partitioning does not.
In some cases, facilitated drug transport can utilize concentration
gradients established by the cell through active transport process where molecules
are moved against the concentration gradient. Active transport processes, which
usually require ATP hydrolysis, might also transport a drug species that
sufficiently mimics a native molecule being transported. Drug uptake in the colon
by an active transport carrier could also utilize some counterflow transport,
similar to the principle by which the Na+ ion gradient drives the uptake of the
glucose molecule through the glucose transporter (Mrsny, 1992).
Various factors affecting the absorption of the drug molecules from the
colon include lipid solubility, degree of ionization of drugs and pH at the
absorption site. Also, the factors influencing the residence time are likely to affect
the absorption of drugs from the colon. The rate and extent of drug absorption
from the colon is thought to be slower than small intestine due to the following
reasons:
1.
Although colon has large diameter, yet the absorption surface area of
colonic mucosa is small due to lack of villi, microvilli and circular folds.
The viscosity of the colonic contents is high. The progressive absorption of
fluids as the material passes along the colon results in the gradual
solidifying mass, which reduces the dissolution rate of particular drug and
also makes more difficult for a drug to diffuse from the lumen to the site of
absorption.
2.
The mucus layer at the epithelial surface presents a formidable physical
barrier
because of specific and
non-specific drug binding e.g.
cephalosporins, penicillin and aminoglycoside antibiotics bind to the
negatively charged mucus
22
REVIEW OF LITERATURE…
3.
The mucosal permeability of colon is low due to tight junctioning of
epithelial cells in the colon.
4.
The ecosystem of the colon is highly complex. It involves both aerobic and
anaerobic micro-organisms. As a result, the bioavailability of systemically
active drugs may be reduced by proteolytic and other activities in the colon.
(Niibuchi et. al., 1986).
Figure 2.7 Epithelial transport in the colon
Colonic absorption of drug molecules may be improved by using following
strategies:

Co-administration of absorption enhancers.

Inhibition of proteolytic enzymes in the colonic lumen.
The permeability of the epithelium to the drugs can be enhanced by the
use of chemical enhancers, which promote the absorption (Zhou and Li Wan,
1991). A wide range of compounds have been used as absorption enhancers.
These are calcium ion chelating agents (EDTA), surfactants (polyoxyethylene
lauryl ether, saponins), bile salts and fatty acids. These enhancers increase
transcellular and paracellular transport through one or other following
mechanisms:
23
REVIEW OF LITERATURE…

By disruption of intercellular occluding junction complex function to open
the
paracellular route.

By modifying the epithelial permeability via denaturing membrane
proteins.

By disrupting the integrity of lipid bilayer of colonic enterocytes.
Enhancement of the colonic-absorption by these agents appears to be drug-
specific e.g. mixed micelles composed of either glycocholate or taurocholate,
mono olein, oleic or lauric acid have shown to enhance the absorption of 5fluorouracil, heparin and bleomycin (Muranishi et. al., 1979; Yoshikawa et. al.,
1983).
In spite of the unfavorable conditions, a large variety of drugs are well
absorbed from the colon (Fara et. al., 1989). However, buflomedil (Wilson et. al.,
1991), cimetidine and hydrochlorthiazide (Riley et. al., 1992) are found to be
poorly absorbed from the colon. The majority of drugs with poor colonic
absorption are those that are primarily absorbed by paracellular route.
2.3
COLONIC
DELIVERY
DOSAGE
FORM:
A
TECHNICAL
PROSPECTIVE
Until recently, colon was considered as a site for water reabsorption and
residual carbohydrate fermentation. However, it is currently being viewed as a site
for drug delivery. Colonic drug delivery is not only restricted to treatment of local
disorders but also for systemic drug delivery. This part of GIT is also being
considered as a site for administration of protein and peptide drugs (Reddy et. al.,
1999). This is because colon provides a less hostile environment for drugs due to
low diversity and intensity of digestive enzymatic activities, and a near neutral
pH. Moreover, colon transit time may last up to 78 h, which is likely to increase
the time available for drug absorption. Further, considering that this site is more
responsive to absorption enhancers, its suitability as a site for drug administration
appears promising. Additionally, colonic delivery of drugs may be extremely
useful when a delay in drug absorption is required from a therapeutic point of
view e.g. in case of diurnal asthma, angina, arthritis, etc. While excluding some
approaches, it is convenient to categorize targeted delivery systems into one of
four categories (Friend, 2005):
(1)
pH-dependent systems (Klein et. al., 2005; Ibekwe et. al., 2006 a,b),
24
REVIEW OF LITERATURE…
(2)
Time dependent systems (Steed et. al., 1997),
(3)
Pressure-based systems (Jeong et. al., 2001) and
(4)
Microflora activated systems (Brondsted et. al., 1998; Katsuma et. al.,
2002; Yano et. al., 2002).
Various approaches and challenges for formulation of colon -specific drug
delivery system are shown in Table 2.8. Various advantages and disadvantages of
oral colon-specific drug delivery methods are given in Table 2.10.
2.3.1 pH-dependent systems
The pH in the gastrointestinal tract varies widely (Friend, 1991; Kinget et.
al., 1998). Use of pH-dependent polymers is based on the differences in pH
levels. Enteric-coated dosage forms are designed to remain intact in the stomach
and release the active substance in the intestine. pH sensitive coatings can be used
to deliver the drugs to the colon. Enteric polymers such as cellulose acetate
trimelliate (CAT), hydroxy propyl methyl cellulose phthalate (HPMCP),
polyvinyl acetate phthalate (PVAP), cellulose acetate phthalate (CAP) and
shellac. Eudragits are preferred coating materials for this purpose since they
dissolve at pH ≥ to 5. A list of few commonly used enteric polymers is given in
the Table 2.9
pH-sensitive calcium alginate-coated gelatin microspheres have been
developed by Rao and Ritschel (1992). The calcium alginate coat is obtained by
crosslinking sodium alginate with calcium chloride. The formulation is a pHcontrolled system because the alginate coat is protonated at gastric pH and ionized
at intestinal pH. Drug release from the drug-loaded microspheres occurs
predominantly in the ileocecal region.
Kelm and Manring et al. (1996) developed a dosage form for colonic
delivery wherein enteric polymer coating material dissolved in an aqueous media
ranging in pH from 5 to 6.3. The enteric polymer coating had a coating thickness
of 250 µm. Dextramethasone was sprayed on sugar spheres. These spheres were
then coated with HPMC as barrier coat and subsequently with CAP. Sugar
spheres sprayed with mesalamine were coated with Eudragit L. Similarly,
propranolol base and salmon calcitonin were separately formulated in selfemulsifying vehicles, filled in soft elastin capsules and coated with CAP.
25
REVIEW OF LITERATURE…
Table 2.8 Approaches and challenges for formulation of colon -specific drug
delivery system
Approaches
pH dependent
systems
Time dependent
Systems
Microbially triggerd
systems
Polysaccharide based
delivery systems
Prodrugs
Challenges
Stomach pH (1-3)
Proximal small intestine pH(7.5)
Distal small intestine pH (7.5)
Caecum (6.4)
Transverse colon (6.8)
Descending colon (7.0)
Wide variation in gastric retention
time
Increased transit time in diseases
such as inflammatory bowel
syndrome, ulcerative colitis,
diarrhoea
Gums are hydrophilic and gel
forming
Colonic microflora varies with diet,
age and disease
Microbial degradation of azo
polymers is slow, drug delivery is
thus incomplete and irregular
Principle of delivery
Enteric polymers do not dissolve in
stomach and at pH ≥ 5 they begin
to dissolve
Swelling type polymers that allow
drug release 4-6 h after leaving
stomach
Polysaccharides not digested by
stomach and intestinal enzymes.
Polysaccharides secreted by colonic
bacteria degrade polysaccharides.
Prodrugs cleaved to active moiety
by the bacterial enzymes present in
colon.
Kelm et al. (1997) prepared bisacodyl formulations where bisacodyl
sprayed sugar spheres coated with barrier coat were further coated with CAP or
Eudragit L. Similarly, bisacodyl soft gelatin capsules coated with CAP were
observed to provide an enteric coating.
Torres et al. (1998) formulated multiparticulate microspheres consisting of
hydrophobic core coated with a pH-dependent polymer for colonic specific drug
delivery. Microspheres were loaded with budesonide and coated with enteric coat
of Eudragit S, by emulsion-solvent evaporation technique for the local treatment
of intestinal disorders.
Raheja et al. (2004) formulated mesalamine tablets by coating with
Eudragit S100 to a weight build up of about 3% w/w (about 35 µ coat thickness).
The in vitro dissolution of mesalamine from these tablets was compared with
Asacol DR tablets. It was observed that even under stressed conditions
encountered during variable gastric residence times in stomach and intestine, the
26
REVIEW OF LITERATURE…
formulated tablets provided a more consistent and predictable release profile,
releasing mesalamine only at pH 7.2.
Table 2.9 Enteric polymers utilized in development of modified-release
formulations for colonic delivery
Polymer
Threshold pH
Eudragit® L 100
6.0
Eudragit® S 100
7.0
Eudragit® L –30D
5.6
Eudragit® FS 30D
6.8
Eudragit® L 100-55
5.5
Polyvinyl acetate phthalate
5.0
Hydroxy propyl methyl cellulose
phthalate
4.5-4.8
Hydroxy propyl methyl cellulose
phthalate 50
HPMC 55
5.2
5.4
Cellulose acetate trimelliate
4.8
Cellulose acetate phthalate
5.0
The plasma concentration of mesalamine was significantly higher after oral
administration of formulated tablets as compared to that after commercially
available formulation. The extent of Asacol DR tablets was found to be 575.87
nghr/ml, which increased to1980.26 nghr/ml for Eudragit S100 coated tablets.
Many commercial drug formulations for the oral treatment of inflammatory bowel
disease (such as Asacolitin®, Claversal®, Salofalk® or Budenofalk®) are coated
with pH-sensitive enteric coating polymers such as Eudragit® L or S. These
polymers have a dissolution pH of between 6 and 7, and are intended to release
the drug as soon as the intestinal pH exceeds 6 or 7, respectively.
Biswajit et al. (2004) developed a suitable matrix type transdermal drug
delivery system (TDDS) of dexamethasone using povidone, ethylcellulose and
27
REVIEW OF LITERATURE…
Eudragit. Invitro results showed that PVP–EC polymers were better suited than
PVP–Eudragit polymers for the development of TDDS of dexamethasone.
Kawashima et al (2005) prepared the nanoparticles for the treatment of
inflammatory bowel disease using tacrolimus entrapped in to pH-sensitive
microspheres.
2.3.2 Time – Dependent systems
Time-controlled systems are useful for synchronous delivery of a drug
either at pre-determined lag time such that patient receives the drug when needed
or at a pre-selected site of the GI tract (Savastano et. al., 1997). The lag time
usually starts after gastric emptying because most of the time-controlled
formulations are enteric coated.
Drug release from these systems is not pH dependent. These systems take
advantage of the relatively constant transit time through the small intestine and are
particularly useful in the therapy of diseases, which depend on circadian rhythms.
The lag time observed with the TIME-CLOCK™ system (Pozzi, et. al.,
1994; Wilding et. al., 1994) is caused by slow hydration of the hydrophobic
coating layer, which consists of wax, Tween-80 and HPMC. Drug release from
the coated tablets is pH-independent and there is little influence of agitation on the
lag time of drug release. Disaggregation of the tablets occurs in the proximal
colon after 5.5 hours.
Savastano et al. (1997) developed an osmotic delivery device for release
of the active ingredient to preselected region of gastrointestinal tract. The delivery
system consisted of solid core containing Metoprolol fumarate, delay jacket
coated over the core (e.g. dextrates, sodium alginate), followed by a
semipermeable membrane (cellulose acetate, CAP) and finally, an enteric coat.
Metoprolol fumarate core tablets were compression coated with dextrate followed
by film coating with cellulose acetate and finally with Eudragit (enteric polymer).
The amount of metoprolol released was negligible till 5 hr and 82.7% till 24 hr
when dissolution studies were carried out in 0.1N HCl for 0-2 hr followed by
phosphate buffer (pH7.5) for 2-24 hr. A unique composition wherein dissolution
of outer layer activated the process of swelling/ dissolution/ erosion of the
intermediate coating layer was described by Poli et al. (2001). Posatirelin tablets
coated with HPMC E50 LV were finally coated with CAP. These tablets did not
release posatirelin at pH lower than 5 for at least 2 hr. An increase in the pH of
28
REVIEW OF LITERATURE…
dissolution media to pH 7.5 also could not release the drug for subsequent 4 hr.
An additional exposure for 45min to pH 7.5 released the drug.
Takada (1997) described a similar time-controlled formulation in the form of
capsules and bilayered tablets. The release time of the drug from formulations is
controlled by disintegration lag-time which depends on the balance between the
tolerability and thickness of a water-insoluble membrane and the amount of a
swellable excipient such as low substituted hydroxypropyl cellulose (L-HPC) and
sodium starch glycolate. The shell of the capsule formulation is made up of ethyl
cellulose (EC), approximately 120 µm in thickness, which contains micropores at
the bottom of body. The fill material is composed of a solid dispersion
formulation of the drug filled into a capsule body also made of EC, and a tablet
containing L-HPC made by direct compression. Finally, a cap made up of EC is
attached to the body of outer EC capsule (Figure 2.8). After oral administration,
GI fluid permeates through the micropores and causes swelling of swellable
excipients. This causes an inner pressure, which pushes the drug container. Then
the disintegration of the capsules occurs with the breakdown of the capsule cap. In
this way, the disintegration of time-controlled capsule is dependent on the balance
between the swelling pressure of formulated L-HPC and the strength or
tolerability of the EC capsule.
Gazzaniga et al, (2001) formulated delayed release tablets of Antipyrine
using hydrophilic swellable polymer to achieve time or site specific release of drug.
Mastiholimath et al, (2006) developed pulsatile device to achieve time
and site specific release of theophylline. The device consists of insoluble hard
gelatine capsule body, filled with eudragit microcapsules of theophylline and sealed
with hydrogel plug.Invitro release studies of pulsatile device revealed that
increasing the hydrophilic polymer content resulted in delayed release of
theopyhlline from microcapsules.
A pressure-controlled drug delivery system that relies on the high pressure
in the distal colon produced by peristalsis has been introduced by Niwa et al. (1995)
and Takaya et al. (1997).
29
REVIEW OF LITERATURE…
Figure 2.8 Time-controlled capsule for colonic delivery
2.3.3 Pressure-Controlled Drug Delivery Systems
ethylcellulose coating, is induced by the pressure and thus the destructive force
produced by peristaltic waves, and depends on the thickness of the ethylcellulose
film. The capsule is filled with a solution of the drug and this should be
advantageous in view of the small amount of fluid in the distal colon, which could
compromise drug dissolution and absorption (Takaya et. al., 1998; Takada and
Murakami 2005).
The ability of Pressure controlled colon-delivery capsules (PCDCs) to
deliver a drug locally into the distal bowel was investigated in humans using
caffeine as a model drug. From this study it was indicated that the thickness of
ethyl cellulose film was an important factor in controlling the disintegration of the
formulation (Muraoka et. al., 1998; Jeong et. al., 2001).
Pan et al., (2007) formulated microbial triggered colon-targeted osmotic pump
chitosan with a very corticosteroid, budesonide.
2.3.4 Microflora - Activated Systems
These systems are based on the exploitation of the specific enzymatic
activity of the microflora (enterobacteria) present in the colon. The colonic
bacteria are predominately anaerobic in nature and secrete enzymes that are
capable of metabolizing substrates such as carbohydrates and proteins that escape
the digestion in the upper GI tract (Basit and Bloor, 2003).. Most common
mechanisms of microbial activation in the colon are azo-reduction and glycosidic
bond hydrolysis. Azoreductases induce reduction of azo-bonds selectively while
polysaccharidases degrade the polysaccharides. Few enzymes are listed in Table
2.11.
30
REVIEW OF LITERATURE…
Table 2.10 Advantages and Disadvantages of Various Oral Colon-Specific
Drug Delivery Methods
Method
Advantages
Disadvantages
References
Timedependent
systems
Small intestine
transit time is fairly
consistent
Substantial variation in
gastric retention times
Davis et. al., 1986
Yang et. al., 2002
Ashford and Fell ., 1993
Watts and Illum, 1997
pHdependent
systems
Formulation well
protected in the
stomach
Transit through the colon
is in patients with colon
disease
pH levels in the small
intestine and colon vary
between and within
individuals
pH levels in the end of
small intestine and
caecum are similar
Pressure
controlled
systems
Drug release
mechanism is
independent of pH
Microfloraactivated
systems
Good site-specificity
With prodrugs and
polysaccharides
Poor site-specificity
High viscosity of luminal
contents in colon affects
the drug dissolution.
Disintegration of
formulation can occur in
the small intestine.
Diet and disease can affect
Colonic microflora.
Enzymatic degradation
may be excessively slow
Few have been accepted
for use in relation to
medicines.
Table 2.11 Enzymes present in colon
Reducing Enzymes
Hydrolytic Enzymes
Nitroreductase
Esterases
Azoreductase
Amidases
N-oxide reductase
Glycosidases
Sulphoxide reductase
Glucuronidase
Hydrogenase
Sulfatase
29
Friend, 1991
Ashford et. al., 1993b
Kinget et. al., 1998
Yang et. al., 2002
Ashford et. al., 1993b
Mastiholimath et al., 2007
Hu et. al., 1999
Takaya et. al., 1995
Rubinstein et al.2006
Yang et. al., 2002
REVIEW OF LITERATURE…
Basit et al, (2008) prepared pellets of theophylline using Eudragit S or
amylose/ ethylcellulose, Invivo results showed Tmax ranging from 5-9h, AUC 8.855.0 mcgh/ml and drug release started in small intestine and in case of bacteria
dependent polymer Tmax 8-10h, AUC 16.5-47.9 mcgh/ml.
a)
Covalent linkage of the drug with the carrier
It involves the formation of covalent linkage between drug and carrier in
such a manner that upon oral administration the moiety remains intact in the
stomach and small intestine. This approach chiefly involves the formation of
prodrug, which is pharmacologically inactive derivative of a parent drug molecule
that requires enzymatic transformation in the biological environment to release the
active drug. The problem of stability of certain drugs from the adverse
environment of the upper g.i.t can be eliminated by prodrug formation, which is
converted in to parent drug molecule once it reaches in to the colon. Site specific
drug delivery may be accomplished by the utilization of some specific property at
the target site, such as altered pH or high activity of certain enzymes relative to
non-target sites for the prodrug conversion.
i)
Azo-polymers
The azo compounds are extensively metabolized by the intestinal bacteria,
both by intracellular enzymatic component and extracellular reduction (Kopecek
1992). The use of these azo compounds for colon-targeting has been in the form
of hydrogels as a coating material for coating the drug cores and as prodrugs (Van
den Mooter et. al., 1997). In the latter approach, the drug is attached via an azo
bond to a carrier. This azo bond is stable in the upper GIT and is cleaved in the
colon by the azo-reductases produced by the microflora. Polymers containing azo
groups have been used as colon-specific film coating (Saffaran et. al., 1986) to
deliver 5-ASA to colon (Schacht et. al., 1996). The susceptibility of polymeric
material to colonic microflora was found to depend upon the concentration of
hydroxyethylmethacrylate (HEMA) polymer (Van den Mooter et. al., 1992).
ii)
Azo bond conjugates
Sulphasalazine, which was used for the treatment of rheumatoid arthiritis,
was later known to have potential in the treatment of IBD (Klotz, 1985). This
compound has an azo bond between 5-ASA and sulphapyridine (SP) (Figure
30
REVIEW OF LITERATURE…
2.9a). In the colon, the azoreductases cleave the azo bond releasing the drug, 5ASA and the carrier SP. With the knowledge that the adverse effects associated
with sulphasalazine are due to SP, an investigation started for the choice of a
suitable carrier for 5-ASA with minimum adverse effects. SP was replaced by paminohippurate in ipsalazide and by 4-aminobenzoyl-β-alanine in balsalzide. In
another approach two molecules of 5-ASA have been joined together to form an
ultimate prodrug disodium azodisalicylate (olsalazine), in which one molecule of
5-ASA is used as a carrier for the other (Figure 2.9b). Under normal GIT
conditions and bacterial flora, olsalazine delivers twice the amount of 5-ASA as
compared to sulphasalazine (Travis et. al., 1994).
Figure 2.9
Sodium salt of (a) Sulphasalazine and (b) Olsalazine prodrugs of 5-ASA (Klotz,
1985)
Veronese et al., (2009) Prepared conjugate system by coupling of drug
mesalamine to polyethylene glycol to azo linkage. The cleavage of azo moiety by
the azo reductase selectivity in the colon, will release mesalamine in the GI tract.
The advantage of PEG as polymeric carrier (Greenward, 2001; Pasut and
Veronese,2007) are the absence of toxicity and immunologically non absorption
along the GI tract.
iii)
Glucuronide and sulphate conjugates
Bacteria of the lower GIT, however, secrete β-glucuronidase (Scheline,
1968) and can deglucuronidate a variety of drugs in the intestine. Thus, the
deglucuronidation process results in the release of the active drug again and
enables its reabsorption. Simpkins et al. (1988) reported colon delivery of
glucuronide conjugates of narcotic antagonist naloxone and nalmefene for local
action to prevent constipation usually associated with the use of opioid analgesics.
Ursodeoxycholic acid (UDCA), primarily used for the treatment of liver
diseases, has been found to have a protective role in colonic carcinogenesis. But
31
REVIEW OF LITERATURE…
when UDCA is given orally, it is absorbed from the intestine and is
biotransformed in the liver and does not reach the colon. Rodrigues et al. (1995)
found that sulfation of UDCA increases the hydrophilicity of the molecule and
prevents its absorption from the intestine thereby facilitating colonic delivery.
Goto et al., (2000) developed sensitive enzyme linked immunosorbent assay for
determining the total amount non - amidated, glycine and tarine - amidated and 3sulfates using a newly established monoclonal antibody.
Figure 2.10 Structure of the three PEG-drug conjugate and release of
mesalamine (a) following azoreductase incubation: linear monoazo, mPEGPABA-NN-SA (b), biazo, PEG-(PABA-NN-SA) 2 (d), branched mPEG2PABA-NN-SA (c)
iv)
Glycoside conjugates
Certain drugs can be conjugated to different sugar moieties to form
glycosides. The colonic microflora produces a wide range of glycosidases,
capable of hydrolyzing glycosides and polysaccharides (Friend and Tozer, 1992).
Various naturally occurring glycosides, e.g. the sennosides, are activated by
colonic microflora to generate rhein anthrones, which gives the better laxative
effect than sugar free aglycones (Kobashi et. al., 1980). Friend and Chang (1984)
32
REVIEW OF LITERATURE…
prepared dexamethasone-21-β-glucoside (Figure 2.11) and prednisolone-21- β glucoside for delivery of these steroids to the colon.
Figure 2.11 Dexamethasone-21- β -D-glucoside (Friend and Chang,
1984)
(Arrow shows site of action of glycosidase)
Minko et. al., (2003) targeted the drugs for colon delivery by using
anticancer drugs lectins and neoglycoconjugate.
v)
Dendrimer-conjugates
A variation on the azo polymer approach relies on dendrimers as the
carrier. 5-ASA was released from these carriers in rat caecal contents although at
a
rate
considerably
slower
than
that
observed
from
sulphasalazine
(Wiwattanapatape et. al., 2003).
Kono et al., (2008) prepare pH sensitive system polyamidoamine
(PAMAM) dendrimers that grafts with poly (Ehyleneglycol) by making
PAMAM-glutamic residues for attaching adriamycin (Anticancer drug using
amide bond) results showed that slight release at pH 7.4 , remarkable extent of
adriamycin released was induced at pH 5.5 which correspond to the pH of colon.
vi)
Amino-acid conjugates
Proteins and amino acids (A.A.) have polar groups like the −NH2− and
−COOH−. These polar groups are hydrophilic and reduce the membrane
permeability of A.A and proteins. Nakamura et al. (1992) studied the conjugation
of drug molecule to these polar A.A and prepared prodrugs for colon-drug
delivery. The salicyluric acid (i.e. the glycine conjugate of salicylic acid) was
prepared (Figure 2.12a). However, this prodrug was absorbed into the circulation
from the upper GIT and was therefore unsuitable as a colon drug carrier. So, the
hydrophilicity and length of the carrier amino acid was increased, decreasing the
membrane permeability of the conjugate and salicyclic- glutamic acid conjugates
33
REVIEW OF LITERATURE…
were prepared which gave good results as a colon-specific carrier for salicyclic
acid (Figure 2.12b).
Figure 2.12 Glycine and glutamic acid conjugates of salicylic acid. (a)
Salicyluric acid. (b) Salicyl-glutamic acid conjugate. (Dotted line shows the
site of cleavage)
Kataoka et al., (2009) prepared polymeric micelles from poly
(ethylene glycol)-poly(amino acid) copolymer. Chemical modification in the core
forming block allows to release the drug in the colon.
vii)
Cyclodextrin conjugates
Cyclodextrins have been used as pharmaceutical carriers due to their
stability against non-enzymatic and enzymatic degradation in various body fluids,
biocompatibility, and safety profile. A clinical study has shown clear evidence
that β-cyclodextrin (- CyDs) is poorly digested in the small intestine but is
almost completely degraded by the colonic microflora (Flourie et. al., 1993).
Amide and ester pro drugs of β- CyD were prepared (Figure 2.13). Amide
conjugate showed no hydrolysis in contents of stomach, small intestine, caecum,
and colon of rats. The ester conjugate showed less than 10% of drug release in the
contents of stomach, small intestine and their tissue homogenates, but a
significant hydrolysis in contents of caecum and colon.
Redenti et al., (2001) made used of peculiar properties of cyclodextrin and
their derivatives as carrier for oligonucleotides agents.
Uekama et al, (2002) prepared controlled release system using
cyclodextrin D in which an anti inflammatory, ketoprofin is covalently bound to
one of the hydroxyl group of α- cyclo dextrin. In vivo results showed delayed
release pattern after oral administration to rats.
34
REVIEW OF LITERATURE…
Figure 2.13 Cyclodextrin-Biphenylacetic acid conjugate (Flourie et.
al., 1993)
(B)
Polysaccharides in colon drug delivery
Polysaccharide-based formulations are very common since they can be
selectively degraded by a colonic enzyme and are natural polymers with proven
safety profile. Polysaccharides are easily available, inexpensive and have
availability in a variety of structures with varied properties (Hovgaard and
Brondsted, 1996). They can be easily modified chemically and bio chemically and
are highly stable, safe, non- toxic, hydrophilic and gel forming and in addition
biodegradable. There are various polysaccharides for colon targeted drug delivery
(Table 2.8). Various enzymes that are involved in the degradation of some of
these polymers are amylase, chitosanase, pectinase, inulinase, xylanase,
dextranase, and galactomannanase.
i)
Amylose
Amylose is a poly (1-4-α-D-glucopyranose) that consists of D-
glucopyranose residues linked by α- (1-4) bonds (Figure 2.14). Amylose
ethylcellulose film coating has been investigated for colon targeted drug delivery
(Siew et. al., 2000). Milojevic et al. (1996a) prepared and evaluated in vitro
potential of amylose-ethocel coating system for colon-targeted delivery. In vitro
release of 5- ASA from coated pellets with amylose and EC (1:4) was retarded in
simulated gastric and small intestinal fluid over a period of 12 hr. It was
fermented and drug was released in 4 h in simulated colonic environment.
Polysaccharides for colon drug delivery are given in Table 2.12.
Basit et al., 2003 developed enzyme based system for the in vitro
assessment of colonic digestion of amylase films and coatings and to compare
with human faecal bacteria in this amylase and ethyl cellulose were mixed in
35
REVIEW OF LITERATURE…
different ratios and cast as isolated films, as well as spray coated on mesalamine
loaded pellets. Four commercial amylase enzymes were individually screened for
their ability to digest amylose casted films. The enzyme from the
Table 2.12 Polysaccharides for colon drug delivery
Source of Polysaccharide
Example
Plant origin
Amylose, Pectin (Semde et. al.,
2000; Ahrabi et. al., 2000), Guar
gum (Siew et. al., 2000; Milojevic
et. al., 1996a,b)
Animal origin
Chitosan, Chondroitin sulphate
Microbial origin
Dextran, Xanthan gum
Algal Origin
Alginates
Figure 2.14 Structure of amylase
bacterium Bacillus licheniformus was found to be most active against amylose
casted films. Digestion directly proportional to amylase content in the films. In
terms of product performance, drug release from coated pellets was accelerated in
the presence of an enzyme.
ii)
Pectin
Pectins are nonstarch linear polysaccharides that consist of α-1,4 D-
galacturonic acid and 1,2 D-rhamnose with D-galactose and D-arabinose side
chains having average molecular weights between 50,000 to 150,000 (Figure
2.15). Pectin is one of the most widely investigated polysaccharides in colonspecific drug delivery. Pectin in the form of matrix tablets (Ahrabi et. al., 2000),
compression coatings (Ashford et. al., 1993), ethylcellulose-pectin film coatings
(Wakerly et. al., 1996a, 1997; Ahmed, 2005; He et. al., 2007), calcium pectinate
beads (CPG) or zinc pectinate beads (ZPG) (El-Gibaly, 2002; Chambin et. al.,
36
REVIEW OF LITERATURE…
2006), prodrugs (Xi et. al., 2005), and calcium pectinate capsules (Xu et. al.,
2005) has been used as a potential carrier for the site-specific delivery of drugs to
the colon. Mixtures of pectin and chitosan have also been used as compression
coatings (Fernandez-Hervas and Fell, 1998) and film coatings (Macleod et. al.,
1999a, b; Marianne et. al., 2003; Ofori-Kwakye and Fell, 2001, 2003a, 2003b;
Ofori-Kwakye et. al., 2004) by spray drying tablet were obtained with different
complex ratios of pectin:chitosan and vancomycin (Luppi et, al.,2008) for colon
delivery.
Figure 2.15 Structure of pectin
TimucinUgurlu et. al., (2007) formulated Nisin containing Pectin/HPMC
compression coated tablets. Nisin was a 34 amino acid residue long, heat stable
peptide belonging to lantibiotics. Results showed that pectin/HPMC envelope was
found to be good delivery system for delivery of the nisin to the colon.
Quing et al., (2008) prepared pletin/ethylcelluose film-coated and
uncoated pellets using 5-florouracil. Results showed that uncoated pellets mainly
distributed in upper GI tract, while coated pellets mainly distributes in the caecum
and colon.
iii)
Guar Gum
Guar gum derived from the seeds of Cyamopsis tetragonolobus is a
naturally occuring galactomannan polysaccharide. It is made up of a linear chain
of β-D-mannopyranose joined by β - (1-4) linkage with α -D-galactopyranosyl
units attached by 1, 6-linkage in the ratio of 1:2 (Figure 2.16). Phosphated crosslinked low swelling guar-gum hydrogels were prepared and analysed in vitro and
in vivo for their potential as colon drug carriers (Gliko-Kabir et. al., 2000). It was
concluded that guar gum is suitable for preparation of colon-specific formulations
and is particularly suitable as a carrier of drugs that are not very soluble in water.
A mixed coating comprising guar gum and xanthan gum (20:10) has been
37
REVIEW OF LITERATURE…
reported to provide colon-specific delivery of 5-Fluorouracil (5-FU) for the
treatment of colorectal cancer (Sinha et. al., 2004).
Krishniah et al., (2002) prepared 5-Fluorouracil fast disintegrating core
tablets using guar gum as carrier. In vitro results showed that compression coated
tablets using guar gum most likely to provide targeting of 5-FU for local action,
since only 2.38% of the drug released in physiological environment of the
stomach and small intestine.
Krishniah et al., (2002) formulated guar gum based colon targeted tablets
of mebendazole. Results of the study indicates that guar gum based colon targeted
tablets of mebendazole did not release in the stomach and small intestine but
delivered the drug in the colon resulting in the slow absorption of the drug and
making availability for the local action in the colon.
Aminabhavi et
al.,
(2004)
formulated tablets
by
incorporating
antihypertensive drug, diltiazem HCl, using polyacramide–grafted–guar gum.
Drug release found to be better in case of hydrolyzed pAAm-g-GG as compare to
unhydrolyzed co-polymer.
Rubinstein et al., (2006) prepared films of chitosan and guar gum for the
local delivery of celecoxib, which was used as local adjuvant for the therapy of
colorectal cancer.
Abraham et al., (2007) prepared pH sensitive alginate- guar gum hydrogel
cross linked with glutraldehyde for the controlled delivery of protein drugs.
Figure 2.16 Structure of guar gum
iv)
Chondroitin sulphate
Chondroitin sulphate is a mucopolysaccharide found in animal connective
tissues especially in cartilage. Chemically, it consists of D-glucuronic acid linked
to N-acetyl-D-galactosamide (Wastenson, 1971; Toledo and Dietrich, 1977)
38
REVIEW OF LITERATURE…
which is sulphated at C-6 (Figure 2.17). Chondroitin sulfate could be used as
carrier for colon targeted delivery of bioactive agents. Chondroitin sulphate was
crosslinked with 1,12 diaminododecane using dicyclohexylcarbodiimide as a
catalyst and formulated in a matrix with indomethacin as a drug marker. It was
concluded that the release of indomethacin depended upon the biodegradation
action of the caecal content (Rubinstein et. al., 1992a, 1992b).
Figure 2.17 Structure of chondroitin sulphate
Murdan et al., (2002) formulated chondroitin sulphate hydrogels for
electro-controlled delivery of peptides and proteins.
Whitelock et al., (2005) prepared perlecan from human epithelial cells that
is a hybrid system of heparan/chondroitin/keratin sulphate proteoglycan.
v)
Dextran
This class of polysaccharide consists of linear chains of α-Dglucose
molecules; 95% of the chains consists of 1:6-α-linked linear glucose units while
the side chains consist of 1:3-α-linked moieties (Figure 2.18). They are obtained
from microorganisms of the family of Lactobacillus (Leuconostoc mesenteroides).
Dextran ester prodrugs of ketoprofen and naproxen using dextran were shown to
release the drug specifically in the colon region of pig (Larsen et. al., 1989, 1991;
Harboe et. al., 1989). Hovgaard and Brondsted (1995) studied the suitability of
dextran hydrogels for colon targeted delivery of hydrocortisone.
Gil et al., (2006) prepared propranolol HCL extended release capsules by
use of dextran:HPMC in 4:10w/w with cetyl alcohol.Invitro results showed that
Higuchi(diffusion) and Hixon-crowell(erosion) kinetic profiles were achieved and
that is main release mechanism of PPL from capsules.
Basan et al., (2007) formulated biodegrable dextran hydrogels by
crosslilking dextran with epichlorohydrin for the invitro colon-specific delivery of
salmon calcitonin.
39
REVIEW OF LITERATURE…
Figure 2.18 Structure of dextran
Pitarresi et al., (2007) prepared novel hydrogels with methacrylated
dextran and methacrylated α,β-poly(N-2-hydroxyethyl)-DL-aspratamide by
photocrosslinkage under the λ at 313nm.These crosslinked polymers used for the
treatment of IBD by incorporating Beclomethasone dipropionate.
Varshosaz et al., 2009 prepared conjugate of Dextran-Budesonide for the
treatment of ulcerative colitis using dimethylamino pyri as succinate spacer.
vi)
Alginates
Alginates, natural hydrophilic polysaccharide derived from seaweed,
consist of 1→4, linked D-mannuronic acid and L-glucuronic acid residues (Figure
2.19). Giavasis et al. (2002) prepared alginate beads and coated them with dextran
acetate. In the absence of dextranase, minimal drug release occurred and the
release was significantly improved in the presence of dextranase.
Bajpai et al., (2006) prepared multilayered beads by complex coacervation
of calcium alginate and chitosan for the release of vitamin B2 in the media of
varying pH at 37ºC in the colon.
Mldenovska et al., (2007) formulated chitosan–Ca-alginate microparticles
for the colon specific delivery of mesalamine.
Figure 2.19 Structure of alginates
Gorasinova et al., (2008) prepared Eurogit S 100 coated chitosan-Caalginate micro particles loaded with budesonide. Release data suggests that
40
REVIEW OF LITERATURE…
influence of erosion and biodegradation of polymer matrix on drugs release from
microparticles.
2.4
COLONIC DELIVERY DOSAGE FORM: EVALUATION
A successful colon-specific drug delivery system is one that remains intact
in the physiological environment of the stomach and small intestine but releases
the drug in the colon. The following techniques are commonly employed for the
evaluation of colonic drug delivery systems.
a) In vitro dissolution testing
Dissolution testing has been an integral component in pharmaceutical
research and development of solid dosage form. The method used should
stimulate the environment to which the dosage form being developed will be
exposed in the gastrointestinal tract. It provides important information on
formulation selection, the critical processing variables, and in vitro /in vivo
correlations of quality assurance during clinical manufacturing. In the United
States Pharmacopoeia (USP), 4 dissolution apparatus are recommended
accommodate different activities and dosage forms: basket method, paddle
method, Bio-Dis method, and flow through cell method.
Dissolution tests related to colon specific drug delivery systems may be
carried out using the conventional basket method (Rudolph et al., 2001). Parallel
dissolution studies in different buffers may be undertaken to characterize the
behaviors of formulations in different pH levels. Rudolph et al., 2001 carried out
dissolution tests of colon- specific formulation in various media simulating pH
conditions at various locations in the gastrointestinal tract. The media chosen
were, for e.g. pH 1.2 to stimulate gastric fluid, pH 6.8 to stimulate jejunal region
of the small intestine, and pH 7.4 to stimulate the ileal segment. The ability of the
colonic delivery system to release the drug in the colon was tested in vitro by
incubating in buffer medium, in the presence of either enzymes e.g. pectinase
(Ashford et. al., 1993), dextranase (McLeod et. al., 1994), or animals such as rat
(Rubinstein et. al., 1992b), guinea pig ((Larsen et. al., 1989), rabbit (Kopeckova
et. al., 1994) caecal contents. Prasad et. al., 1998 concluded that the buffer
medium with rat caecal content (4%w/v) obtained after 7 days of enzyme
induction proves the best condition for in vitro evaluation.
Rat caecal contents were usually prepared immediately prior to initiation
of drug release study due to anaerobic nature of caecum. Rats were anaesthetized
41
REVIEW OF LITERATURE…
and the caecum was exteriorized for collection of the contents. The caecal
contents were diluted with phosphate –buffered saline (PBS, pH 7). This step was
conducted under CO2 or nitrogen to maintain an anaerobic environment
(Rubinstein et. al., 1992b, 1993).
Another in vitro method involves the incubation of the drug delivery in a
fermentor with commonly found human colonic bacteria like Streptococcus
faecium (Kopecek, 1992) or Bacteroide ovatus (Rubinstein et. al., 1993). In a
suitable medium under anaerobic conditions and the amount of drug released at
the different intervals is found out. Different strategies used for drug release
studies of colonic drug delivery systems are given in Table 2.13.
b) In vivo evaluation of Colon targeted drug delivery:
In vivo tests in humans are important in developing controlled – release
drug delivery systems.
Animal models
There are different animal models used in evaluating in vivo performance
of colon specific drug delivery systems. Guinea pigs were used to evaluate colon
specific drug delivery from a glucoside prodrug of dexamethasone (Friend et. al.,
1991). Other animal models used include rat (Van den Mooter et. al., 1995) and
pig (Harboe et. al.,1989).
c) Instrumental Methods for Evaluation of Colon Specific Drug Delivery
(i)
Gamma scintigraphy
It is the most useful, up to date technique to evaluate in vivo behavior of
dosage forms in animals and humans. Gamma scintigraphy has become the most
popular means of investigating the gastrointestinal performance of pharmaceutical
dosage forms, especially site-specific dosage forms. (Wilding et al., 1991, 2001).
The procedure involves labeling a formulation with a suitable gamma-emitting
radionuclide such as 99mTechnetium or 111Indium which is then taken by a subject
who is positioned in front of gamma camera. By means of gamma scintigraphic
imaging, information can be obtained regarding time of arrival of a colon-specific
drug delivery system in the colon, time of transit through the stomach and small
intestine and disintegration. Information about the spreading or dispersion of a
formulation and the site at which release from it takes place can also be obtained.
42
REVIEW OF LITERATURE…
Table 2.13 Different strategies used for drug release studies of colonic drug delivery systems
Drug release in different pH medium
S.No. Code
HCl buffer
pH 7.4 buffer
0.1 SGF PBS SCF SB
M/L
HCl
Y
N
N
N
N
Y
(2h)
(3h)
pH
1.2
pH
7.5
buffer
N
pH 6.8 PBS
pH 7.0
SCF
With
Without SCF
(rat
rat
rat
caecal)
ceacal
ceacal
Y
N
N
N
(19h)
pH
6.4
PBS
pH
6.5
buffer
pH
4.5
buffer
Miscellaneous remarks
N
N
N
N
1.
A
2.
B
Y
(2h)
N
N
N
N
Y
(3h)
N
Y
(16h)
N
N
N
N
N
N
N
3.
C
N
Y
(2h)
N
N
N
N
N
Y(19h)
Y
(19h)
N
N
N
N
N
N
4.
D
Y
(2h)
N
N
Y
(2h)
N
N
N
N
Y
(2h)
N
N
N
N
Y
(2h)
N
5.
E
Y
(2h)
N
N
Y
(3h)
N
N
N
N
N
Y
(6h)
N
N
N
N
N
6.
F
N
Y
(2h)
N
Y
(2h)
N
N
N
N
Y
(1h)
N
N
N
Y
(15h)
N
Drug release studies are also
carried out in presence of
Galactomannase (9.68 *10-3
U/ml) added to pH 6.5 PBS
7.
G
Y*
(2h)
N
N
N
N
N
Y**
(3h)
N
N
N
N
N
N
Y***
(3h)
N
43
REVIEW OF LITERATURE…
8.
H
N
N
N
N
N
N
N
N
N
*SGI fluid consisting of NaCl (2g), Hcl (7ml) and pepsin (3.2g)
**SIF fluid consisting of K2H2Po4 (6.8g), 0.2N NaoH and pancreatin (10g)
***Mixture of SGI and SIF (39:61)
SB-Sorenson’s buffer
PBS-Phosphate buffer saline
SCF-Simulated colonic fluid
A
Guar Gum (Krishnaiah et al., 2001)
B
Konjac glucomannan,KGM (Meimei et al., 2007)
C
Xanthan gum (Felipeet al., 2008)
D
Boswellia gum (Irit et. al., 1998)
E
Gellam gum (Fatmanur et. al., 2004)
F
Locust bean gum (Raghavan et. al., 2002)
G
Chitosan (Raghavan et. al., 2002)
H
Alginate (George et. al., 2007)
44
N
N
Y
(24h)
N
N
Drug release studies are also
carried out in presence of
Galactomannase (0.175 and 2
U/ml for I1 and I2
respectively) and αgalactosidase (0.033 U/ml) in
same buffers
REVIEW OF LITERATURE…
Gamma scintigraphy has also been used to determine gastrointestinal
transit times and sites of disintegration of calcium pectinate tablets intended to
allow colon-specific drug delivery (Adkin et. al., 1997). Gamma scintigraphy was
used to investigate the suitability of Eudragit S coated tablets for drug delivery to
the colon. A good correlation was found between in vitro and in vivo studies
(Ashford et. al., 1993b).
In vivo evaluation of
99m
Tc-DTPA and
99m
Tc-Sulphur colloid as traces in
the colonic drug delivery system was done by gamma scintigraphy in human
volunteers (Krishnaiah et. al., 2002a). The results showed that DTPA is a suitable
tagging agent for Tc in the evaluation of colonic drug delivery systems containing
water-soluble drugs.
Information about gastrointestinal transit and the release behaviour of
dosage forms can be obtained by combining pharmacokinetic studies and gamma
scintigraphic studies (pharmacoscintigraphy). Gamma scintigraphy has been used
to identify the site of release from a PulsincapTM formulation intended to release
drug after five hours lag time (Stevens et. al., 2002). A good correlation was
found between release times determined scintigraphically and pharmacokinetic
profiles. A correlation between pharmacokinetic and gamma scintigraphic data
was also found, when times and anatomical locations of break-up of colonspecific formulation were determined, by Sangalli et al., 2001.
(ii)
Radiotelemetry
This technique involves administration of a capsule that consists of a small
pH probe interfaced by a miniature radio transmitter which is capable of sending a
signal indicating the pH of the environment to an external antenna attached to the
body of the subject. Furthermore, the dosage form must contain significant
amount of buffer salts, the release of which produces a change in the
gastrointestinal pH that is detected by the pH capsule indicated the change in the
dosage form. This prevents the evaluation of commercially available products
which do not contain buffer salts.
(iii)
Roentgenography
In this technique radio- opaque material is incorporated in solid dosage
form which enables it to be visualized by X-rays. By incorporating barium
sulphate into pharmaceutical dosage form, it is possible to follow movement,
45
REVIEW OF LITERATURE…
location and integrity of dosage form after oral administration by placing the
subject under fluoroscope and taking a series of X-rays at various time points.
This method is first used by Losinsky & Diver (1993).
(iv)
Endoscope Technique
This technique is employed by Hey et al., 1979. It is an optical technique
in which a fiberscope (gastroscope) is used to directly monitor the behaviour of
the dosage form after digestion.This technique involves administration of capsule
that consists of small pH probe interfaced with a miniature radio transmitter which
is capable of sending a signal indicating the pH of the environment to an external
antenna attached to the body of the subject. So it is necessary to physically attach
the dosage form to the capsule which in turn affects the behaviour of the dosage
being studied. Furthermore, the dosage form must contain significant amount of
buffer salts, the release of which produces a change in gastrointestinal pH. This
prevents the evaluation of commercially available products which do not contain
buffer salts.
2.5.
CHITOSAN: A BIODEGRADABLE POLYMER
Chitin, next to cellulose, is the second common polysaccharide on the
earth. Chitin, the source material of chitosan, is prepared from the shells of crabs,
shrimps, other arthropods, fungi, yeasts, squid pens and so on (Becker et. al.,
2000). Chitosan, a deacetylated product of chitin, is a high molecular weight
cationic heteropolysaccharide. It is composed mainly or fully of β- (1, 4)-2-deoxy2-amino-d-glucopyranose and partially or none of β-(1, 4)-2-deoxy-2-acetamidod-glucopyranose units (Nam et. al., 2001). Unlike chitin, chitosan is readily
soluble in various acidic solutions, such as formic and acetic acids. The structure
of chitin and chitosan are depicted in Figure 2.18
One of the most important parameter in the characterization of chitosan is
the degree of acetylation (DA), defined as the ratio of the number of formed NH2
groups to the initial number of NHCOCH3 groups present in chitin (Martı´nezRuvalcaba, 2001). Depending on the source and preparation procedure, its
molecular weight may range from 300 to over 1000 kD with a DD from 30% to
95% (Dornish et. al., 2001; VandeVord et. al., 2002). The important physical and
chemical properties are listed in Table 2.14.
Chitosan is a linear polymer where amino groups are readily available for
chemical reactions and salt formation with acids. Chitosan, derived from chitin
46
REVIEW OF LITERATURE…
which is the main structural element of the cuticles of crab and shrimp, has been
exploited for wide biomedical applications such as wound dressing, drug delivery
vehicle, and tissue engineering owing to nontoxicity, nonimmunogenicity,
biocompatibility and
H O H 2C
O
HO
HO H2C
HO
O
O
O
C h itin
H O H 2C
NH
C
CH 3
HO
H
O
O
O
NH
C
CH3
n
HO
H
O
C h itin -D e a c e ty la s e
H O H 2C
NH
C
CH3
OH
O
HO
H O H 2C
HO
O
O
H O H 2C
NH2
H
HO
O
NH2
O
n
C h ito s a n
HO
H
NH2
OH
Figure 2.20 Structure of chitosan and chitin
biodegradability (Suh et. al., 2000; Madihally et. al., 1999; Lloyd et. al., 1998).
Chitosan can easily be complexed or cross-linked with anions, oppositely charged
drugs, polymers and other materials. This property of chitosan finds use in diverse
applications such as preparation of beads and microspheres for drug delivery,
biosorption of metal ions, cross linking of films by different ions and polymers,
immobilization of enzymes and microbes etc. Few important pharmaceutical
applications of chitosan are enlisted in Tables 2.15.
(A)
Properties
(i)
Degree of ionization/dissociation
The dissociation constant (pKa) of chitosan ranges from 6.3 to 7.0. It
exhibits maximum degree of ionization at lower pH i.e. between 1 to 4 and as the
pH increases above 6.0, the ionization of amine group decreases sharply (Yalpani
and Hall, 1984). Thus, adjustment of pH of solutions of chitosan plays an
important role while cross-linking it with functional groups present in other
polymers.
47
REVIEW OF LITERATURE…
(ii)
Swelling index
The swelling capacity of chitosan in all the media has been related to the
concentration of the cross-linking agent and hence, to the degree of cross-linking
(Kim et. al., 1992).
In addition, the swelling behaviour is greatly affected by pH of the
medium. In acidic medium, chitosan swells to a greater extent and eventually
dissolves. This is the reason that after swelling, non cross-linked chitosan
spontaneously dissolves in 0.1N HCI. Whereas, de-swelling occurs in buffer of
pH 7.4 (Remunan-Lopez and Bodmeier, 1997). Effect of various acids on
viscosity and pH of chitosan solution is tabulated in Table 2.16.
(iii)
Crystalline Structure
X-ray diffraction data has shown that chitosan exhibits three forms:
hydrated, dehydrated and non-crystalline structures (Yui et. al., 1994).
Intramolecular O(3’)H…O(5), intermolecular NH…O(6) hydrogen bonding and
hydrogen bridging involving water molecules exist in these forms.
(B)
Mechanisms of cross linking
Cross–linked chitosan can be classified as (Figure 2.19, Table 2.17):
1)
Covalently cross-linked chitosan (irreversible cross-linking)
(i)
Chitosan cross-linked with itself
(ii)
Hybrid polymer networks (HPN)
(iii)
Semi or full inter penetrating polymer network (IPN)
(iv)
Ionically cross-linked chitosan (reversible cross-linking)
48
REVIEW OF LITERATURE…
Table 2.14 Physical and chemical properties of chitosan
Property
Parameter
Attributes
Morphology
Semicrystalline polymer in solid state
Molecular weight
300-1000 kDa
Particle size
Less than 30 m
Density
1.35-1.40 g/cc
pH
6.5-7.5
pKa
6.2-7.0
Acetyl value
< 4-4.5
Degree of deacetylation
30-95%
Solubility
Insoluble in water, alkaline solutions (pH <
Physical
6.5) and organic solvents
Soluble in dilute formic acid, citric acid, lactic
acid, sulphuric acid, acetic acid, hydrochloric
acid.
Low degree of deacetylation (#40%) are
soluble up to a pH of 9, whereas amine groups
leading to faster highly deacetylated chitosans
(>85%) are soluble salt form of chitosan is
neutralized, it can form only up to a pH of 6.5.
Viscosity
High Mol.Wt. (600,000)-800-2000cps
Low Mol.Wt. (150,000)-20-200 cps
Chemical
Reactive groups
Hydroxyl and amino
Charge density
High at pH < 6.5
Surface charge
Positive
Ionic interactions
Chelates transition metals
Forms gels with polyanions
Adheres to negatively charged surfaces
Covalent interactions
Forms covalent bonds with carboxylic,
hydroxyl, tripolyphosphate, pyrophosphate
etc.
(Source:-Rinaudo et. al., 2006)
49
REVIEW OF LITERATURE…
Table 2.15 Pharmaceutical applications of chitosan
Application
Suggested Mechanism
Reference
Directly compressible
Crystalline structure
Felt et. al., 1998; USFDA,
vehicle
1999
Controlled release
Cross-linking with drugs and
matrix
other ions or polymers
Wet granulation
Binding property
-do-
Illum, 1998; Paul and Sharma,
2000
Gels
Forms water soluble gels with
-do-
acidic substances
Films
Forms flexible membranes
-do-
Tablet coating
Film former
Felt et. al., 1998
Emulsions
Emulsifying agent
Illum, 1998; Paul and Sharma,
2000
Wetting agent
Reduces surface tension
-do-
Microspheres
Cross linking with ions and
Felt et. al., 1998; Illum, 1998
and
ionic molecules to form
microparticles
spherical agglomerates
Bioadhesion
High cohesive force
Felt et. al., 1998; Illum, 1998
Peptide delivery
Cross-linking with amino acids
Lueben et. al., 1997
Immobilisation
-Glucosidase of Aspergillus
Bissett and Sternberg, 1978
of enzymes
immobilized on chitosan using
glutaraldehyde
Transmucosal
Binding with mucin
Felt et al, 1998
Decreases drug crrystallinity
Sawayanagi et al., 1982b
Cross-linked chitosan insoluble
Tozaki et al., 1997b
drug transport
Fast releasing dosage
forms
Colon drug release
in acidic medium and
biodegradation by chitosanase
enzyme in colon
Cosmetics
Demulscent and soothing
Lang et al., 1985
Wound Healing
Rapid dermal regeneration
Luo et al., 2004
Antimicrobial
By shrinkage of cell membrane
Chen et al., 2005
leads to cell death
50
REVIEW OF LITERATURE…
Table 2.16 Effect of acids on viscosity (cps) and pH of chitosan solution (1%
w/v)
Acid
Acetic
Citric
Oxalic
Property influenced by different concentrations of acid (%
v/v)
1% concentration
5% concentration 10%
concentration
Viscosity
pH
Viscosit pH
Viscosit pH
y
y
260
4.1
260
3.3
260
2.9
35
3.0
195
2.3
215
2.0
12
1.8
100
1.1
100
0.8
Figure 2.21 Schematic representation of different type of cross-linking in
chitosan molecules. (a) chitosan cross-linked with itself (b) hybrid polymer
network (c) semi-interpenetrating network (d) ionic cross-linking (Source:Gurny et al.,2004)
51
REVIEW OF LITERATURE…
Table 2.17 Cross linking behaviour of chitosan
S.No.
1
Type
of Type of network
cross
linking
Two
structural
Chitosan
units
involved
cross-linked
may or may not
belong to same
with itself
chitosan
Cross linker
References
Dialdehydes via Schiff Angeli’s et. al., 1998;
base.Glyoxal,Glutaraldeh
yde,Diethyl
Hirano
et.
squarate, Monteiro
oxalic acid and genipin
al.,
and
1990,
Airoldi,
1999 , Patel and Amiji,
1996),
Aly,
1998).
Ballantyne and Jordan,
2001, Murata-Kamiya et.
al., 1997, Monteiro and
Airoldi, 1999
2
Hybrid
polymer
networks
(HPN)
3
One
structural
units belong to
chitosan and other
belongs to other
polymeric chain
Semi or full In this network,
one structural unit
inter
entrapped in to
another polymer
penetrating
Poly ethylene glycol Airoldi and Monteiro,
diacrylate, scleroglucan,
2000, Kim et. al., 1995,
gelatin, collagen or a
silylating
agent
via Crescenzi et. al., 1997,
glutaraldehyde
Crescenzi et. al., 1995
Polyether, poly vinyl
pyrrolidone, silk fibroin,
PEO, poly (N-isopropyl
acrylamide, PEG
polymer
Guan et. al., 1996; Yao et.
al., 1998; Lee et. al.,
2000, Risbud et. al.,
2000,Khalid et. al., 1999,
Wang et. al., 2000 and
Lee et. al., 2000.
network
(IPN)
4
Ionically
cross-linked
chitosan
(reversible
crosslinking)
In this network, Metallic ions
ionic
interface
formed between
chitosan
with
negatively charged
components
as
chitosan
is
a
polycation
polymer.
52
Guival
et.
al.,
2001,
Brack et. al., 1997, Mi et.
al., 1999, Shu et. al., 2001
REVIEW OF LITERATURE…
(C)
Factors affecting ionic cross-linking density
The crosslinking density is the main parameter influencing important properties of
ionically and covalently cross-linked chitosan. Important properties of chitosan
being influenced by cross-linking density are mechanical strength, swelling and
drug release (Remunan-lopez and Bodmeier, 1997). Cross linkers for cross linking
chitosan are given in Table 2.18.
(i)
Size of cross linker
The smaller the molecular size of the crosslinker, the faster is the
crosslinking reaction, since its diffusion is easier (Mi et. al., 1999).
(ii)
Global charge on cross linker
The global charge densities of chitosan and cross-linker must be
sufficiently high in order to allow interaction. This means that the pH during
cross-linking reaction must be in the vicinity of the pKa interval of chitosan and
the cross-linker.
(iii)
Charge density and swelling
Cross linkers having a high charge density, such as tripolyphosphate, may
produce a high cross-linking density. Indeed, in order to allow a pH dependent
swelling with such cross-linkers, cross linking should be incomplete (Remunanlopez and Bodmeier, 1997). If the pH decreases the charge density of the crosslinker, the crosslinking density decreases. This leads to swelling. In addition,
swelling is favoured by protonation and repulsion of free ammonium groups
present in chitosan.
2.6
Role of Chitosan in the Colonic Delivery Dosage Forms
Chitosan was used in oral drug formulations to provide sustained release
of drugs. Recently, it was found that chitosan is degraded by the microflora that is
available in the colon. As a result, this compound could be promising for colonspecific drug delivery (Tozaki et. al., 1997b).
A microparticulate system consisting of chitosan-Ca-alginate matrix in
which 5-aminosalicylic acid was dispersed was designed. The chitosan-alginate
complex eroded slowly in the upper segments of GIT and controlled the release in
the colon where pH value ranged between 6.5 to 7.0 (Tapia et. al., 2004). In
addition, chitosan is degraded by the microflora that is available in the colon
(Shin-ya et. al., 2001; Sardar et. al., 2003).
53
REVIEW OF LITERATURE…
Table 2.18 Cross linkers for cross linking chitosan
Type of cross
linker
Ionic
Covalent
Polymeric
Examples
References
Metallic anions such as mo (vi) or pt
(ii), -glycerophosphate
Tripolyphosphate
Draget et. al., 1992
Chenite et. al., 2001
Mi et. al., 1997
Dialdehydes,Glyoxal,Glutaraldehyde,
Diethyl Squarate, Oxalic Acid,
Genipin, Poly Ethylene Glycol,
Diacrylate, Scleroglucan, Gelatin,
Collagen
Polyacrylic acid, Sodium Salt,
Carboxymethylcellulose, Xanthan,
Carrageenan, Alginate, Pectin,
Heparin, Hyaluronan, Sulphated
Cellulose, Dextran Sulfate, NAcylated Chitosan/ Chondroitin
Sulphate, Sodium Alginate, KappaCarrageenan And Polyacrylic Acid
Airoldi and Monteiro, 2000,
Kim et. al., 1995, Crescenzi
et. al., 1997, Crescenzi et.
al., 1995, Angeli’s et. al.,
1998, Hirano et. al., 1990,
Monteiro and Airoldi, 1999,
Patel and Amiji, 1996, Aly,
1998, Ballantyne and
Jordan, 2001, MurataKamiya et. al., 1997,
Monteiro and Airoldi, 1999
Peniche and ArguellesMonal, 2001, ArguellesMonal and Peniche, 1988,
Rusu-Balaita et. al., 2003;
Vasiliu et. al., 2005, Kubota
and Kikuchi, 1998;
Goycoolea et. al., 2000,
Daly and Knorr, 1988;
Ohtahara et. al., 1989
A multiparticulate system consisting of hydrogel beads was formed by
chitosan and tripolyphosphate (TPP) for the delivery of protein (Zhang et. al.,
2002). The cross linking of chitosan with TPP resulted in reduced solubility of
chitosan, thereby resulting in lesser protein release during upper GI transit. At the
same time, the cross-linking and reduced
solubility did not affect the degradability of chitosan by microbial flora in the
colon. Another multiparticulate system comprising of chitosan microspheres
coated with Eudragit L100 or S100 for the colonic delivery of metronidazole for
the treatment of amoebiasis was developed (Chourasia and Jain, 2004).
Locust bean gum and chitosan in the ratios of 2:3, 3:2 and 4:1 developed
as colon-specific drug delivery systems were evaluated using in vitro and in vivo
methods (Raghavan et. al., 2002). The studies revealed that the system was
54
REVIEW OF LITERATURE…
capable of protecting the drug in the physiological environment of stomach and
small intestine but susceptible to colonic bacteria, hence the drug release occurred
in the colon. An interpolymer complex of pectin and chitosan was used to coat
paracetamol and indomethacin tablets and it proved valuable both in restricting
drug release in the upper gastro-intestinal tract and allowing rapid release in the
colon (Fernandez-Hervas and Fell, 1998).
Suzuki et al. (1998) prepared hard capsules of chitosan with enteric
polymers for colon targeted drug delivery. Tozaki et al. (1997b) prepared capsules
of chitosan, for specific delivery of insulin to the colon. Chitosan capsules were
coated with the hydroxylpropyl methylcellulose phthalate and contained, apart
from insulin, various additional absorption enhancers and enzyme inhibitors. It
was found that the capsules specifically disintegrated in the colonic region. Tozaki
et al. (1999) used rats to study the colon specificity of chitosan capsules bearing
R-68070, a thromboxane synthetase inhibitor used for the treatment of chemically
induced ulcerative colitis. Chitosan was reacted separately with succinate and
phthalic anhydrides and resulted in the formation of semisynthetic derivatives,
chitosan succinate and chitosan phthalate. The matrices of diclofenac sodium were
prepared and incorporated into tablets. The in vitro studies revealed very slight
release of drug under acidic conditions while improved drug release profiles were
observed under basic conditions suggesting suitability for colon targeted drug
delivery (Aiedeh et. al., 1999).
Cores of acetaminophen were coated with
chitosan as inner coating layer and gastric acid resistant material phytin as an
outer coat (Tominaga et. al., 1998). Phytin protected the core from gastric pH and
dissolved in the small intestine. Chitosan protected the core in the small intestine
and released the core upon biodegradation in the colon. A chitosan dispersed
system, composed of acetaminophen reservoir and the outer drug release
regulating layer dispersing chitosan powder in hydrophobic polymer, Eudragit RS
has been developed for colon-specific drug delivery by Shimono et al. (2002).
Liu et al. (2007) developed Chitosan-based controlled porosity osmotic
pump of budesonide for colon-specific delivery system. Insulin nanoparticulate
systems by using chitosan, triethylchitosan and dimethyl-ethylchitosan for colon
delivery were prepared by the polyelectrolyte complexation method (Bayat et. al.,
2008). Rai et al. (2005) have prepared microspheres of chitosan hydrochloride to
deliver albendazole specifically to the colon.
55
Review of Literature…
2.7 Modified Gums: Approaches and applications in drug delivery
Natural gums are polysaccharides consisting of multiple sugar units linked together to
create large molecules. Gums are frequently produced by higher plants as a result of
their protection mechanisms following injury. They are heterogeneous in
composition. Upon hydrolysis they yield simple sugar units such as arabinose,
galactose, glucose, mannose, xylose or uronic acids etc.
The polysaccharide gums represent one of the most abundant industrial raw materials
and have been the subject of intensive research due to their sustainability,
biodegradability and biosafety. Many natural gums form three dimensional
interconnected molecular networks known as ‘gels’. The strength of the gel depends
on its structure and concentration, as well as on factors such as ionic strength, pH and
temperature. The linear polysaccharides occupy greater volume than branched
polymers of comparable molecular weight. Hence, at the same concentration,
comparable linear polysaccharides exhibit greater viscosity. Therefore, it is difficult
for the heterogeneous gum molecules to move freely without becoming entangled
with each other (and any other large molecules also present). Also, the natural gums
are often known for their swelling properties. Such properties are due to entrapment
of large amounts of water between their chains and branches. Hence, they could be
classified depending upon their origin, gelation etc (Table 2.19). The chemical
structures of some pharmaceutically useful gums are shown in Fig. 2.22.
Natural gums are used in pharmaceuticals for their diverse properties and
applications. They have good adhesive and laxative properties and are used in dental
preparations. They are used as binders and disintegrants in solid dosage forms. In
liquid oral and topical products they are used as suspending,
56
Review of Literature…
-D-Galp
1
2
-D-Xylp
1
-D-Xylp
1
-D-Xylp
1
6
4)--D-Glcp-(1
6
4)--D-Glcp-(1
6
4)--D-Glcp-(1
Tamarind kernel gum
-D-Galp
1
-D-Galp
1
6
4)--D-Manp-(1
4)--D-Manp-(1
6
4)--D-Manp-(1
Guar gum
-D-Galp
1
-D-GlcpA
1
4
2)--L-Rhap-(1
3
4)--D-GalpA-(1
-D-Galp
1
2)--L-Rhap-(1
2
4)--D-GalpA-(1
Gum Karaya
-L-Rhap
1
4)--D-GalpA-(1
2
4)--D-Galp-(1
4)--D-GalpA-(1
Okra gum
-D-Galp
1
4)--D-Manp-(1
6
4)--D-Manp-(1
4)--D-Manp-(1
Gum Cassia
Figure 2.22: Chemical structure of some pharmaceutically useful gums.
57
Review of Literature…
Table 2.19: Classification of polysaccharide gums.
S.No. Basis
1.
Origin
Class
Seed gums
Example
Guar gum (guar beans), Karaya gum
(Sterculia gum)
Gum tragacanth (Astragalus shrubs),
Chicle gum (From Chicle tree), Konjac
glucomannan (From Konjac plant),
Gum Arabic (Acacia tree), Gum ghatti
(sap of Anogeissus tree), Locust bean
gum ( carub tree), Mastic gum (mastic
tree)
Plant exudates
Microbial exudates
(Fermentation)
Gellan gum, Xanthan gum, Tara gum
(tara tree) , spruce gum (spruce tree)
Sea weed
Sodium alginate, Alginic acid
2.
Gelation
behaviour
Cold set gels (form Gellan gum
gels on cooling the
solution )
Heat set gels (form Konjac glucomannan
gels on heating the
solution)
3.
Chemical
structure
Reentrant gels (from Xyloglucan
which
galactose
residues
are
removed)
Galactomannans
Fenugreek gum, guar gum, locust bean
gum
Glucomannans
Konjac glucomannan
Uronic
acid Xanthan gum
containing gums
58
Review of Literature…
thickening and/or stabilizing agents. Natural gums are preferred over comparable
synthetic materials due to their non-toxicity, low cost and availability. Most of the
natural gums are safe enough for oral consumption in the form of food additives or
drug carriers. Gums are metabolised by the intestinal microflora and ultimately
degraded to their individual component sugars (Fig. 2.23). In addition, enzymes
available in the intestine can cleave the gums at specific sites. For example, αgalactosidase can hydrolyse terminal non reducing galactose residues to produce free
α–D-galactose.
However, there are certain problems associated with the use of gums. These include
uncontrolled rates of hydration, pH dependent solubility, thickening, drop in viscosity
on storage, and the possibility of microbial contamination. Chemical modification of
gums not only minimizes these drawbacks but also enables their use for specific drug
delivery purposes. In light of the above, the present article is aimed at providing a
comprehensive review of the various modifications made on gums to make them
suitable for modified drug delivery applications.
Figure 2.23: Biodegradation of polysaccharides in the intestine.
59
Review of Literature…
2.7.1. Modifications of Gums
1. Carboxymethylation/carbomoylethylation of gums
Carboxymethylation of gums increases their hydrophilicity and solution clarity and
makes them more soluble in aqueous systems. Modification of tamarind kernel
powder, cassia tora gum and guar gum were investigated by Goyal, Kumar, &
Sharma, 2007; Sharma, Kumar, & Soni, 2004 and Sharma, Kumar, & Soni, 2003a.
The general scheme of carboxymethylation is outlined in Fig. 2.24. Regardless of the
carboxymethyl content, the aqueous gum solutions were characterised by nonNewtonian pseudoplastic behaviour.
Guar gum (GG) was derivatised with monochloroacetic acid to produce
carboxymethyl guar gum (CMGG). Carboxymethyl guar gum microbeads were
prepared by dropping the solution of CMGG in a solution of divalent or trivalent
metal ions. Out of Ca2+ and Ba2+ ions the Ba2+ ions were found to cross-link more
efficiently than Ca2+. The Ba2+ cross-linked products were able to protect drug release
under gastric pH conditions while Ca2+ ion cross-linked products released the
encapsulated drug when exposed to pH 7.4 i.e. intestinal pH (Thimma, &
Tammishetti, 2001). Therefore, Ba2+ cross-linked CMGG beads were envisaged to
possess potential for gastrointestinal drug delivery. Although, the beads produced by
cross-linking Ba2+ with sodium
Figure 2.24: Carboxymethylation (CM) of gums.
60
Review of Literature…
alginate are not reported to be safe, the literature does not reveal any safety studies
pertaining to Ba2+ cross-linked CMGG beads. Therefore, toxicity as well as safety
investigations are required for Ba2+ cross-linked CMGG beads. Out of the various
cations that were investigated, only trivalent ions (Al3+, Fe3+) were found to produce
beads with smooth morphology and provided greater than 75% drug retention at
much lower concentrations as compared to divalent metal ions (Ba2+, Ca2+, Cu2+ and
Cd2+). The cross-linking efficiency of trivalent ions was found to be higher and this
was suggested to be due to their higher valency. The trivalent ions could easily
conjugate with at least two cationic sites of sodium carboxymethyl guar gum to effect
cross-linking without subjecting the polymer to any folding that may be necessary to
accommodate divalent ions. Furthermore, the beads formed by cross-linking CMGG
with divalent metal ions were found to be soft and rubbery while with trivalent
cations, the beads were soft and brittle which may be due to extensive cross-linking
of CMGG by the latter, even at low concentrations (Thimma, & Tammishetti, 2001).
Jiangyang, Wang, Liu, & He (2008) reported a method wherein a polyelectrolyte
complex was formed between COO- groups of Konjac glucomannan (KG) and NH3+
groups of chitosan. The polyelectrolyte beads were prepared via electrostatic
interaction and characterised by IR and DSC analysis. The maximum swelling index
of the beads was found at pH 1.2. The polyelectrolyte beads released only 65% of
bovine serum albumin (BSA) at pH 5.0 whereas 81% and 73% BSA was released at
pH 1.2 and 7.4, respectively in 3 h. Thus, the carboxymethyl konjac glucomannan
chitosan beads could be anticipated to be suitable for use as a polymeric carrier for
site specific bioactive drug delivery.
Carboxymethylation of cashew gum:Cashew tree gum was carboxymethylated in aqueous alkaline medium using
monochloroacetic acid (MCA) as the etherifying agent. In order to provide a better
comparison with a carboxymethylated hydrogel, a polysaccharide with a low degree
of carboxymethylation (DS) was prepared. The reaction conditions as described by
Silva et al. (2004) are briefly described herein. The purified cashew gum (5.00 g) was
mixed with water (5 ml) until a homogeneous paste was formed. Sodium hydroxide
solution (10 M, 2.7 ml) was added and the mixture was kneaded for 10 min. This was
61
Review of Literature…
followed by the mixing of monochloroacetic acid (2.62 g) thoroughly with the paste.
The mixture was heated at 55 ºC, for 3 h. The system was neutralised with
hydrochloric acid (1 M) and dialysed against distilled water until all remaining
reagents/salts were eliminated (~ 4-5 days).
Carbamoylethylation of cassia tora gum and guar gum was carried out with
acrylamide in the presence of sodium hydroxide under various reaction conditions
(Sharma, Kumar, & Soni, 2003a, 2004). The optimum conditions for preparing
carbamoylethyl cassia tora gum (3.24 % N) comprised acrylamide (1.12 mol), sodium
hydroxide (1.25 mol) and cassia tora gum (0.197 mol) at 30 °C for 1 h. The optimum
reaction conditions for carbamoylethylation of guar gum were: acrylamide (1.0 mol),
sodium hydroxide (0.75 mol) and guar gum (0.061 mol) at 30 °C for 2 h (total
reaction volume = 500 ml). Rheological properties of carbamoylethyl cassia tora gum
solutions showed non-Newtonian pseudoplastic behaviour regardless of the %N. At a
constant rate of shear the apparent viscosity of carbamoylethyl cassia tora gum
solutions increased with the increase in %N of the product. Similar results were
obtained with carbamoylethyl guar gum.
Although, carboxymethylation carbamoylethylation of natural gums can be
accomplished relatively easily, the degree of substitution is usually low. This method
is expected to be more suitable for gums containing (1,4)-linked units because
carboxymethylation/ carbomoylethylation occurs primarily at free –CH2OH groups
(i.e. the C6 position of units) due to steric reasons. The steric hindrance by -OH
groups present in the gum needs to be considered while attempting such
modifications in order to achieve significant degrees of substitution.
2. Gums grafted with acrylic acid or its derivatives
Grafting of acrylic acid or its derivatives on gums has been used for modifying the
swelling characteristics, film forming properties and drug release properties of the
later. The different methods used for grafting other moieties on gums are summarised
in Fig. 2.25.
Poly (acrylic acid) (PAA) and its derivatives are typical pH-responsive
polyelectrolytes, which have been widely used for drug delivery to specific regions of
the gastrointestinal tract (Ganorkar, Liu, Baudys, & Kim, 1999). However, high water
solubility limits their use for delivering drugs to a certain extent, because the drug
62
Review of Literature…
release takes place before the dosage form reaches the absorption site (Needleman, &
Smales, 1995). In order to overcome the above drawback, PAA is usually crosslinked with organic cross- linkers to form interpenetrating networks (IPNs) and
copolymers. However, the conventional chemically cross-linked hydrogels have
many limitations with respect to morphology and properties, e.g., morphological
inhomogeneity, mechanical weakness, limited swelling at equilibrium, and slow
response to stimuli (Siegel, Falamarzian, Firestone, & Moxley, 1988). It is well
understood that PAA has carboxylic acid groups which can be utilised for
intermolecular interactions including electrostatic interactions, hydrogen bonding or
dipole-ion interactions with other polymers. Many investigators have shown that
there are strong interactions between PAA and natural ionic polysaccharides in
aqueous solutions. Therefore, this interaction has been advantageously utilised for
developing pharmaceutical preparations. For example, chitosan-PAA polyelectrolyte
hydrogel for use in controlled drug release formulations has attracted considerable
attention, due to its simplicity, feasibility and mild conditions (De la Torre,
Enobakhare, Torrado, & Torrado, 2003; Shim, & Nho, 2003).
Chemical modification of tamarind kernel powder (TKP) and cassia tora gum through
grafting has received considerable attention for imparting new functional groups for
different applications. Goyal, Kumar, & Sharma (2008b) developed a method for
graft copolymerisation of acrylamide onto TKP. This was carried out in an aqueous
medium using a ceric ammonium nitrate-nitric acid initiation system. The maximum
grafting efficiency was found to be 93.66%. Also, cassia tora gum was used for graft
copolymerisation of acrylamide using ceric ammonium nitrate-nitric acid as redox
initiator (Sharma, Kumar, & Soni, 2002).
Grafting of gums with other polymers or ions requires availability of –COO- and/or –
CH2OH groups in the gum. The main advantage of these grafted gums is that the
resultant molecule can be designed to yield a compound with the desired drug release
profile. The grafted molecule could be selected in a way that it does not solubilise
while the gum solubilises at a particular pH. In this way, a predetermined drug release
profile could be obtained.
63
Review of Literature…
Figure 2.25
Strategies for grafting Xanthan gum: (a) acrylic acid; (b) acryloyl
chloride; (c) maleic anhydride, A-Xan; Xanthan gum, TEA;
Triethanolamine, DMF; Dimethyl formamide, DEC; N’-[3(dimethylaminopropyl)]-N-ethylcarbodiimide hydrochloride, MA;
Maleic anhydride, ACT; Acetone.
3. Konjac glucomannan or its derivatives
Konjac glucomannan (KGM) is a non-ionic polysaccharide found in the tubers of
Amorphophallus konjac, which mainly grows in China and Japan. KGM has long
been used as a health food in China and Japan. KGM is regarded as a non-calorie
food, the role of which has been displayed in weight loss and cholesterol reduction
(Abdulmnem et al., 2008). The Food Chemicals Codex in the United States lists
Konjac flour as a food additive (Zhang, Xie, & Gan, 2005). Moreover, low cost,
excellent film-forming ability, good biocompatibility, biodegradability, as well as
gel-forming properties entitle KGM to be a novel polymeric material. KGM exhibits
promising application in various fields like packing and preservation (Luo, & Feng,
2004), formulating controlled drug release dosage forms (Wang, & He, 2002) and as
a wood adhesive (Umemura, Inoue, & Kawai, 2003).
Chemically, it consists of (1,4)-linked
-D-mannose and
-D-glucose in a molar
ratio of 1.6:1 (Kato, & Matsuda, 1969) with about 1 in 19 units being acetylated
64
Review of Literature…
(Maekaji, 1978). The KGM backbone possesses 5–10% acetyl-substituted residues,
and it is widely accepted that the presence of substituted groups confers solubility to
the glucomannan in aqueous solution. If the molecules of KGM lose their acetyl
groups with the aid of alkali, the aqueous solution is transformed into a thermally
stable gel. This gelation is promoted by heating. The addition of alkali to a KGM
dispersion plays an important role in solubilising it in addition to facilitating chain
deacetylation (Williams et al., 2000). The molecular weight (length of the main chain
backbone) (Zhang et al., 2001) and the acetyl group content in the KGM molecule
(Huang, Kobayashi, & Nishinari, 2001; Huang, Takahashi, Kobayashi, Kawase, &
Nishinari, 2002) serve as determinants of its gelation characteristics. The
physicochemical properties, however, have not been fully elucidated mainly because
of the difficulty in obtaining easily soluble and well-fractionated KGM samples.
Increasing demand for materials and products from renewable resources makes it
important to develop new functional properties of KGM through physical or chemical
modification. Previously, several KGM derivatives were prepared by grafting (Xiao,
Gao, Li, & Zhang, 1999), carboxymethylation, palmitoylation, sulfation (Kobayashi,
Tsujihata, Hibi, & Tsukamoto, 2002, Zhang, Xie, & Gan, 2005) and their properties
and applications assessed. The applications of KGM have been also extended greatly
from food and food additives to various fields, such as colon delivery (Luo, & Feng,
2004), field-flow fractionation (Benincasa, Cartoni, & Fratte, 2002), ion exchange
and adsorption (Luo, & Feng, 2004) etc. The reaction scheme for the grafting of
KGM with PAA is shown in Fig. 2.26A.
Cross-linking of konjac glucomannan by organic borate:KGM powder was dispersed in distilled water at room temperature for 1 h, heated to
80ºC and maintained at this temperature for 1 h. Following cooling to room
temperature, resultant KGM solution was equilibrated at room temperature for 2
days. After this time, borax solution (200 µL) was added to KGM solution (3.0 g)
using a microsyringe, and the solutions were thoroughly mixed by manual stirring
using a teflon muddler. The resulting gel was centrifuged at 3000 rpm for 30 min to
remove visible bubbles for rheological measurements (Gao, Guo, Wu, & Wang,
2008). All the systems were found to have a pH of 9.0 due to self-buffering by borax
(Fig. 2.26B).
65
Review of Literature…
4. Cynoethylation of gums
Goyal, Kumar, & Sharma (2008a) prepared cyanoethyl tamarind kernel powder
(CTKP) using acrylonitrile in the presence of sodium hydroxide under different
reaction conditions. The results suggested that optimum CTKP (DS = 0.49) was
obtained when 0.008 mol (1.3 equivalent/OH group) of acrylonitrile was reacted at 30
ºC for 45 min using 0.026 mole TKP (0.07 mole of OH group) in 100 ml of water.
Further, the CTKP was observed to exhibit non- Newtonian pseudoplastic behaviour,
relatively high viscosity, cold water solubility, and good solution stability and clarity,
as compared to unmodified TKP. In another investigation, cyanoethyl cassia tora gum
(DS = 0.44) was produced by mixing 0.608 mol acrylonitrile and 0.625 mol sodium
hydroxide at 30ºC for 4 h (Sharma, Kumar, & Soni, 2003b).
2.7.2. Cross-linking of gums
1. Cross-linking with glutaraldehyde
Natural gums being hydrophilic swell in the presence of dissolution media. Hence,
there is a possibility of the entrapped drug leaking out prior to arrival of the drug at
its site of absorption. Thus, there is a need to reduce the enormous swelling of the
gums by cross-linking.
(i) Cross-linking of alginate-guar gum with glutaraldehyde:Alginate guar gum hydrogels were prepared with distinct alginate to guar gum
percent weight ratios. Guar gum solution was prepared, the required amount of
alginate was added and stirred well to form a uniform mixture. To this mixture
glutaraldehyde was added to a final concentration of 0.2% (v/v), blended, and
precipitated (in 0.5% w/v CaCl2) to form beads. The beads were washed with distilled
water to remove any residual glutaraldehyde and calcium chloride, and lyophillised
(George, & Abraham, 2007). This is depicted in Fig. 2.27A.
Glutaraldehyde has been used extensively for cross-linking polymers containing
hydroxyl groups. It was observed that with an increase in the concentration of
glutaraldehyde there was an increase in the cross-link density and as a result there
was a decrease in buffer uptake. Drying of the hydrogel to form discs introduced
irreversible changes in the hydrogel. Thus, it was observed that when discs were
formed by physical entanglements in the polymer network, it resulted in a change in
66
Review of Literature…
A
B
OH
HO
O
O
O
HOH2C
OH
O
HO
CH2OH
B(OH)4-
CH2OH
CH2OH
O
O
B
O
O
HO
OH
CH2OH O
O
4)--D-Manp-(1
4)--D-Glcp-(1
O
CH2OH
O
O
HO
OH
Figure 2.26
A) Grafting of konjac glucomannan (KG) with poly acrylic acid
and B) complexation with Borax ions (B(OH)4-).
67
Review of Literature…
A
B
C
Figure 2.27
Cross linking of gums with A) glutaraldehyde; B) Tri sodium
trimetaphosphate and C) grafting of guar gum with
polyacrylamide (pAAm-g-GG) followed by crosslinking with
glutaraldehyde.
68
Review of Literature…
the degree of swelling. However, it was observed that high amounts of glutaraldehyde
were required for the cross-linking reaction suggesting that the cross-linking
efficiency was low. This could be attributed to a) low reactivity of guar gum hydroxyl
groups as a result of limited water solubility b) glutaraldehyde polymerisation during
the cross-linking process and c) possible masking effect of the hexose units of the
branched polymer. However, the cross-linked products retained the ability of guar
gum to be degraded in vitro by a mixture of galactomannase and α-galactosidase
(Kabir, Yagen, Penhasi, & Rubinstein, 1998).
In another investigation carried out by Soppirnath, Kulkarni, & Aminabhavi (2000)
interpenetrating network microspheres of polyvinyl alcohol and guar gum were
prepared. These microspheres were cross-linked with glutaraldehyde. The aldehyde
groups of glutaraldehyde reacted with the hydroxyl groups of the polymers to form
acetal cross-links. The IR spectra
exhibited a corresponding peak at 1251 cm-1. Similarly, DSC studies showed an
increase in ∆H value. This increase in the ∆H value may be attributed to the high
amount of energy required to break the highly cross-linked polymeric network
structure. This also suggested the formation of a highly crystalline polymeric matrix
due to increase in cross-linking agent density. The crystalline nature of the polymeric
matrix dictates its water uptake. An increase in cross-linking leads to formation of a
dense macromolecular network. Therefore, a decrease in molecular transport of liquid
within such polymeric matrices was observed, resulting in reduced swelling. The in
vitro release of nifedipine from these microspheres was observed to depend on the
extent of cross-linking. This was due to the fact that the solvent uptake by the
microspheres decreased with increased cross-linking. Thus, the drug release
continued for several hours. The release of the drug from these microspheres
increased initially due to the polymer relaxation process as water penetrated and
converted the glassy polymer into a rubbery one. However, the latter part of the
release profile from the fully swollen polymer was due to a diffusion process.
(ii) Cross-linked microspheres of polyacrylamide grafted guar gum (pAAm-g-GG) by
water-in-oil (w/o) emulsification method:5.0% (w/v) polymer solution (20 ml) was prepared and acidified with 5 ml dilute
sulfuric acid. In order to cross-link the polymer, 2.5, 5 or 7.5 ml of 25% (w/v)
69
Review of Literature…
glutaraldehyde solution was added to the polymer solution separately. These
solutions were then emulsified into 100 ml of light liquid paraffin with 2% (w/v)
Tween 80. The hardened microspheres were filtered and washed repeatedly with
hexane and water to remove liquid paraffin, unreacted glutaraldehyde and any
adhered Tween 80. The hydrogel microspheres were then dried under vacuum at 40ºC
overnight and kept in a desiccator until further use (Soppirnath, & Aminabhavi,
2002). The scheme is depicted in Fig. 2.27C
2. Phosphate cross-linking of natural gums.
The high swelling characteristics of natural gums in matrices which leads to burst
release does not make them suitable for delivering drugs to distal parts of the gut.
Such high swelling can be prevented by phosphate cross-linking (Kabir, Yagen,
Penhasi, & Rubinstein, 2000a; Kabir, Yagen, Baluom, & Rubinstein, 2000b; Dulong
et al., 2004). Generally, phosphate cross-linked gums are prepared by dissolving
trisodium trimetaphosphate (STMP) in sodium hydroxide solution (1 M, pH = 11) at
room temperature for 30 min, followed by addition of gum under continuous stirring
(Fig. 6B). The dispersion is then stirred slowly to allow maximum swelling of the
gum. The mixture is finally poured into a Petri dish and dried. The dried hydrogel
obtained is rinsed several times with distilled water to remove unreacted STMP, gum,
and other soluble agents and dried to constant weight and stored until further use
(Fig. 2.27B).
3. Cross-linking with ions
Preparation of barium ion-cross-linked sodium alginate–CMGG beads:Sodium alginate and carboxymethyl guar gum (CMGG) were dissolved in distilled
water at a concentration of 4% (w/v). The polymer solution was then added drop-wise
into the gelation medium (BaCl2 solution of definite composition (w/v), 250 mL) at
room temperature. The beads, thus formed, were cured in the gelation medium for 20
min and then taken out, washed with distilled water and then allowed to dry to
constant weight at 30 ºC (Bajpai, Saxena, & Sharma, 2006).
4. Miscellaneous methods
Cross-linking of cashew gum with epichlorohydrin
70
Review of Literature…
Cashew gum was mixed with sodium hydroxide solution (5 M, 2 ml) and distilled
water until a homogeneous paste was formed. Epichlorohydrin (volume in the range
of 0.4–0.86 ml) was then added to the mixture and kneaded to afford proper
homogenisation. The mixture was heated at 40ºC for 24 h, followed by a second
heating time of 15 h at 70ºC (Figs. 2.28). The cross-linked gel was washed with
distilled water, dialysed for 72 h against distilled water and finally, freeze-dried
(Silva, Feitosa, Maciel, Paula, & Paula, 2006).
Radiation-induced polymerisation of sterculia gum:Sterculia gum and definite concentration of monomers were dissolved in distilled
water (10 ml). The reaction mixture was irradiated with c-rays in a 60Co γ-chamber
for 24 h with a total dose of 53.14 kGy. The polymers thus formed were stirred for
two hours in a 1:1 mixture of distilled water and ethanol to remove remaining soluble
fractions, and were then dried in an oven at 40 ºC (Singh, & Vashistha, 2008).
Cross-linking of gums requires availability of active functional groups in their basic
structure. Hence, gums such as guar gum, cashew gum or sterculia gums that possess
free alcoholic and/or carboxylic units seem to be a good choice for modification by
cross-linking. However, it is essential to investigate the vulnerability of the crosslinking to different pH in order to use the modified molecule for site specific
delivery.
2.7.3. Mechanism of cross-linking: modification of gums.
Natural gums are generally soluble in water. This is due to the presence of an
excessive number of –OH moieties which form hydrogen bonds with water
molecules. Hence, these natural gums cannot be used for controlling drug release.
Moreover, the –OH moieties are unable to form strong ionic interactions with counter
ions. Therefore, these gums need to be modified by derivatisation. A wide variety of
functional groups can be attached to natural gums to make them more suitable for
controlling the release of drugs from dosage forms. For example, attachment of
carboxyl groups, carboxymethyl groups, polyacrylamide groups, phosphate groups
etc, have all been extensively investigated for such purposes.
Interestingly, the properties of cross-linked gum derivatives depend mainly on their
cross-linking density, namely the ratio of moles of cross-linking agent to the moles of
polymer repeating units. Moreover, a critical number of cross-links per chain are
71
Review of Literature…
required to allow the formation of a network. The types of interactions forming the
network depend on the nature of the cross-linker. Therefore, cross-linked gums or
their derivatives can be classified as:
(i)
(ii)
(iii)
Hybrid polymer networks (HPN)
Semi or full inter-penetrating polymer networks (IPN)
Ionically cross-linked gums or their derivatives (reversible cross-linking)
R
OH
OH
6
-D-Galp
1
-D-Galp
1
HO
HO CH2 O
OH
CH2OH
O
R
6
-D-Galp
1
6
-D-Galp
1
-D-Galp-(1
3
-D-Galp-(6
1
3
-D-Galp-(6
1
1)--D-Galp
3
-D-Galp
1
.....
3)--D-Galp-(1
6
OH
R
3)--D-Galp-(1
6
Epichlorohydrin
(NaOH, pH 10)
1
-D-Glcp
1
-D-Galp
3
6
3)--D-Galp-(1
3)--D-Galp-(1
OH
-D-Galp
1
3
-D-Galp
1
6
3)--D-Galp-(1
O
O
HO
CH2OR
O
O
3
-D-Galp-(6
1
3)--D-Galp-(1
6
6
3)--D-Galp-(1
R
3)--D-Galp-(1
6
1
-D-Galp-(3
6
1
-D-Galp-(3
6
1
-D-Galp
1
-D-Galp
6
OH
1
-D-Galp-(3
6
1)--D-Galp-(6
3
1)--D-Galp
HO
O
O
HO
1
-D-Galp
O
R
1
-D-Glcp
OH
O
HO
O
O
O
OH
Figure 2.28
Scheme of cashew gum crosslinking with epichlorohydrin.
1. Interaction chemistry
Cross-linked gum or its derivative formed by HPN involves reaction between a
structural unit of a gum or its derivative chain and a structural unit of a polymeric
chain of another type. In addition, cross-linking of two structural units of the same
type and/or belonging to the same polymeric chain cannot be excluded. Semi or full
IPNs contain a non-reacting polymer added to the gum or its derivative solution
before cross-linking. This leads to the formation of cross-linked gum or its derivative
72
.....
R
Review of Literature…
network in which the non-reacting polymer is entrapped. It is also possible to further
cross-link this additional polymer in order to have two entangled cross-linked
networks, forming a full IPN, where microstructure and properties can be quite
different from its corresponding semi-IPN (Song et al., 2001).
In each of the three types of structures, ionic bonds are the main interactions that
form the network but other interactions cannot be excluded. Indeed, secondary
interactions, such as formation of hydrogen bridges and hydrophobic interactions
cannot be totally ruled out.
Ionically cross-linked gum (or its derivative) networks can be divided into two groups
depending on the type of cross-linker used (anions or anionic molecules). However,
most of their characteristics and properties are identical. A network is formed in the
presence of negatively charged entities, which form bridges between the positively
charged polymeric chains.
Interactions between the positively charged groups of the cross-linker and the
negatively charged groups of the gum derivatives mainly contribute towards ionic
interactions inside the network. Their nature depends on the type of cross-linker.
Metallic ions induce the formation of co-ordinate covalent bonds between negatively
charged groups of gum derivatives. This type of bonding is stronger than the
electrostatic interactions formed by anionic cross-linking molecules. Besides the
negatively charged groups (e.g. –COO-, -CH2COO- etc.) of gum derivatives, other
groups along the gum derivative chains such as hydroxyl groups can also react with
ionic cross-linkers. Moreover, additional interactions can occur inside the network.
Such interactions include hydrophobic interactions or inter chain hydrogen bonds due
to reduced electrostatic repulsion after neutralisation of gum derivatives by crosslinker (Berger et al., 2004; Knapczyk, Majewski, Pawlik, & Wisniewska, 1994;
Knapczyk, 1994).
Ionic cross-linking is a simple and mild procedure. In contrast to covalent crosslinking, no auxillary molecules such as catalysts are required (Peppas, 1986). This is
of great interest for medical and pharmaceutical applications due to regulatory
consideration for safety aspects of catalysts and residual solvents in dosage forms. In
fact, ionic cross-linking can be ensured by the classical method of preparing a cross73
Review of Literature…
linked network by adding the cross-linker in either solubilised or dispersed form to
the gum or its derivative solution. Gum or its derivative can be cross-linked by
simply dipping their pieces into a solution of the cross-linker (Brack, Tirmizi, &
Risen, 1997) or by adding the gum derivative solution into a solution of the crosslinker (Knapczyk, Majewski, Pawlik, & Wisniewska, 1994; Monteiro, & Airoldi,
1999).
2. Factors affecting ionic cross-linking density
As in covalent cross-linking, the cross-linking density is the main parameter
influencing important properties of ionically cross-linked derivatives of gums.
Important properties of these derivatives being influenced by cross-linking density
are mechanical strength, swelling and drug release (Argüelles-Monal, Goycoolea,
Peniche, & Higuera-Ciapra, 1998). Therefore, it is important to understand the
reaction conditions influencing the cross-linking density in order to modulate the
properties of the network.
Size of cross linker:
Cross-linking reactions are mainly influenced by the size of the cross-linker. The
smaller the molecular size of the cross-linker, the faster is the cross-linking reaction,
since its diffusion is easier (Wang, Fang, & Hu, 2001).
Charge density and swelling:
Particular attention should be paid to cross-linkers possessing a high charge density,
such as tripolyphosphate, that may result in a high cross-linking density. Indeed, in
order to allow a pH-dependent swelling with such cross-linkers, cross-linking should
be incomplete (Argüelles-Monal, Goycoolea, Peniche, & Higuera-Ciapra, 1998). This
can be achieved by a short reaction time and a low cross-linker concentration (Brack,
Tirmizi, & Risen, 1997). Another approach for obtaining optimised networks
(mechanically stable but with high swelling and drug release) is to combine different
cross-linkers such as trisodium trimetapolyphosphate (Brack, Tirmizi, & Risen,
1997).
74
Review of Literature…
Ionic interactions between chains of the gums cause swelling, which depend on the
cross-linking density set during the formation of the network (Knapczyk , Majewski,
Pawlik, & Wisniewska, 1994; Wang, Fang, & Hu, 2001). Therefore, the cross-linking
density is modified by external conditions after administration, mainly by the pH of
the medium (Yao, Peng, Goosen, Min, & He, 1993). Swelling can occur in both
acidic and basic conditions. In the case of the cross-linking of chitosan with gum or
its derivatives, pH decreases the charge density of cross-linker and hence the crosslinking density decreases, which leads to swelling. In addition, swelling is favoured
by protonation and repulsion of free amino groups present in chitosan. If the decrease
in pH is too large, dissociation of ionic linkages and dissolution of the network may
occur, which eventually leads to faster release of drug molecules (Brack, Tirmizi, &
Risen, 1997). If the pH increases, the protonation of chitosan decreases and induces a
decrease in cross-linking density, thus, allowing swelling. If the pH becomes too
high, amino groups of chitosan become neutral and ionic cross-linking is inhibited. If
the cross-linking density becomes too low, interactions are no longer strong enough
to avoid dissolution and the ionic cross-linker itself is released into the medium.
Microgels prepared using polyacrylamide grafted guar gum (pAAm-g-GG) showed
increased swelling when the pH of the medium was changed from acidic to alkaline
(Soppimath, Kulkarni, & Aminabhavi, 2001). For a polymer containing ionic groups,
the swelling forces may be greatly enhanced as a result of localisation of charges on
the polymer chains. When neutralised with alkali, either partially or totally, the
negatively charged carboxylic groups attached to polymer chains set up an
electrostatic repulsion, which tends to expand the chain network. However,
exceedingly large electrostatic repulsions would prevail when carboxylic ions are
present in the presence of hydroxyl ions in the solution. The carboxylic groups are
ionised at higher pH (i.e. near to 7), while at lower pH, they are protonated. The pK
value of the carboxylic group of the hydrolysed pAAm-g-GG is 4.6. The counter ion
concentration inside the gel network increases upon ionisation of the gel. However,
due to the higher concentration of the counter ions in the solution inside the gel, an
osmotic pressure difference exists between the internal and external solutions of the
gel network. This osmotic pressure is balanced by the swelling of the gel.
75
Review of Literature…
Additionally, the presence of salt ions induces a hydration sheath surrounding the
polymer, with a consequent reduction in the degree of ionisation and equilibrium
swelling. This movement of ions across the membrane to attain electroneutrality
created by the osmotic pressure difference closely resembles the Donnan membrane
equilibrium. This effect has also been observed by Durmaz, & Okay (2000) while
studying the swelling of acrylamide/2-acryamido-2-methylpropane sulfonic acid
sodium salt-based hydrogels.
3. Ionic cross-linkers
Ionic cross-linking of gums or their derivatives requires multivalent counter ions such
as metal ions (Ca2+, Ba2+), anionic molecules (trisodium trimetapolyphosphate,) or
polymers (polyvinyl alcohol, polyacrylic acid, polyacrylamide).
2.7.4. Pharmaceutical applications of cross-linked or derivatised gums
Cross-linked or derivatised gums are widely being investigated for the design of new
delivery systems with tailor-made drug release profiles. An additional advantage of
biodegradability confers the property of complete drug release to the dosage form due
to the degradation of gums by colonic bacteria and enzymes present in the distal
portion of the gastro-intestinal tract. The versatility of gums in designing dosage
forms can be judged from the investigations summarised in Table 2.20. The strategies
employed for developing selected dosage forms are discussed below.
I. Hydrogels:Spherically cross-linked hydrogels of polyacrylamide-grafted guar gum were
prepared by the emulsification method (Soppimath, Kulkarni, & Aminabhavi, 2001).
These were selectively derivatised by saponification of the –CONH group to the –
COOH group. The derivatised microgels were responsive to the pH and ionic strength
of the external medium. The swelling of the microgels increased when the pH of the
medium was changed from acidic to alkaline. Transport parameters, solvent front
velocity and diffusion coefficients, were calculated from measurement of the
dimensional response of the microgels under variable pH conditions. The variation in
pH changed the transport mechanism from Case II (in 0.1 M hydrochloric acid) to
non-Fickian (in pH 7.4 buffer), and these processes were polymer relaxation76
Review of Literature…
controlled. Ionic strength exerted a profound influence on the swelling of the
microgels. Swelling was reversible and pulsatile with the changing environmental
conditions. The pH-sensitive microgels were loaded with diltiazem hydrochloride and
nifedipine. The release was relatively quicker in pH 7.4 buffer than that observed in
0.1 M HCl and the release followed non-Fickian transport in almost all the cases.
Huang, Yu, & Xiao, (2007a) synthesised a polyelectrolyte hydrogel combination
based on cationic guar gum (CGG) and acrylic acid monomer by photo initiated free
radical polymerisation. Fourier transform infrared spectroscopy (FT-IR), scanning
electron microscopy (SEM), and differential scanning calorimetry (DSC) confirmed
that the formation of the polyelectrolyte hydrogel could be attributed to the strong
electrostatic interaction between cationic groups in CGG and anionic groups in
polyacrylic acid (PAA). Swelling experiments indicated that CGG-PAA hydrogels
were highly sensitive to pH of the environment. Ketoprofen-loaded CGG-PAA
matrices demonstrated drug release mainly by non-Fickian diffusion in pH 7.4 buffer
solution. However, for tablets, the drug release in pH 7.4 buffer solution was mainly
affected by polymer erosion. The pH of the dissolution medium appeared to have a
strong effect on the drug transport mechanism. At more basic pH values, Case II
transport was observed, indicating that the drug release mechanism was highly
influenced by macromolecular chain relaxation (Huang, Lu, & Xiao, 2007b). Gels
composed of KG, copolymerised with acrylic acid (AA) and cross-linked by N, Nmethylene-bis-(acrylamide) (MBAAm) were prepared. The influence of various
parameters on the equilibrium swelling ratios of the hydrogels was investigated
(Chen, Liu, & Zhuo, 2005). The results revealed that swelling ratio was inversely
proportional to the content of MBAAm. Also, it was possible to modulate the degree
of swelling of the gels by changing the cross-linking density of the polymer. The
swelling ratio of the gels responded to variation in environmental pH. The results of
degradation tests revealed that the hydrogels retained the enzymatic degradation
character of KG and could be degraded by 52.5% in 5 days using Cellulase E0240.
In-vitro release of 5-aminosalicylic acid (5-ASA) in the presence of Cellulase E0240
in pH 7.4 phosphate buffer at 37°C reached 95.19% after 36 h and was controlled by
77
Review of Literature…
Table 2.20: Pharmaceutical applications of gums in drug delivery
S.No.
Natural Gum
Model Drug
Dosage
form
Remarks
Reference(s)
Dexamethasone
Tablets

72-82% of Dexamethasone was Kenyon et al.
(1997)
delivered to colon.
340 mg per 420 Indomethacin
mg
tablet
(77.19%)
Matrix
tablets

In-vitro drug release without rat
caecal medium was 29.2% which
was increased upto 49.7% and
59.64% with 2% and 4% of rat
caecal medium, respectively.
100 mg coating Indomethacin
over 80 mg core
tablet (125%)
Tablets

In-vitro Cumulative percent drug Krishnaiah,
release with rat caecal medium Satyanarayana,
Rama Prasad,
(2%, 21 h study) was ≤ 20%.
&
Narsimha
In-vivo Scintigraphy study showed Rao (1998)
intact tablet in small intestine (2 h),
the
commencement
of
disintegration of the coat (4 h),
distribution of broken pieces of the
tablet in ascending colon, hepatic
flexure, transverse colon and
splenic flexure (8 h)
A. Guar Gum
1.
3.
97.3%

4.
20%
Albendazole
Matrix
tablets

78
In-vitro drug release without rat
caecal medium was 20.9% which
was increased upto 43.9% with rat
caecal medium (4%)
Prasad,
Krishnaia, &
Satyanarayana
(1998)
Krishnaiah,
Nageswara
Rao, Bhaskar
Reddy,
Karthikeyan, &
Satyanarayana
Review of Literature…
(2001a)
5.
20%
Mebendazole
Matrix
tablets

In vitro drug release without rat
caecal medium was 44.3% which
increased upto 97.5% With Rat
caecal medium (4%)
Krishnaiah,
Dinesh Kumar,
Bhaskar,
&
Satyanarayana
(2001b)
6.
20%
Mebendazole
Matrix
Tablets

In-vitro drug release in SCF Krishnaiah, &
(Sorenson’s buffer pH7.4) was Srinivas (2008)
found to be 57% which was
increased upto 97% when SCF
with 4% rat caecal medium was
used.

Studies
also
showed
that
pretreatment of rats with 5-FU at a
dose of 0.3 mg/kg or less did not
affect drug release
7.
65%
Ornidazole
Matrix
tablets

In vitro drug release without rat
caecal medium was 73.42% which
increased upto 96.8%With Rat
caecal medium (4%)
8.
80%
5 FU
Tablets

In vitro drug release without rat Krishnaiah,
caecal medium was found to be Satyanarayana,
Dinesh Kumar,
50.85%
Karthikeyan, &
In-vivo studies in healthy human Bhaskar
volunteers showed Cmax (216 (2003a)
ng/ml) at T max (7.6h)

79
Krishnaiah,
Muzib, Indira
Rao, Srinivasa
Bhaskar,
&
Satyanarayana
(2003c)
Review of Literature…
9.
260
mg Tinidazole
compression
coating on 200
mg core tablets
(130%)
Tablets
10.
50%
Calcium
Sennosides
Matrix
tablets

In-vitro drug release without rat Momin (2004)
caecal medium was 43%which
increased upto 96%With Rat caecal
medium (4%)
11.
350 mg total coat Mesalazine
weight
Tablets

Guar gum (Supercol U-NF) grade
was found to be suitable for
colonic drug delivery, which shows
11.86% drug release after 6 h.

X-ray investigations
revealed
colonic arrival time of 3-8 h for 6
volunteers and up to 24 h for 2
volunteer
Matrix
tablets

In vitro drug release without rat
caecal medium was 47.3% which
increased upto 99.5% with Rat
caecal medium (4%)
Al-Saidan,
Krishnaiah,
Satyanarayana,
& Rao (2005)
β- Matrix
tablets

In vitro drug release without rat
caecal medium was 63.5% which
increased upto 82% with Rat
caecal medium (4%)
Shyale,
Chowdary,
Krishnaiah
(2005)
12.
13.
60%
20%
Rofecoxib
Albendazole
cyclodextrin


80
In vitro drug release was found to Krishnaiah,
Bhaskar,
be 67.50%
Satyanarayana,
In-vivo studies healthy human &
Latha
volunteers showed Cmax=2158 (2003b)
ng/mL at 14.9h and AUC was
found to be 57740 ng/ml/h
Demiroz,
Acartürk,
Sevgi, & Oznur
(2004)
&
Review of Literature…
14.
50%
Albendazole
Albendazole
cyclodextrin
and Matrix
β- tablets


15.
16.
150mg coating Ondansetron
per 213 mg core
tablet (70.42%)
44%
Indomethacin
Matrix
tablets
Pellets

&
In-vitro drug release was found to Demiroz,
Takka (2006)
be 19.8% over 8 h.

After galactomannase enzyme
addition(19.6 U/L) the drug release
was increased up to 84.90% after
10 h

In vitro drug release with GG/ Ji, Xu, & Wu
Eudragit FS 30D double coated (2007)
pellets was found to be 66.56%
(After 7 h) which increased up to
100.2 %, when drug release was
studied in presence of enzymes.

In-vivo study was carried out in
Beagle dogs which showed
Cmax=1291.51
ng/ml
and
Tmax=5.41 h with double coated
pellets while Eudragit FS 30D
coated
pellets
showed
Cmax=3296.87
ng/ml
and
Tmax=2.46 h

In vitro drug release for uncoated Ravi, Mishra,
Kumar
GG formulated tablets when &
(2008)
unincubated SCF was used was
found to be 60% at end of
(Coatd with
Eudragit FS
30D)
17.
75%
Diltiazem
Matrix
tablets
(coated with
81
In vitro drug release without rat Shyale,
Chowdary,
caecal medium was 67.7 %
Krishnaiah, &
In-vivo studies healthy human Bhat (2006)
volunteers showed Cmax (916.49
ng/mL) at 12 h
Review of Literature…
double
coating
of
Inulin
and
shellac)
dissolution study (11 h),which was
increased up to 80% with
incubated medium

With 2% inulin coating 28% was
released after 11 h

Non-fickion
observed

In vitro drug release with Eudragit
L 100 coated tablets (with MAA-gGG:Xanthum gum,3:1) was found
to be (86.6%).

The Eudragit L 100 coated tablets
(with
MAA-g-GG:Xanthum
gum,1:3)
cause
maximum
retardation in drug release

During one step drug release George,
studies (Separate release studies at Abraham
pH 1.2 and 7.4 for 10 h) only 20 % (2007)
BSA released after 10 h at pH 1.2.
While two step drug release studies
(pH 1.2 for 2 h followed by study
B. Guar gum methacrylate conjugates
18.
Graft copolymer Verapamil (VRP) Hydrogel
of
GG
with and
Microspheres
acrylamide
by Nifedipine(NFD)
crosslinking with
glutaraldehyde
drug
release
was Soppirnath, &
Aminabhavi,
(2002)
(5% W/V)
19.
Methacrylic acid- Metronidazole
g-Guar gum
Tablets
(MAA-g-GG)
Mundargi,
Patil,
Agnihotri, &
Aminabhavi
(2007)
C. Guar gum-alginate conjugates
20.
Guar
gum- BSA
Alginate
combination
cross linked with
glutaraldehyde
Hydrogels
82
&
Review of Literature…
at pH 7.4 up to 10 h) showed 94%
BSA was released after 10
D. Crosslinked Guar Gum
I. Crosslinking with glutaraldehyde
21.
Indomethacin,
Discs
Sodium salicylate
and Budesonide

In-vitro drug release studies
showed that Sodium salicylate
shows complete drug release
within 120 min. while Indomethcin
and Budesonide showed negligible
drug release for 10 h, which
enhanced after galactomannase
addition.
22.
Metronidazole
Microsphere

In vitro drug release without rat Chourasia,
caecal medium was 31.23% which Jain (2004)
was increased with Rat caecal
medium (2% and 4%) upto 47.72%
and 61.65% respectively which
further increased up to 59.35% and
76.72% after enzyme induction.
23.
Methotraxate
Microsphere

In vitro drug release Without rat Chourasia et al.
caecal medium was 38.9% which (2006)
was icreased with Rat ceacal
medium(6%) upto 73.20% which
further increased up to 91% after
enzyme induction
24.
Ibuprofen
Hydrogel
Discs

Cumulative percent in vitro drug Das, Wadhwa,
release( 2 h SGF , 3 H SIF) was & Srivastava
(2006)
found to be 2-5%
83
Kabir, Yagen,
Penhasi,
&
Rubinstein
(1998)
&
Review of Literature…
II Crosslinked with tri sodium trimetaphosphate (STMP)
25.
Hydrocortisone
Hydrogels

Increase in degree of cross linking
caused decrease in extent of
degradation during In-vitro drug
release in presence of rat ceacal
medium.
Kabir, Yagen,
Penhasi,
&
Rubinstein
(2000a)
Caffeine,
Matrix
tablets

&
Within physiologic ionic strength Talukdar,
range the swelling of XG matrix Kinget (1995)
tablets shows a reciprocal relation
of In-vitro release with salt
concentration.
Drug release was influenced by
ionic strength and buffer conc.
Drug release depends on swelling
behavior.
E. Xanthan gum
26.
Indomethacin,Sodi
um Indomethacin


F. Xanthan gum derivatives
27.
Combination
Cimetidine
with
Konjac
glucomannan,KG
M
Matrix
tablets


(20%TWG)

28.
XG:GG
5-FU
Matrix

84
XG shows more In-vitro drug
release than konjac glucomannan
Β-mannase accelerates drug release
from matrix tablets prepared by
Konjac glucomannan but no effect
on tablets prepared by XG.
Matrix tablets with a single
polysaccharide (either XG or
KGM) could not retard drug
release from tablets effectively
while XG: KGM complex does.
In-vitro drug release was found to
be 42.6% which was increased
Jiangyang,
Wang, Liu, &
He (2008)
Sinha, Mittal,
Bhutani,
&
Review of Literature…
(10:20)
tablets
upto 67.2% with 2% and 80.34% Kumaria
with 4% rat caecal medium.
(2004)
29.
Combination
Diltiazem
with
Konjac
glucomannan
(both American
and
Japanese
varities)
Tablets

In vitro drug releasewith Japanese
KGM drug release was complete
within 24 h in the presence of βmannase. There was a smaller
effect on release from formulation
of American KGM.
30.
Xanthum
5-FU
gum:Boswellia
gum (3:1) with
300 mg total coat
weight
AND
Boswellia
gum:HPMC (2:3)
with 275 mg total
coat weight
Compressed
coated tablets

In-vitro drug release was found to Sinha, Singh,
be 80.2% which was increased Singh, & Binge
upto 98.22% with 2% rat caecal (2007)
medium
Studies also showed that XG play
a major role in retardation of drug
release

Manceñido,
Landin, Lacik,
&
Pacheco
(2008)
G. Khaya gum and albizia gum
31.
Khaya
(300mg)
Albizia
(400mg)
gum Paracetamol and Tablets
and Indomethacin
gum
85

In vitro drug release (After 12 h) of Odeku, & Fell,
coated with khaya gum(400 mg)
was 33.09% for Indomethacin and (2005)
36.10% for Paracetamol.

In vitro drug release with rat caecal
media (of Indomethacine only) of
tablets coated with Khaya gum
(400mg) was 98.68% while albizia
gum (400mg) coated tablets
Review of Literature…
showed 94.34% drug release

Tablets
coated
with
Khaya:Albizia(1:1)
mixture(400mg) showed 97.34%
drug release
Beads
(Coated with
Eudragit S
100)

Uncoated tablets showed 32.27%
In-vitro drug release at pH 7.4 with
galactomannase as compared to
coated tablets (28%)
Singh,
Trombetta, &
Kim
(2004);
Singh, & Kim
(2007)
Compression
coated tablets

4:1 ratio showed best In-vitro drug
release.
In-vivo
study
(Human):Cmax=28.25µg/ml,Tmax=16
h,AUC =498.62 µg.h/ml
Raghavan,
Muthulingam,
Josephine,
Jenita, & Ravi
(2002)
H. Crosslinked gellam gum
32.
A.(I)With
Azathiopurine
Calcium
or
Deacylated
gellam
gum
crosslinked with
calcium
I. Locust bean gum
33.
Locust
bean Mesalazine
gum:Chitosan

(4:1)
86
Review of Literature…
the swelling and degradation of the hydrogels. The results suggested that although,
KG was biodegradable, safe and water soluble, it could not prevent drug release due to
its sensitivity to large variations in pH encountered in the gastrointestinal tract. On the
other hand, poly(acrylic acid) is pH dependent, but is a non-biodegradable polymer.
Therefore, combining KG and poly(acrylic acid) was envisaged for combining the
useful properties of these polymers.
Reis, Guilherme, Cavalcanti, Rubira, & Muniz (2006) prepared pH-responsive
hydrogel from arabic gum (AG) chemically modified with glycidyl methacrylate
(GMA). Water uptake tests of arabic gum-glycidyl methacrylate (AG-MA) hydrogels
showed significant pH dependence, which affected the water absorption transport
mechanism. In the studied pH range, it was found that the transport of water in AGMA hydrogel was controlled by Fickian diffusion and polymer relaxation (anomalous
transport). At high pH values, the water transport profile became more dependent on
polymer relaxation. This effect was attributed to the increase in the ionised groups of
glucuronic acid segments, which contributed to electrostatic repulsion among the
groups and resulted in expansion of the gel polymer network. AG-MA hydrogels
exhibited pH-responsive behaviour, demonstrating them to be appropriate materials
for delivering drugs to specific regions in the gut.
Kabir, Yagen, Penhasi, & Rubinstein (2000a) developed guar gum (GG) cross-linked
with increasing amounts of trisodium trimetaphosophate to reduce its swelling
properties and for use as a vehicle in oral delivery formulations for targeting drugs to
distal portions of the small bowel. Swelling of native GG in artificial gastrointestinal
fluids was reduced from 100 to 120-fold to 10–35-fold depending on the amount of
cross-linker used, showing a bell-shape dependency. GG lost its non-ionic nature due
to the cross-linking procedure and became negatively charged. This was demonstrated
by methylene blue (MB) adsorption studies and swelling studies in sodium chloride
solutions with increasing concentrations in which the hydrogel network collapsed. The
adsorption of MB was also used to characterise the degree of the GG cross-linking,
from which the effective network density was calculated. In addition, effective
87
Review of Literature…
network density was calculated from elasticity measurements. Both measurements
showed that the cross-linking density (but not swelling) of the new products was
linearly dependent on the amount of trisodium trimetaphosphate in the reaction.
Further, phosphate cross-linked guar gum was evaluated for targeting hydrocortisone
to the colon. It was found that phosphate cross-linked guar gum loosely cross-linked
with 0.1 equivalents of sodium trimetaphosphate (STMP) was able to prevent the
release of 80% of the loaded hydrocortisone for at least 6 h in PBS (pH 6.4). When a
mixture of α-galactosidase and β-mannanase was added to the buffer solution, an
enhanced hydrocortisone release was observed. In-vivo degradation studies in the rat
cecum showed that despite the chemical modification of GG, its vulnerability to being
degraded by enzyme was retained in a cross-linker concentration-dependent manner.
Eight days of GG diet prior to the study increased the α-galactosidase activity in the
cecum of the rat three-fold, compared to its activity without the diet. However, this
increase in enzyme activity was unable to improve degradation of the different
phosphate cross-linked GG products. The overall α-galactosidase activity in the rat
cecum was found to be extracellular, while the activity of β-mannanase was found to
be bacterial cell-wall associated (Kabir, Yagen, Baluom, & Rubinstein, 2000b). The
observation that GG cross-linked with STMP could be biodegraded enzymatically and
was able to retard the release of a low water-soluble drug suggested that it could
potentially be used as a vehicle for colon-specific drug delivery
Meimei, Jiangyang, Wang, & He (2007) prepared hydrogel systems using KG crosslinked with STMP for targeting to the colon. Interestingly, a bell-shaped dependency
for swelling degrees in both solutions (simulated gastric test solution, pH 1.0
hydrochloric acid solution as well as simulated intestinal test solution, pH 7.4
phosphate buffer solutions) was obtained on employing increasing amounts of STMP.
In vitro release of hydrocortisone was studied in the presence and absence of βmannanase. KG cross-linked with STMP was able to retard the release of
hydrocortisone and could be biodegraded enzymatically. Further, hydrocortisone
release was observed to depend on the cross-linking density and controlled by
degradation of the hydrogels.
88
Review of Literature…
II. Microspheres
Poly-acrylamide-grafted guar gum (pAAm-g-GG) hydrogel microspheres for
controlling the release of calcium channel blockers like verapamil hydrochloride and
nifedipine were prepared by Soppirnath, Kulkarni, & Aminabhavi (2000) as well as by
Soppirnath, & Aminabhavi (2002). The presence of amide functional groups in these
microgels could be used for introducing the required ionic functionalities. The -CONH
moieties of polyacrylamide were converted to –COOH moieties resulting in an ‘ionic
micro gel’, which by definition refers to a spherical micron size and covalently crosslinked high molecular mass matrix having the fixed groups bound to its backbone
(Eichenbaum, Kiser, Shah, Simon, & Needham, 1999). Such a change in functionality
has many advantages over the neutral acrylamide-based polymers (Tripathy, & Singh,
2000, Pradip, Kulkarni, Ghandi, & Modudgi, 1991). For instance, the introduction of
polyelectrolyte functional groups changes the pAAm-g-GG matrix into a polyanionic
polysaccharide network and the weakly ionic functional group on the polymeric chain
makes them pH-responsive. This approach was advantageous because microgels in
micron size exhibited rapid response to the changing environment such as pH and
ionic strength.
III. Tablets
Toti, & Aminabhavi (2004) prepared pAAm-g-GG by employing three different ratios
of guar gum to acrylamide (1:2, 1:3.5 and 1:5). Amide groups of these grafted
copolymers were converted into carboxylic functional groups. Tablets prepared by
incorporating diltiazem hydrochloride exhibited release that continued up to 8 h and
12 h, respectively, for pAAm-g-GG and hydrolysed pAAm-g-GG copolymers. The
release was found to be dissolution-controlled in the case of unhydrolysed copolymer.
However hydrolysed copolymers showed swelling controlled release initially (i.e. in
0.1 M HCl), but at a later stage, it became dissolution-controlled in pH 7.4.
Hydrolysed pAAm-g-GG matrices were found to be pH sensitive and could be used
for intestinal drug delivery.
89
Review of Literature…
IV. Colon specific drug delivery
Kabir, Yagen, Penhasi, & Rubinstein (1998) prepared guar gum microspheres by
cross-linking with glutaraldehyde to reduce the swelling properties of guar gum. The
researchers also reported that there was no effect of cross-linking on guar gum
degradation in the presence of colonic enzymes. Chaurasia et al. (2006) prepared
microspheres of metronidazole by using glutaraldehyde as cross-linking agent for guar
gum. It was observed that in vitro release in different pH media was affected by
changes in guar gum concentration as well as glutaraldehyde concentration. Sinha,
Mittal, Bhutani, & Kumaria (2004) prepared rapidly disintegrating core tablets coated
with a mixture of xanthan gum and guar gum. It was found that the xanthan gum:guar
gum mixture (10:20) coated tablets were able to deliver the drug to the colon. Das,
Wadhwa, & Srivastava (2006) prepared glutaraldehyde cross-linked guar gum
hydrogel discs and showed that cross-linking resulted in significant reduction in the
swelling of the guar gum. Studies also showed that percentage drug release increased
with increasing glutaraldehyde concentration. Kabir, Yagen, Penhasi, & Rubinstein
(2000a) also prepared discs of hydrocortisone by using trisodium trimethophosphate
(STMP) cross-linked guar gum and showed that even increased α-galactosidase
activity (induced by eight days of guar gum diet) was unable to accelerate the
degradation of different guar gum cross-linked products.
Prasad, Krishnaia, & Satyanarayana (1998) and Krishnaiah, Satyanarayana, Rama
Prasad, & Narasimha Rao (1998) prepared tablets of indomethacin by direct
compression and coated them with guar gum. Studies showed that guar gum matrix
tablets containing more than 80% gum were capable of delivering indomethacin to the
colon. Momin (2004) observed that matrix tablets containing 50% w/w guar gum were
suitable for targeting of sennosides for local action in the colon. These results were
complementary to studies conducted by Ji, Xu, & Wu (2007), which suggested pellets
prepared with 44% w/w guar gum and coated with Eudragit FS 30D to be suitable for
colonic drug delivery. Krishnaiah, Satyanarayana, Dinesh Kumar, Karthikeyan, &
Bhaskar (2003a) prepared guar gum based matrix tablets of 5-FU and investigated the
tablets for in-vivo study in humans. These tablets showed delayed T max, absorption
90
Review of Literature…
time, decreased Cmax and absorption rate constant compared to immediate release
tablets. Mundargi, Patil, Agnihotri, & Aminabhavi (2007) prepared tablets of
metronidazole by using various polysaccharides or by graft copolymerisation using
methacrylic acid (MAA) with guar gum, xanthan gum, pectin or carrageenan. In vitro
release of metronidazole was observed to be enhanced from tablets containing grafts
of xanthan gum and MAA-g-GG as well as GG with MAA-g-GG. Ji, Xu, & Wu
(2007) observed that pH and enzyme dependent colon targeted tablets of indomethacin
could be prepared using film coating of guar gum and Eudragit FS 30D. Studies
suggested that the use of guar gum to a weight gain of 44% was enough to provide a
lag time of 3.1 h for releasing 10% drug, which provided a basis for dissolution of the
guar gum coating in the proposed colon targeted system.
Talukdar, & Kinget (1995) prepared xanthan gum matrix tablets by using three drugs
having different properties i.e. caffeine as a soluble drug, indomethacin as an insoluble
acidic drug and the sodium salt of indomethacin as a soluble acidic drug. These
studies revealed the dependence of drug release on the swelling of the polymer matrix,
which in turn was influenced by the ionic strength and buffer concentration. Sinha,
Mittal, Bhutani, & Kumaria (2004) prepared rapidly disintegrating core tablets coated
with a mixture of xanthum gum and guar gum. It was found that the XG:GG mixture
(10:20) coated tablets were able to deliver drug to the colon.
Gellan gum has been investigated as a possible carrier for colonic drug delivery.
Singh, & Kim (2007) investigated potential interactions among a model drug
(azathioprine; AZA), polymers, and a divalent metal ion, which were utilised for
developing a novel multiparticulate formulation for colonic drug delivery. The authors
prepared beads by ionotropic gelation of deacylated gellan gum (DGG) in the
presence of Ca2+ ions, followed by coating with Eudragit® S-100. The results of FT-IR
studies suggested interactions of DGG with AZA and Eudragit® S-100, and provided
evidence for interactions of AZA and DGG with Ca2+ ions, which was also supported
by results of DSC studies.
Gums are abundantly found in nature. They are cheaper than the synthetic polymers
available for various purposes. In addition, they are well tolerated by the human body
91
Review of Literature…
because they are easily degraded to monosaccharides by colonic bacteria. However,
the highly swellable nature of their putative form often restricts their use for delivering
drugs to distal parts of the gastrointestinal tract.
The present discussion revealed different approaches that have been investigated for
modifying the properties of gums. The modified gums were observed to be useful for
preparing various dosage forms with modified drug release profiles. Unlike the dosage
forms prepared using synthetic polymers, the dosage forms prepared with modified
gums do not suffer from the disadvantage of incomplete drug release. This is due to
their susceptibility to degradation by colonic microflora.
Certain
modifications
like
carboxymethylation
and
carbomoylethylation
by
replacement of few free-OH groups increase the aqueous solubility of gums. Hence,
the resultant moiety is not suitable for delaying the drug release. Therefore, these
groups need to be cross-linked with the oppositely charged anions. These cross-linked
structures are resistant to dissociation in acidic pH but slowly degrade in the intestine,
thus providing sustained release of drugs from dosage forms during the transit in the
gastrointestinal tract. Similarly, phosphorylation of gums with sodium metaphosphate
is reported to be useful in designing dosage forms for colonic drug release.
However, the degree of substitution is the main concern in almost all types of
modifications. A low degree of substitution leads to less cross-linking density, which
in turn results in premature release of drug in the gastrointestinal tract. Therefore,
gums containing galactomannan are ideally suited for modification since they contain
carboxylic groups, which are more amenable to cross-linking than hydroxyl groups.
The abundance of gums, their economic cost and biodegradability have compelled
formulation scientists to design approaches for making them suitable for modifying
the drug release of dosage forms.
2.8 5-Fluorouracil
5-Fluorouracil (5-FU) has been in use for the treatment of cancer for more than
two decades. It is a fluorinated antimetabolite of uracil (Figure 2.7). It slows tumour
cell growth by inhibiting thymidine formation, thereby inhibiting protein synthesis by
getting
92
Review of Literature…
Figure 2.29:Structure of 5-fluorouracil
incorporated into RNA. The physical properties of 5-FU are summarized in Table
2.21.
Transdermal delivery of 5-FU
It has been found that slow intravenous administration of anti-cancer drugs prolong
their effect and theoretically maximizes their exposure to dividing cells (Chabner et
al., 1996). Similarly, reducing the rate of injection to slow continuous infusion has
been found to decrease the toxic effects of 5-FU. But, the parenteral administration in
the form of continuous infusion entails certain risks and therefore, necessitates
hospitalization and close medical supervision (Chien, 1992). Rapid intravenous
administration gives peak plasma concentration with in minutes and the drug is so
rapidly cleared from the blood that the drug level is undetectable after 2-3h (Chabner
et al., 1996). Availability of 5-FU following oral administration is only 28% due to
high degradation particularly in the liver and intestinal mucosa by the enzyme
dihydropyrimidine dehydrogenase. Therefore, transdermal delivery of 5-FU seems to
be the best mode of systemic delivery so as to obtain maximum effectiveness with
least associated toxicity. Moreover, 5-FU is reported to be stable against skin enzymes
(Sugibayashi et al., 1985) which makes it a good candidate for transdermal delivery.
Furthermore, the literature revealed the use of 5-FU as model polar drug for
evaluating the mechanism of penetration enhancers such as terpenes, azone, fatty
acids, amines, etc. (Goodman and Barry, 1988; Aungst et al., 1990; Williams and
Barry, 1991; Lee et al., 1993; Yamane et al., 1995; Abdullah et al., 1996; Moghimi et
al., 1998).
93
Review of Literature…
The in vitro permeation characteristics of 5-FU and flurbiprofen were investigated
through three layers of the human nail plate. Most of the lipids in the human nail plate
are present in the dorsal and ventral layers. With respect to 5-FU permeation through
each single layer, the permeability coefficient of the intermediate layer was higher
than those of other single layers. The diffusion coefficients of 5-FU and flurbiprofen
in the dorsal layer were lowest through any single layer. Hence, it was suggested that
the human nail plate behaved like a hydrophilic gel membrane rather than a lipophilic
partition membrane and that the upper layer functioned as the main nail barrier to drug
permeation due to its low diffusivity(Kobayashi et al., 1999). Further it was observed
that addition of penetration enhancer in a lotion formulation, Belanyx (containing
urea, propylene glycol) improved the efficacy of a low
concentration of 5-FU (1% w/v) in psoriatic finger nail lesions. However, Belanyx
without enhancer did not increase the efficacy of 5-FU in psoriatic nail dystrophy in
this study population (de Jong et al., 1999).
The effect of volatile oils from Rhozoma Apiniae officinarum, Pericarpium
zanthoxyli, Herbal asari, cineol and ethanol extracts on the percutaneous penetration
of 5-FU was studied. The results suggested that these volatile oils (1%-3% v/v)
enhanced the percutaneous absorption of 5-FU (Shen et al., 2000).
Levy et al. (2001) compared the percutaneous absorption of 3H-labeled fluorouracil
from three fluorouracil (0.5%) formulations incorporated in to a porous microsphere
delivery system with that from a commercially available fluorouracil (5%)
formulation. The flux of 5-FU from microsphere systems was 20 to 40 times greater
than that from the conventional
formulations. Higher percentage of absorbed
fluorouracil was found to be retained in the skin in 24 h after application of these
formulations.
The stratum corneum layer of skin gets partly ablated by an erbium-Yag laser-treated
skin. The permeation of 5-FU was 53-133 fold higher than that across intact skin (Lee
et al., 2002). Electroporation exerted a disruptive influence on the stratum corneum.
However, combining electroporation with erbium: Yag laser led to partial ablation
resulting in enhanced skin permeation of 5-FU (Fang et al., 2004). The systemic
delivery of percutaneously applied 5-FU across athanol-perturbed rat skin treated with
94
Review of Literature…
either 600 µg or 1200 µg beta chloroalanin (beta-CA, an inhibitor of sphigosin
synthesis) was found to be significantly greater as compared to that after oral
administeration or after application of lower percutaneous doses of beta- CA (Gupta et
al., 2004). This suggested that 5-FU permeated through the polar pathway. The
passive permeation of 5-FU was observed to depend upon the pH of the donor
solution. Its permeation was enhanced by approximately 3, 4 and 24-fold,
respectively, when isopropyl myristate, lauryl alcohol and azone were incorporated in
to the donor solution. Azone appeared to be better enhancer for 5-FU, indicating its
permeation from the polar pathway (Singh et al., 2005c).
Table 2.21:The physical properties of 5-FU
Sr. No.
Properties
Description
1.
Emperical Formulae
C4H3FN2O2
2.
Molecular weight
130.08
3.
Appearance, colour and White to practically white, odourless, crystalline
odor
powder
4.
Melting point
282 °C to 283 °C
5.
Log P
-2.987
(n-octanol/ water)
6.
Solubility
1 gm in 8 ml water, 170 ml alcohol, 55 ml
methanol, insoluble in chloroform, ether and
benzene. Soluble in aqueous solutions and
solubility increases with increase in pH.
7.
Pka
8.1
Colo-rectal delivery of 5-FU
5-Fluorouracil (5-FU)' was synthesized in 1957 and since then has become an
established antineoplastic agent used clinically in the treatment of various human solid
tumors. The biochemical mechanisms of action for 5-FU have been studied
extensively (Cohen et al., 1958; Dannberg et al., 1958) with particular emphasis on
thymidylate synthetase inhibition (Hartmann and Heidelberger, 1961) and
95
Review of Literature…
incorporation of 5-FU into RNA (Fernandes and Bertino, 1980). Metabolic
modulation to enhance either of these two actions has failed to show a causal link with
therapeutic efficacy ((Fernandes and Bertino, 1980; Spiegelman et al., 1980) and the
relative importance of each remains controversial. Neither mechanism excludes an
antitumor effect separate from these antimetabolic actions.
The concept that 5-FU acts primarily as a cytotoxic drug affecting rapidly dividing
cells has lead to the use of high doses that are active against not only tumor cells, but
also cells of the gastrointestinal mucosa and the hematopoietic system (Makinodan, et
al., 1970; Mitchell and
DeConti, 1970). It has been assumed that 5-FU is
immunosuppressive because of the inhibitory effects seen at these high doses
(Mitchell and DeConti, 1970). Studies of the effect of 5-FU and other fluorodinated
pyrimidines on the rodent immune response have been conflicting (Santos and Owens,
1964). Merrit and Johnson (1963) demonstrated significant augmentation of the
murine immune response when 5-FUDr (the deoxyriboside of 5-FU) was given before
antigen administration. Conversely, however, 5-FU and 5-FUDr produced
immunosuppression when given after antigen administration. Blomgren et al. (1965)
have demonstrated that delayed hypersensitivity may be augmented by 5-FU. Uy et al.
(1965) failed to show immunosuppression by either 5-FU or 5-FUDr of the mouse
anti-sheep erythrocyte response. Similar lack of immunosuppression has been
demonstrated in rabbits (Sterzl, 1961). The disparate effects of 5-FU on rodent
immune responses can be attributed to differences in dosage and in timing of
administration in relation to antigen stimulation. These factors are often of
criticalimportance in the immune response to any agent.
Although, 5-FU is a widely used antineoplastic agent, the cytotoxicity of 5-FU is not
limited to tumor tissue. Hematopoietic cells and normal epithelial cells of the GI tract
are susceptible to 5-FU-induced cytotoxicity, which produces severe leucopenia and
intestinal toxicity leading to lethal translocation of intestinal microflora (Kucuk et al.,
2005). In addition, because of the short plasma half-life of 10–20 min, high doses have
to be administered repeatedly by IV route to reach therapeutic drug levels (Peters et
al., 1993). Moreover, the clinical use of 5-FU is limited by its GI (stomatitis and
myelotoxicity) toxicity (Fraile et al., 1980). The oral bioavailability in humans is
96
Review of Literature…
reported to be only 28% (Gilman, 1996). The reported severe systemic toxic effects
and very short plasma half-life make this drug particularly suitable for local delivery
at the site by a suitable drug delivery system thus, exposing the diseased tissues in a
continuous and sustained manner (Koole et al., 1998). Colorectal delivery of 5-FU is
expected not only to reduce the systemic side effects, but can also be expected to
provide an effective and safe therapy for colon cancer with reduced dose and duration
of therapy.
Intravenous administration of 5-fluorouracil for colon cancer therapy produces severe
systemic side-effects due to its cytotoxic effect on normal cells. Krishnaiah et al.,
(2002) developed novel tablet formulations for site-specific delivery of 5-fluorouracil
to the colon without the drug being released in the stomach or small intestine using
guar gum as a carrier. Fast-disintegrating 5-fluorouracil core tablets were compression
coated with 60% (FHV-60), 70% (FHV-70) and 80% (FHV-80) of guar gum, and
were subjected to in vitro drug release studies. Guar gum compression-coated tablets
released only 2.5-4% of the 5-fluorouracil in simulated GI fluids. When the
dissolution study was continued in simulated colonic fluids (4% w/v rat caecal content
medium) the compression-coated FHV-60, FHV-70 and FHV-80 tablets released
another 70, 55 and 41% of the 5-fluorouracil respectively. The results of the study
show that compression-coated tablets containing 80% (FHV-80) of guar gum are most
likely to provide targeting of 5-fluorouracil for local action in the colon, since they
released only 2.38% of the drug in the physiological environment of the stomach and
small intestine. In another study Sinha et al (2007) developed colon-specific
compression coated systems of 5-fluorouracil (5-FU) for the treatment of colorectal
cancer using xanthan gum, boswellia gum and hydroxypropyl methylcellulose
(HPMC) as the coating materials. Core tablets containing 50 mg of 5-FU were
prepared by direct compression. The coating of the core tablets was done using
different coat weights (230, 250, 275 and 300 mg) and different ratios (1:2, 2:1, 1:3,
1:7 and 3:4) of boswellia gum and xanthan gum and different ratios (1:1, 1:2, 2:1, and
2:3) of boswellia gum and HPMC. Among the different ratios used for coating with
boswellia:xanthan gum combination, ratio 1:3 gave the best release profile with the
lowest coating weights of 230 mg (7.47 +/- 1.56% in initial 5 h). Further increase in
97
Review of Literature…
the coat weights to 250, 275 and 300 mg led to drug release of 5.63 +/- 0.53%, 5.09
+/- 1.56% and 4.57 +/- 0.88%, respectively, in the initial 5 h and 96.90 +/- 0.66%,
85.05 +/- 1.01% and 80.22 +/- 0.35%, respectively, in 24 h. Increasing the coat
weights to 250, 275 and 300 mg led to drug release of 6.5 +/- 0.27%, 3.70 +/- 2.3%
and 2.99 +/- 0.72%, respectively, in the initial 5 h and 96.90 +/- 0.66%, 85.05 +/1.01% and 80.22 +/- 0.35%, respectively, in 24 h. In-vitro studies were further carried
out in the presence of 2% w/v rat caecal contents, which led to complete release of the
drug from the tablets. Therefore, this study lays a basis for use of compression coating
of 5-FU as a tool for delaying the release of the drug, which ensures better clinical
management of the disease.
The distribution in the gastrointestinal (GI) tract of Eudragit S-100 encapsulated
colon-specific sodium alginate microspheres containing 5-fluorouracil (5-FU) in rats,
were determined and compared with a control immediate-release (IR) formulation of
5-FU. (Rahman et al., 2008). 5-FU was distributed predominantly in the upper GI tract
from the IR formulation but was distributed primarily to the lower part of the GI tract
from the microsphere formulation. No drug was released in the stomach and intestinal
regions from the colon-specific microspheres. Significantly, a high concentration of
the active drug was achieved in colonic tissues from the colon-specific microspheres
(P < 0.001), which was higher than the IC50 required to halt the growth of and/or kill
colon cancer cells. Colon cancer was induced in rats by subcutaneous injection of 1,2dimethylhydrazine (40 mg kg
(-1)
) for 10 weeks. The tumours induced were non-
invasive adenocarcinomas and were in Duke's stage A. The 5-FU formulations were
administered for 4 weeks after tumour induction. Non-significant reductions in tumour
volume and multiplicity were observed in animals given the colon-specific
microspheres. Enhanced levels of liver enzymes (SGOT, SGPT and alkaline
phosphatase) were found in animals given the IR formulation of 5-FU, and values
differed significantly (P < 0.001) from those in animals treated with the colon-specific
microspheres. Elevated levels of serum albumin and creatinine, and leucocytopenia
and thrombocytopenia were observed in the animals given the IR formulation. In
summary, Eudragit S-100 coated alginate microspheres delivered 5-FU to colonic
98
Review of Literature…
tissues, with reduced systemic side-effects. A long-term dosing study is required to
ascertain the therapeutic benefits.
Paharia et al., (2007) prepare and evaluate Eudragit-coated pectin microspheres for
colon targeting of 5-fluorouracil (FU). Microspheres prepared by using drug:polymer
ratio 1:4, stirring speed 1000 rpm, and 1.25% wt/vol concentration of emulsifying
agent were selected as an optimized formulation. The in vitro drug release study of
optimized formulation was also performed in simulated colonic fluid in the presence
of 2% rat cecal content. Organ distribution study in albino rats was performed to
establish the targeting potential of optimized formulation in the colon. The release
profile of FU from Eudragit-coated pectin microspheres was pH dependent. In acidic
medium, the release rate was much slower; however, the drug was released quickly at
pH 7.4. It is concluded from the present investigation that Eudragit-coated pectin
microspheres are promising controlled release carriers for colon-targeted delivery of
FU.
In another study, Dev et al (2011) statistically optimize a colon specific formulation of
5-Fluorouracil for the treatment of colon cancer. A 32 full factorial design was used
for optimization. Drug release studies were carried out using change over media [pH
1.2, 7.4 and 6.5 in presence of 4% (w/v) rat caecal contents]. The optimized
formulation consisting of pectin (66.67%, w/w) and starch paste (15%, w/w) released
negligible amount of drug at pH 1.2 and pH 7.4 whereas significant (p < 0.05) drug
release was observed at pH 6.5 in presence of 4% (w/v) rat caecal contents.
Roentgenographic studies corroborated the in vitro observations, thus providing the
“proof of concept”. Pharmacokinetic studies revealed significant reduction in systemic
exposure and cytotoxicity studies demonstrated enhanced cellular uptake of drug by
the developed formulation. Shelf life of the formulation was found to be 2.83 years.
The results of the study established pectin-based coated matrix tablet to be a
promising system for the colon specific delivery of 5-FU so as to treat colon
carcinoma.
99