REVIEW OF LITERATURE…shodhganga.inflibnet.ac.in/bitstream/10603/8027/8/08_chapter 2.pdf · REVIEW OF LITERATURE… 12 aerobic. The most commonly isolated species are streptococci,

REVIEW OF LITERATURE…

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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


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Table 2.1 Anatomical features of small intestine and large intestine

Organ Characteristics

Small Intestine

Duodenum It is 25-30 cm long section. It serves as a receiving area for chemicals and partially digested food from stomach.

Jejunum

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

Ileum 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 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.

Ascending 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.

Hepatic flexure

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.

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).

Descending colon

It is about 30 cm long. It traverses inferiorly along the left abdominal wall to the pelvic region.

Sigmoid colon

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.

Rectum

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


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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)


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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).


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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


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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 Solidity or viscosity of content may be affected by diet

Mucus barrier, unstirred layer 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)


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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


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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 Transit time (h) Reference

Stomach

Small intestine

Colon

< 1 (Fasting), > 3 (Fed)

3-4

33 (Men)

47 (Women)

Hardy et al., 1987

Hinton et al., 1969

Hinton et al., 1969

Hinton et al., 1969

Table 2.4 pH in different regions of GIT

Region of GIT pH

Reference

Stomach 1.0-2.5 Evans et al., 1988

Small

intestine

Duodenum 5.1-6.6 Evans et al., 1998;

Ashford, 2002

Jejunum 5.2-6.2 Ashford, 2002

Ileum 6.8-7.8 Evans et al., 1988;

Ashford, 2002

Large

intestine

Caecum 6.4 Bussemer et al., 2001

Ascending colon 5.7 Bussemer et al., 2001

Transverse colon 6.6 Bussemer et al., 2001

Descending colon 7.0 Bussemer et al., 2001

Figure 2.5 Concentration of bacterial flora in different regions of the

gastrointestinal tract


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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.


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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

Infectious Agents

Viruses (eg. measles)

Commensal bacteria Mycobacteria Chlamydia

Genetics

Metabolic defects

Connective tissue disorders Environmental factors

Geographical and socioeconomic factors Diet Smoking

Immune defects

Altered host susceptibility Immune-mediated mucosal damage

Psychological factors

Stress Emotional or physical trauma Occupation

Barrier functions

Impaired intestinal permeability

(Source:- Modified from Dipro, J.T and Schade, R.R, 2005)


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Table 2.6 Colon targeting diseases, drugs and target sites

Target Sites

Disease Conditions Symptoms Drugs and active agents

Topical / Local action

Inflammatory Bowel Disease

Crohn’s disease

Diarrhea, Abdominal pain and cramping, blood in stool, ulcers, reduced appetite and weight loss

Mesalamine, Hydrocortisone, Budenoside, Prednisolone, Sulfasalazine, Olsalazine, Balsalazide, Infliximab

Ulcerative colitis

Inflammation in the rectum, rectal bleeding,

rectal pain

Mesalamine, Sulfasalazine, Balsalazide, Infliximab, Azathioprine and Mercaptopurine

Irritable bowel syndrome

Abdominal pain or cramping, a bloated feeling, flatulence, diarrhea or constipation —people with IBS may also experience alternating bouts of constipation and diarrhea, mucus in the stool

Dicyclomine, Hyoscine, Propantheline, Cimetropium, Mebeverine, Trimebutine, Scopolamine, Alosetron, Tegaserod

Colorectal cancer

A change in bowel habits, narrow stools, rectal bleeding or blood in stool, persistent abdominal discomfort, such as cramps, abdominal pain with bowel movement, unexplained weight loss

5-Flourouracil, Leucovorin, Oxaliplatin, Irinotecan, Bevacizumab and Cetuxim B

Diverticulitis

Formation of pouches (diverticula) on the outside of the colon due to bacterial infection

Bactrim, Flagyl, Sulfatrim Metronidazole

Antibiotic associated

colitis

Overgrowth of Clostridium difficile and its subsequent toxin production

Clindamycin, Broad-Spectrum Penicillins (Eg, Ampicillin, Amoxicillin) and Cephalosporins

Hirschsprung's disease

Severe form of constipation in which bowel movement occurs only once or twice a week

Metronidazole, vancomycin, loperamide, botulinum toxin

Systemic action

To prevent first pass

metabolism of orally ingested

drugs

---- Steroids

Oral delivery of peptides

---- Insulin

Ulcerative

colitis

Ulcerative proctitis, pancolitis, fulminant colitis

Prednisolone Metasulfobenzoate, Tixocortol Pivalate, Fluticasone Propionate, Beclomethasone Dipropionate And Budesonide, Azathiorpine/6-Mercaptopurine.


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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/ethanol-

induced 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


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A number of animal models of colitis have been reperted, but all are T-

helper 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

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

Mouse and rat models with gene overexpression

IL-7 transgenic mice STAT-4 transgenic mice HLA B27 transgenic rat

Models of spontaneous colitis

C3H/HeJBir mice SAMP/Yit mice

Inducible colitis models

Trinitrobenzene sulfonic acid-induced colitis

Oxazolone colitis Dextran sulfate sodium induced

colitis Acetic acid induced colitis Indomethacin induced colitis Carrageenan colitis Peptidoglycan-polysaccharide

colitis Dextran sulfate sodium colitis Adoptive transfer models

CD4+/CD45RB high T-cell transfer colitis

Colitis induced by transfer of hsp60-specific CD8 T-cells


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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 10-

12 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


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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


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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).


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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


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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


23

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:


24

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 5-

fluorouracil, 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),


25

(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 pH-

controlled 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 self-

emulsifying vehicles, filled in soft elastin capsules and coated with CAP.


26

Table 2.8 Approaches and challenges for formulation of colon -specific drug delivery system

Approaches

Challenges Principle of delivery

pH dependent

systems

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)

Enteric polymers do not dissolve in stomach and at pH ≥ 5 they begin to dissolve

Time dependent

Systems

Wide variation in gastric retention time Increased transit time in diseases such as inflammatory bowel syndrome, ulcerative colitis, diarrhoea

Swelling type polymers that allow drug release 4-6 h after leaving stomach

Microbially triggerd systems

Polysaccharide based delivery systems

Prodrugs

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

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


27

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 5.2

HPMC 55 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


28

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


29

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).


30

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.


29

Table 2.10 Advantages and Disadvantages of Various Oral Colon-Specific

Drug Delivery Methods Method Advantages Disadvantages References

Time-dependent systems

Small intestine transit time is fairly consistent

Substantial variation in gastric retention times

Transit through the colon is in patients with colon disease

Davis et. al., 1986

Yang et. al., 2002

Ashford and Fell ., 1993

Watts and Illum, 1997

pH-dependent systems

Formulation well protected in the stomach

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

Poor site-specificity

Friend, 1991

Ashford et. al., 1993b

Kinget et. al., 1998

Yang et. al., 2002

Ashford et. al., 1993b Mastiholimath et al., 2007

Pressure controlled systems

Drug release mechanism is independent of pH

High viscosity of luminal contents in colon affects the drug dissolution.

Disintegration of formulation can occur in the small intestine.

Hu et. al., 1999

Takaya et. al., 1995

Microflora-activated systems

Good site-specificity With prodrugs and polysaccharides

Diet and disease can affect Colonic microflora. Enzymatic degradation may be excessively slow Few have been accepted for use in relation to medicines.

Rubinstein et al.2006 Yang et. al., 2002

Table 2.11 Enzymes present in colon

Reducing Enzymes Hydrolytic Enzymes

Nitroreductase Esterases

Azoreductase Amidases

N-oxide reductase Glycosidases

Sulphoxide reductase Glucuronidase

Hydrogenase Sulfatase


30

Basit et al, (2008) prepared pellets of theophylline using Eudragit S or

amylose/ ethylcellulose, Invivo results showed Tmax ranging from 5-9h, AUC 8.8-

55.0 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


31

2.9a). In the colon, the azoreductases cleave the azo bond releasing the drug, 5-

ASA 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 p-

aminohippurate 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


32

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 3-

sulfates 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, mPEG-

PABA-NN-SA (b), biazo, PEG-(PABA-NN-SA) 2 (d), branched mPEG2-PABA-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)


33

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


34

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.


35

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


36

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 colon-

specific 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.,


37

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 cross-

linked 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


38

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)


39

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.


40

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-Ca-

alginate micro particles loaded with budesonide. Release data suggests that


41

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


42

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.


43

Table 2.13 Different strategies used for drug release studies of colonic drug delivery systems

S.No.

Code

Drug release in different pH medium

HCl buffer pH 7.4 buffer pH 7.5

buffer

pH 6.8 PBS pH 7.0 SCF (rat

caecal)

pH 6.4

PBS

pH 6.5

buffer

pH 4.5

buffer

Miscellaneous remarks pH 1.2

0.1 M/L HCl

SGF PBS SCF SB With rat

ceacal

Without rat

ceacal

SCF

1. A Y (2h)

N N N N Y (3h)

N Y (19h)

N N N N N N N

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


44

8. H N N N N N N N N N 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 α-galactosidase (0.033 U/ml) in

same buffers *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)


45

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 99mTc-DTPA and 99mTc-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 colon-

specific 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,


46

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-deoxy-

2-amino-d-glucopyranose and partially or none of β-(1, 4)-2-deoxy-2-acetamido-

d-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´nez-

Ruvalcaba, 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


47

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

OH O

H O

H

N H

H O H 2C

O

COC H 3

O

H O

H

N H

H O H 2 C

O

COC H 3

O

H ON H

H O H 2 C

COC H 3

O H

OH O

H O

H

N H 2

H O H 2C

O O

H O

H

N H 2

H O H 2 C

OO

H ON H 2

H O H 2 C

O H

n

n

C h itin -D ea ce ty la se

C h itin

C h ito sa n

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.


48

(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)


49

Table 2.14 Physical and chemical properties of chitosan

(Source:-Rinaudo et. al., 2006)

Property

Parameter Attributes

Physical

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 <

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.


50

Table 2.15 Pharmaceutical applications of chitosan Application Suggested Mechanism Reference

Directly compressible

vehicle

Crystalline structure Felt et. al., 1998; USFDA,

1999

Controlled release

matrix

Cross-linking with drugs and

other ions or polymers

-do-

Wet granulation Binding property Illum, 1998; Paul and Sharma,

2000

Gels Forms water soluble gels with

acidic substances

-do-

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

and

microparticles

Cross linking with ions and

ionic molecules to form

spherical agglomerates

Felt et. al., 1998; Illum, 1998

Bioadhesion High cohesive force Felt et. al., 1998; Illum, 1998

Peptide delivery Cross-linking with amino acids Lueben et. al., 1997

Immobilisation

of enzymes

-Glucosidase of Aspergillus

immobilized on chitosan using

glutaraldehyde

Bissett and Sternberg, 1978

Transmucosal

drug transport

Binding with mucin Felt et al, 1998

Fast releasing dosage

forms

Decreases drug crrystallinity Sawayanagi et al., 1982b

Colon drug release Cross-linked chitosan insoluble

in acidic medium and

biodegradation by chitosanase

enzyme in colon

Tozaki et al., 1997b

Cosmetics Demulscent and soothing Lang et al., 1985

Wound Healing Rapid dermal regeneration Luo et al., 2004

Antimicrobial By shrinkage of cell membrane

leads to cell death

Chen et al., 2005


51

Table 2.16 Effect of acids on viscosity (cps) and pH of chitosan solution (1%

w/v)

Acid

Property influenced by different concentrations of acid (% v/v) 1% concentration 5% concentration 10%

concentration Viscosity pH Viscosit

y pH Viscosit

y pH

Acetic 260 4.1 260 3.3 260 2.9 Citric 35 3.0 195 2.3 215 2.0 Oxalic 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)


52

Table 2.17 Cross linking behaviour of chitosan

S.No. Type of cross linking

Type of network Cross linker References

1

Chitosan

cross-linked

with itself

Two structural units involved may or may not belong to same chitosan

Dialdehydes via Schiff

base.Glyoxal,Glutaraldeh

yde,Diethyl squarate,

oxalic acid and genipin

Angeli’s et. al., 1998;

Hirano et. al., 1990,

Monteiro and 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)

One structural units belong to chitosan and other belongs to other polymeric chain

Poly ethylene glycol diacrylate, scleroglucan, gelatin, collagen or a silylating agent via glutaraldehyde

Airoldi and Monteiro,

2000, Kim et. al., 1995,

Crescenzi et. al., 1997,

Crescenzi et. al., 1995

3 Semi or full

inter

penetrating

polymer

network

(IPN)

In this network, one structural unit entrapped in to another polymer

Polyether, poly vinyl pyrrolidone, silk fibroin, PEO, poly (N-isopropyl acrylamide, PEG

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.

4 Ionically cross-linked chitosan (reversible cross-linking)

In this network, ionic interface formed between chitosan with negatively charged components as chitosan is a polycation polymer.

Metallic ions Guival et. al., 2001,

Brack et. al., 1997, Mi et.

al., 1999, Shu et. al., 2001


53

(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 (Remunan-

lopez and Bodmeier, 1997). If the pH decreases the charge density of the cross-

linker, 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 colon-

specific 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).


54

Table 2.18 Cross linkers for cross linking chitosan

Type of cross linker

Examples References

Ionic

Metallic anions such as mo (vi) or pt (ii), -glycerophosphate

Tripolyphosphate

Draget et. al., 1992 Chenite et. al., 2001

Mi et. al., 1997

Covalent

Dialdehydes,Glyoxal,Glutaraldehyde,

Diethyl Squarate, Oxalic Acid, Genipin, Poly Ethylene Glycol,

Diacrylate, Scleroglucan, Gelatin, Collagen

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, Murata-Kamiya et. al., 1997,

Monteiro and Airoldi, 1999

Polymeric

Polyacrylic acid, Sodium Salt, Carboxymethylcellulose, Xanthan,

Carrageenan, Alginate, Pectin, Heparin, Hyaluronan, Sulphated Cellulose, Dextran Sulfate, N-

Acylated Chitosan/ Chondroitin Sulphate, Sodium Alginate, Kappa-Carrageenan And Polyacrylic Acid

Peniche and Arguelles-Monal, 2001, Arguelles-

Monal 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


55

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.

Review of Literature…

56

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,


57

Tamarind kernel gum

-D-Xylp1

64)--D-Glcp-(1 4)--D-Glcp-(1 4)--D-Glcp-(1

-D-Galp1

2-D-Xylp

1

6

-D-Xylp1

6

Guar gum

4)--D-Manp-(1

-D-Galp1

64)--D-Manp-(1 4)--D-Manp-(1

-D-Galp1

6

Gum Karaya

2)--L-Rhap-(1 4)--D-GalpA-(1

-D-Galp1

22)--L-Rhap-(1 4)--D-GalpA-(1

-D-Galp1

4

-D-GlcpA1

3

Okra gum

4)--D-GalpA-(1

-L-Rhap1

24)--D-Galp-(1 4)--D-GalpA-(1

Gum Cassia

-D-Galp1

64)--D-Manp-(1 4)--D-Manp-(1 4)--D-Manp-(1

Figure 2.22: Chemical structure of some pharmaceutically useful gums.


58

Table 2.19: Classification of polysaccharide gums.

S.No. Basis Class Example 1.

Origin Seed gums Plant exudates Microbial exudates (Fermentation) Sea weed

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) Gellan gum, Xanthan gum, Tara gum (tara tree) , spruce gum (spruce tree) Sodium alginate, Alginic acid

2.

Gelation behaviour

Cold set gels (form gels on cooling the solution ) Heat set gels (form gels on heating the solution) Reentrant gels (from which galactose residues are removed)

Gellan gum Konjac glucomannan Xyloglucan

3.

Chemical structure

Galactomannans Glucomannans Uronic acid containing gums

Fenugreek gum, guar gum, locust bean gum Konjac glucomannan Xanthan gum


59

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, α-

galactosidase 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.


60

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 non-

Newtonian 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.


61

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


62

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


63

release takes place before the dosage form reaches the absorption site (Needleman, &

Smales, 1995). In order to overcome the above drawback, PAA is usually cross-

linked 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.


64

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


65

(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).


66

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


67

A

B

4)--D-Manp-(1 4)--D-Glcp-(1O

O

O

CH2OH

O

OH

HOO

CH2OH

OCH2OH

O

O

OO

CH2OH

OHHO

OOH

HO

HOH2C

O

OH

HOO

CH2OH B(OH)4-

B

Figure 2.26 A) Grafting of konjac glucomannan (KG) with poly acrylic acid and B) complexation with Borax ions (B(OH)4

-).


68

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.

A

B

C


69

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)


70

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


71

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 cross-

linking 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


72

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) Hybrid polymer networks (HPN) (ii) Semi or full inter-penetrating polymer networks (IPN) (iii) Ionically cross-linked gums or their derivatives (reversible cross-linking)

3)--D-Galp-(1 3)--D-Galp-(1 3)--D-Galp-(1 3)--D-Galp-(13)--D-Galp-(1 3)--D-Galp-(1 3)--D-Galp-(1 3)--D-Galp-(1..... .....

-D-Galp

6

-D-Galp

1)--D-Galp

1

3

-D-Galp-(31

6

-D-Galp1

1)--D-Galp-(6 1)--D-Galp3

R

6

-D-Glcp1

-D-Galp-(31

6

-D-Galp1

6

-D-Galp-(31

6

-D-Galp1

6

R

6

-D-Glcp1

6

1

-D-Galp-(6

3

1

-D-Galp

3

1

-D-Galp

6

1

R

-D-Galp

6

1

-D-Galp-(6

3

1

-D-Galp

3

1

-D-Galp

6

1

R

6

R

-D-Galp-(1

-D-Galp-(6

6

1

-D-Galp

3

1

R

O

OH

HO

OHCH2OH

O

O

OH

HO

O

O CH2OR

OHOH

HOCH2O

O

OH

O

OHO

O

OHHO

O

OHO

HO

O

Epichlorohydrin(NaOH, pH 10)

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


73

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 cross-

linker (Berger et al., 2004; Knapczyk, Majewski, Pawlik, & Wisniewska, 1994;

Knapczyk, 1994).

Ionic cross-linking is a simple and mild procedure. In contrast to covalent cross-

linking, 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 cross-


74

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 cross-

linker (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).


75

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 cross-

linking 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.


76

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 relaxation-


77

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, N-

methylene-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


78

Table 2.20: Pharmaceutical applications of gums in drug delivery

S.No. Natural Gum Model Drug Dosage form

Remarks Reference(s)

A. Guar Gum

1. 97.3% Dexamethasone Tablets 72-82% of Dexamethasone was delivered to colon.

Kenyon et al. (1997)

340 mg per 420 mg tablet (77.19%)

Indomethacin 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.

Prasad, Krishnaia, & Satyanarayana (1998)

3. 100 mg coating over 80 mg core tablet (125%)

Indomethacin Tablets In-vitro Cumulative percent drug release with rat caecal medium (2%, 21 h study) was ≤ 20%.

In-vivo Scintigraphy study showed 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)

Krishnaiah, Satyanarayana, Rama Prasad, & Narsimha Rao (1998)

4. 20% Albendazole Matrix tablets

In-vitro drug release without rat caecal medium was 20.9% which was increased upto 43.9% with rat caecal medium (4%)

Krishnaiah, Nageswara Rao, Bhaskar Reddy, Karthikeyan, & Satyanarayana


79

(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 (Sorenson’s buffer pH7.4) was 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

Krishnaiah, & Srinivas (2008)

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%)

Krishnaiah, Muzib, Indira Rao, Srinivasa Bhaskar, & Satyanarayana (2003c)

8. 80% 5 FU Tablets In vitro drug release without rat caecal medium was found to be 50.85%

In-vivo studies in healthy human volunteers showed Cmax (216 ng/ml) at T max (7.6h)

Krishnaiah, Satyanarayana, Dinesh Kumar, Karthikeyan, & Bhaskar (2003a)


80

9. 260 mg compression coating on 200 mg core tablets (130%)

Tinidazole Tablets In vitro drug release was found to be 67.50%

In-vivo studies healthy human volunteers showed Cmax=2158 ng/mL at 14.9h and AUC was found to be 57740 ng/ml/h

Krishnaiah, Bhaskar, Satyanarayana, & Latha (2003b)

10. 50% Calcium Sennosides

Matrix tablets

In-vitro drug release without rat caecal medium was 43%which increased upto 96%With Rat caecal medium (4%)

Momin (2004)

11. 350 mg total coat weight

Mesalazine 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

Demiroz, Acartürk, Sevgi, & Oznur (2004)

12. 60% Rofecoxib 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)

13. 20% Albendazole β-cyclodextrin

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)


81

14. 50% Albendazole and Albendazole β-cyclodextrin

Matrix tablets

In vitro drug release without rat caecal medium was 67.7 %

In-vivo studies healthy human volunteers showed Cmax (916.49 ng/mL) at 12 h

Shyale, Chowdary, Krishnaiah, & Bhat (2006)

15. 150mg coating per 213 mg core tablet (70.42%)

Ondansetron Matrix tablets

In-vitro drug release was found to 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

Demiroz, & Takka (2006)

16. 44% Indomethacin Pellets

(Coatd with

Eudragit FS 30D)

In vitro drug release with GG/ Eudragit FS 30D double coated 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

Ji, Xu, & Wu (2007)

17. 75% Diltiazem Matrix tablets

(coated with

In vitro drug release for uncoated GG formulated tablets when unincubated SCF was used was found to be 60% at end of

Ravi, Mishra, & Kumar (2008)


82

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

B. Guar gum methacrylate conjugates

18. Graft copolymer of GG with acrylamide by crosslinking with glutaraldehyde

(5% W/V)

Verapamil (VRP) and Nifedipine(NFD)

Hydrogel Microspheres

Non-fickion drug release was observed

Soppirnath, & Aminabhavi, (2002)

19. Methacrylic acid-g-Guar gum

(MAA-g-GG)

Metronidazole Tablets In vitro drug release with Eudragit L 100 coated tablets (with MAA-g-GG: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

Mundargi, Patil, Agnihotri, & Aminabhavi (2007)

C. Guar gum-alginate conjugates

20. Guar gum-Alginate combination cross linked with glutaraldehyde

BSA Hydrogels During one step drug release studies (Separate release studies at pH 1.2 and 7.4 for 10 h) only 20 % BSA released after 10 h at pH 1.2. While two step drug release studies (pH 1.2 for 2 h followed by study

George, & Abraham (2007)


83

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, Sodium salicylate and Budesonide

Discs 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.

Kabir, Yagen, Penhasi, & Rubinstein (1998)

22. Metronidazole Microsphere In vitro drug release without rat caecal medium was 31.23% which 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.

Chourasia, & Jain (2004)

23. Methotraxate Microsphere In vitro drug release Without rat caecal medium was 38.9% which was icreased with Rat ceacal medium(6%) upto 73.20% which further increased up to 91% after enzyme induction

Chourasia et al. (2006)

24. Ibuprofen Hydrogel Discs

Cumulative percent in vitro drug release( 2 h SGF , 3 H SIF) was found to be 2-5%

Das, Wadhwa, & Srivastava (2006)


84

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)

E. Xanthan gum

26. Caffeine,

Indomethacin,Sodium Indomethacin

Matrix tablets

Within physiologic ionic strength range the swelling of XG matrix 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.

Talukdar, & Kinget (1995)

F. Xanthan gum derivatives

27. Combination with Konjac glucomannan,KGM

(20%TWG)

Cimetidine

Matrix tablets

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.

Jiangyang, Wang, Liu, & He (2008)

28. XG:GG 5-FU Matrix In-vitro drug release was found to be 42.6% which was increased

Sinha, Mittal, Bhutani, &


85

(10:20) tablets upto 67.2% with 2% and 80.34% with 4% rat caecal medium.

Kumaria (2004)

29. Combination with Konjac glucomannan (both American and Japanese varities)

Diltiazem Tablets In vitro drug releasewith Japanese KGM drug release was complete within 24 h in the presence of β-mannase. There was a smaller effect on release from formulation of American KGM.

Manceñido, Landin, Lacik, & Pacheco (2008)

30. Xanthum gum:Boswellia gum (3:1) with 300 mg total coat weight AND Boswellia gum:HPMC (2:3) with 275 mg total coat weight

5-FU Compressed coated tablets

In-vitro drug release was found to be 80.2% which was increased upto 98.22% with 2% rat caecal medium

Studies also showed that XG play a major role in retardation of drug release

Sinha, Singh, Singh, & Binge (2007)

G. Khaya gum and albizia gum

31. Khaya gum (300mg) and Albizia gum (400mg)

Paracetamol and Indomethacin

Tablets In vitro drug release (After 12 h) of coated with khaya gum(400 mg) was 33.09% for Indomethacin and 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

Odeku, & Fell,

(2005)


86

showed 94.34% drug release

Tablets coated with Khaya:Albizia(1:1) mixture(400mg) showed 97.34% drug release

H. Crosslinked gellam gum

32. A.(I)With Calcium or Deacylated gellam gum crosslinked with calcium

Azathiopurine

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)

I. Locust bean gum

33. Locust bean gum:Chitosan

(4:1)

Mesalazine 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)


87

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 AG-

MA 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


88

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 cross-

linked 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 β-

mannanase. 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.


89

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 cross-

linked 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.


90

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 Tmax, absorption


91

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


92

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


93

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).


94

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


95

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 odor

White to practically white, odourless, crystalline powder

4. Melting point 282 °C to 283 °C

5. Log P

(n-octanol/ water)

-2.987

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


96

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


97

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


98

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 1,2-

dimethylhydrazine (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


99

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.

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