Chapter: One

Introduction and aim of the thesis

Stomach ulcer

Zollinger- -
Ellison tumor
in pancreas

Duodenal-
ulcers due to
hyperacidity

Duodenal ulcer

5
1.1.0 Introduction
Nonsteroidal anti-inflammatory drugs (NSAIDs) are one of the most frequently
used medications worldwide to treat pain, fever and chronic inflammatory diseases, such
as arthritis. NSAIDs exert their therapeutic activity by inhibiting cyclooxygenase-derived
prostaglandin synthesis, but this mechanism of action is inherently responsible for the
gastrointestinal (GI,1'5 renal,6'8 hepatic9 side effects observed in patients undergoing a

long-term treatment. The most common side effects associated with NSAID therapy are
upper GI irritation, ulceration, dyspepsia, bleeding, and in some cases death.10 It is clear
that NSAID-induced toxicity is a serious public-health problem contributing significantly
to morbidity and mortality.

All NSAIDs are believed to inhibit the biosynthesis of prostaglandins by

inhibiting the group of enzymes called cyclooxygenases (COX).11 In early 1990’s, two

isoforms of COX were discovered, a constitutive COX-1 and inducible COX-2. The
COX-1 enzyme is located in normal tissues and cytoprotective, physiologically important
for GI and renal functions. They promote inflammation, pain, and fever, support blood
clotting function of platelets, and protect the lining of the stomach from the damaging
effects of acid. On the other hand COX-2 is pathological, found primarily in inflamed
tissues,12'15 Thus, non-selective COX inhibitors cause inhibition of both the isoforms,

producing GI and renal side effects due to inhibition of COX-1. While selective
inhibition of COX-2 could block the prostaglandin production at the site of inflammation
without affecting the beneficial prostaglandin in normal tissues such as stomach and
kidney.16'20 This led to the development of selective COX-2 inhibitors with improved
pharmacological profile mid reduced gastric toxicity.21 But selective COX-2 inhibitors,
Rofecoxib mid Celecoxib were withdrawn from the market (Figure 1) due to its
cardiovascular side effects of chronic use.19

The cydo-oxygenase pathway

The normal process begins with arachidonic acid, a dietary unsaturated fatty acid
obtained from animal fats. This acid is converted by the enzyme cyclooxygenase to
synthesize different prostaglandins.

6
 ,0

F
’F

Rofecoxib Celecoxib

Figure 1: Structures of Rofecoxib and Celecoxib

COX-1 enzyme is constitutive, means its concentration in the body remains stable. It is
present in most tissues and converts arachidonic acid into prostaglandins. These
prostaglandins in turn stimulate normal body functions, such as stomach mucus
production and kidney water excretion, as well as platelet formation. The location of the
COX-1 enzyme dictates the function of the prostaglandins it releases, i.e. COX-1 in the
stomach wall produces prostaglandins that regulate gastric acid secretion (stimulate
mucous production). Thus COX-1 is important for the production of prostaglandins of
homeostatic maintenance, such as platelet aggregation, the regulation of blood flow in the
kidney and stomach, and the regulation of gastric acid secretion response.

Figure 2: The cyclo-oxygenase pathway

7
Pathogenesis of NSAID-induced gastroduodenal mucosal injury

Inhibition of COX-1 activity is considered a major contributor to NSAID GI toxicity. In

contrast, the COX-2 enzyme is inducible isoenzyme. It is not normally present in cells
but its expression can be increased dramatically by the action of macrophages, the
scavenger cells of the immune system. The COX-2 plays most important role in pain and
inflammatory processes since it involved in producing prostaglandins for an
inflammatory. Clinical use of most of the available acidic NSAIDs is strongly limited by
their GI side effects which range in both severity and frequency from relatively mild to
more serious and potentially life threatening states, such as GI ulceration and
haemorrhage.22 It is a well accepted fact that the GI side effect of acidic NSAIDs is a
result of two different mechanisms.23'26

a) Local effect on GI tract

The first mechanism involves a local action comprising of a direct contact effect and an
indirect effect on the GI mucosa.23'26 The direct effect can be attributed to the local

inhibition of prostaglandin (PG) synthesis in the GI tract The indirect effect can be
attributed to a combination of an ion-trapping mechanism of NSAIDs in mucosal cells
and back diffusion of H1" ions from the lumen into the mucosa. Topical irritation by the
free carboxylic group of die NSAIDs is considered an important factor in establishing
superficial stomach erosion, particularly in the corpus region of the stomach.

b) Systemic effects

The second mechanism is based on the generalized systemic action occurring after
absorption and can be manifested even after intravenous dosing.26"27 The systematic

effects are manifested due to inhibition of synthesis of gastric prostaglandins like PGL
and PGE2.

Considerable attention has been focused on the development of bioreversible

derivatives, such as prodrugs, to temporarily mask the acidic group of NSAIDs as a
promising means of reducing or abolishing the GI toxicity due to the local action
mechanism. Prodrugs are pharmacologically inactive derivatives of active agents, which
undergo chemical and/or enzymatic biotransformation resulting in the release of active

B
drug after administration. The metabolic product (i.e. parent drug) subsequently elicits
the desired pharmacological response.28"29

Most of NSAID prodrugs have been prepared by derivatization of carboxyl group.

The esters have been dominated prodrug research because they have the ideal
characteristic of exhibiting reasonable in vitro chemical stability which allows them to be
formulated with adequate shelf lives. In addition, by virtue of their ability to fimction as
esterases, esters are suitably labile, in vrvo.30-31

The prodrug is able to overcome one or more of the barriers to drug delivery more
efficiently thantbe parent (hug (Figure 2).
These barriers include:
i) physicochemical properties
ii) pharmacokinetic properties
I) Physicochemical properties
a) Poor aqueous solubility; Prevents the drug horn being administered in the form of
injectables. Gives rise to dissolution rate-limited (and variable) oral bioavailability
b) Low lipophilicity: Limits the design of lipid-based formulations
c) Chemical instability: Prevents the drug from being incorporated into adequate dosage
forms

Figure 2: An Illustration ofthe Prodrug Concept

9
ii) Pharmacokinetic properties
a) Incomplete absorption across biological membranes (gastrointestinal mucosa and
blood-brain barrier)
b) Unfavourable metabolism
c) Low and variable bioavailability (extensive first-pass metabolism).

1.2.0 Aim of the thesis

Nonsteroidal anti-inflammatory drags (NSAIDs) have been widely used for

management of inflammation, pain and nociception. Gastric intolerance caused by most
of the NSAIDs used today restricts them for long-term therapeutic use. This side effect
produced by NSAIDs (COX-I inhibitors) are believed to involved by two different
mechanism: inhibition of prostaglandin synthesis in stomach, responsible for inducing
mucus production and a local action exerted by direct contact of the drags with gastric
mucosa due to the acidic nature of the NSAIDs.32 Several approaches have been proposed
to modify the parent NSAIDs molecule in order to reduce their gastric toxicity. The use
of latentiation as molecular modification strategy provides NSAID prodrags with
improved safety profiles.33'34 The prodrag approach afforded compounds with better anti­

inflammatory, differentiated pharmacokinetic profile and reduced gastric ulcerogenic

activity.35'39 Using the prodrag approach, one strategy that could be useful is to

temporarily mask the carboxylic function of the NSAIDs and the prodrag hydrolyzes in
vivo to release the active parent NSABD.40'42

The aim of the thesis was to synthesize novel ester prodrugs of nonsteroidal
anti-inflammatory drugs (NSAIDs) and evaluate their ulcerogenic, and anti­
inflammatory properties, enzymatic bioconversion rate, and physicochemical
properties in comparison with pareant drug.

Another aim of the thesis was to synthesize novel ester prodrugs of fenofibric
acid and bezafibrate, and evaluate their hypolipidemic activity (anticholesterol
activity) in comparison with parent drug.

10
the gastric mucosa should be reduced if at all possible. There are different prodrug
approaches for reduction of GI side effects and ulcerogenicity of NSAIDs

i) Esters and Amide prodrugs ofNSAIDs,

ii) Anhydride prodrugs of NSAIDs,
iii) Mutual prodrugs of NSAIDs, and
iv) Nitric oxide releasing prodrugs

Ester prodrugs are most often used to enhance the lipophilicity, and thus the passive
membrane permeability of water soluble drugs by masking charged group such as
carboxylic acids and phosphates. The ester bond is readily hydrolyzed in body by enzyme
esterases found in the blood, liver, and other organs and tissues.

The use of prodrugs has provided some optimism to reduce adverse effects. For
example, prodrugs such as nabumetone and etodolac confer added gastric mucosal
protection by not significantly inhibiting gastric prostaglandin synthesis. Post marketing
surveillance data and short term endoscopic studies indicate that the incidence of
gastroduodenal erosive injury is lower (< 1%) with both of these agents.46

Based on information, we decided to synthesize novel ester prodrugs of NSAIDs

which are currently on the market and to evaluate their physicochemical, anti­
inflammatory and pharmacokinetic properties. The objective of the invention is to
minimize the gastrointestinal (GI) tract side effects associated with orally administered
NSAIDs, to prevent the localization of NSAIDs in the gastric mucosa with maintaining
anti-inflammatory properties of parent drugs.

b) Anti-hyperlipoproteinemic prodrugs (fibrates)

2-Methyl-2-phenoxypropionic acid (fibric acid) derivatives are called fibrate4749

and clinically useful in hyperlipidemic patients who shows middle to high level of

triglyceridemia and cholesterol in the blood. Fibrates are frequently prescribed to reduce

triglycerides, increase good cholesterol (HDL) and reduce bad cholesterol (LDL). The

12
1.3.0 Background of the invention
Research work was carried out on two therapeutic areas i.e. nonsteroidal anti­
inflammatory and anti-cholesterol drugs.
i

a) Non-steroidal antiinflammatory drugs (NSAIDs)

Non-steroidal anti-inflammatory drugs (NSAIDs) are prescribed extensively
throughout the world for the treatment of pain, fever and inflammation as a result of acute
injuries, rheumatoid arthritis, and osteoarthritis.43-44 Most commonly used NSAIDs are
aspirin, indomethacin, diclofenac sodium, ibuprofen, sulindac, and peroxicam. The
NSAIDs are generally broken down into following chemical classes.

Aryl acetic acids i.e. indomethacin,

Aryl propionic acids i.e. such as ibuprofen
Salicylates i.e. aspirin,
P-Aminophenols i.e. acetaminophen,
Fenamic acids i.e. mefenamic Acid,
Oxicams i.e. piroxicam, and
Pyrazolidinediones i.e. phenylbutazone.

A significant percentage of patients taking NSAIDs were reported some type of

adverse gastrointestinal effects, ranging from dyspepsia to generalized abdominal
discomfort. In a minority of users, severe complications, including gastric and duodenal
ulcerations, gastrointestinal bleeding or perforation, and mucosal injury to either the
small or large intestine, develop. The organs most commonly affected by ulceration in
NSAID users are the stomach (12 to 30%) and duodenum (2 to 19%), though there is
some risk of injury to the esophagus, small bowel, and colon,45

In the presence of gastric acid, weak acid NSAIDs such as indomethacin and
ketoprofen diffuse freely across the gastric mucosal barrier and become ionized and
sequestered in the mucosal cells, an occurrence that leads to cytotoxicity. NSAIDs cause
local damage through inhibition of cyclooxygenase, and may also exert a direct toxic
effect upon the mucosal cells. Thus to reduce ulcerogenicity, localization of NSAIDs in

II
reductions in triglycerides are generally in the range of 30 to 50 percent. Several fibrates

are available on the market i.e. bezafibrate, clofibrate, ciprofibrate, gemfibrozil,

fenofibrate etc. We have synthesized prodrug of commonly prescribed fenofibrate and

bezafibrate and evaluated in-vivo and in-vitro for their physicochemical properties in

comparison with reference compounds i.e. fenofibrate and bezafibrate (Figure 3).

Figure 3: Chemical structure of fenofibric acid (1), fenofibrate (2) and bezafibrate (3)

Fenofibrate is a prodrug major metabolite of fenofibrate in plasma is fenofibric acid,

an active species which has an elimination half-life of approximately twenty hours.
Fenofibrate is usually orally administered with maximum dose 120 mg per day for patient
suffering from hypertriglyceridemia. A patient is often instructed to take the drug at meal
times or with food. Fenofibrate is nearly insoluble in water and requires special
pharmaceutical formulation comprising a micronized drug in order to ensure good bio­
availability, especially after oral administration. Accordingly fenofibrate has been
prepared in several different formulations. It is poorly and variably absorbed when taken
with food. After oral administration, during a meal, about 60% of the dose of the
conventional form is effectively absorbed and found in the blood as fenofibric acid. The
main drawback of lipid lowering agent fenofibrate is low bioavailability when taken
orally on an empty stomach and requires micronization to improve bioavailability.
The main objective of the invention was to synthesize fenofibric acid ester
prodrugs and bezafibrate ester prodrugs, and evaluate their hypolipedemic
activities in comparison with the reference drugs which are currently prescribed as
hypoplipidemic agents.

13
References

(1) Cryer, B. Am. J Gastroenterol. 2005,100,1694-1695.

(2) Go, M. F. Gastrointest. Endosc. Clin. N. Am. 2006,16, 83-97.
(3) James, M. W.; Hawkey, C. J. Br. J. Clin. Pharmacol. 2003,56,146-155.
(4) Lazzoroni, M.; Porro, G. B. Aliment. Pharmacol. Ther. 2004,20 (2), 48-58.
(5) Naesdal, J.; Brown, K. Drug Saf. 2006,29,119-132.
(6) Mounier, G.; Guy, C.; Berthoux, F.; Beyens, M. N.; Ratrema, M.; Ollagnier, M.
Therapie 2006, 61, 255-266.
(7) Schneider, V.; Levesque, L. E.; Zhang, B.; Hutchinson, T.; Brophy, J. M. Am. J.
Epidemiol. 2006,164, 881-889.
(8) Zadrazil, J. Vnitr. Lek. 2006,52,686-690.
(9) Adebayo, D.; Bjamason, I. Postgrad. Med. J. 2006,82,186-191.
(10) Thomsen, R. W.; Riis, A.; Munk, E.M.; Norgard, M.; Christensen, S.; Sorensen,
H. T. Am. J. Gastroenterol. 2006,101,2704-2710.
(11) Allison, M. C.; Howatson, A. G.; Torrance, C. J.; Lee, F. D.; Russell, R. I. G. N.
Engl. J. Med. 1992,327,749-754.
(12) Hla, T.; Nelson K. Proc. Natl. Acad. Sci. U.S.A 1992,89,7384-7388.
(13) Xie, W,; Chipman, J. G.; Robertson, D. L.; Erikson, R. L.; Simmons, D. L.
Proc. Natl. Acad. Sci. USA. 1991,88,2692-2696.
(14) Kujubu, D. A.; Fletcher, B. S.; Vamum, B. C.; Lim, R. W.; Herschman, H. R. J.
Biol. Chem. 1991,266,12866-12872.
(15) Masferrer, J. L.; Siebert, K.; Zweifel, B.; Needleman, P. Proc. Natl. Acad. Sci.
U.S.A. 1992,88,3917-3921.
(16) Meade, E. A.; Smith, W. L.; Dewitt, D. L.Drug Dev. Res. 1992,25,249-265.
(17) Xie, W.; Robertson, D. L.; Simmons, D. L. Drug Dev. Res. 1992, 733, 263.
(18) Cao, C.; Matsumara, K.; Yamagata, K.; Wantanabe, Y. Brain Res. 1996, 733,
263-272.
(19) Reuter, B. K.; Asfaha, S.; Buret, A.; Sharkey, K. A.; Wallace, J. L. J. Clin.
Invest. 1996,98,2076-2085.
(20) Warner, T. D.; Giuliano, F.; Vojnovic, I.; Bukada, A.; Mitchell, J. A.; Vane, J.
R. Proc. Natl. Acad. Sci. U.S.A. 1999,96, 7563-7568.

14
(21) Roth, R. N.; Weiss, L. D. Clin. Dermatol 1994,12,141-156.
(22) Champion, G. D.; Feng, P. H.; Azuma, T.; Caughey, D. E.; Chan, K. H.;
Kashiwazaki, S.; Liu, H. C.; Nasution, A. R.; Hobunaga, M.; Prichanond, S.;
Torralba, T. P.; Udom, V.; Yoo, M. C. Drugs 1997,53, 61-69.
(23) Allan, H. P.; Fletcher, M. Drug 1990,40,1-11.
(24) Schoen, R. T.; Vender, R. J. Am. J Med. 1989,86,449-458.
(25) Mitchell, J. A.; Warner, T. D. Br. J Pharmacol 1999,128,1121-1132.
(26) Wallace, J. L.; Cirino, G. Trends Pharmacol. Sci. 1994,15,405-406.
(27) Wallace, J. L.; Keenan, C. M.; Granger, D. N. Am. J. Physio. Gastrointest. Liver
Physiol. 1990,259, G462-G467.
(28) Alert, A. Nature 1958,182,421-423.
(29) Bundgaard, H. Drug Future 1991,16,443-458.
(30) Bundgaard, H. Adv, Drug. Rev. 1989,3,39-65.
(31) Bundgaard, H. In: Design ofProdrugs', Ed; Elsevier, New York, Plenum Press
1986,49-68.
(32) Bandarage, U. K.; Chen, L.; Fang, X.; Garvey, D. S.; Glavin, A.; Janero, D. R.;
Letts, L. G.; Mercer, G. J.; Saha, J. K.; Schroeder, J. D.; Shumway, M. J.; Tam, S.
W. J. Med. Chem. 2000,43,4005-4016.
(33) Wermuth, C. G. J. Med. Chem. 2004,47, 1303-1314.
(34) Silva, A. T. A.; Chung, M. C.; Castro, L. F.; Guido, R. V.; Ferreira, E. I. Min
Rev. Med. Chem. 2005,10, 893-914.
(35) Zhao, X.; Tao, X.; Wei, D.; Song, Q. Eur. J. Med. Chem. 2006,41,1352-1358.
(36) Shanbhag, V. R.; Crider, A. M.; Gokhale, R.; Harpalani, A.; Dick, R. M. J.
Pharma Sci. 1992,81,149-154.
(37) Kumakura, S.; Mishima, M.; Kobayashi, S.; Shirota, H.; Abe, S.; Yamada, K.;
Tsurufuji, S. Agent. Action. 1990,29,286-291.
(38) Ribeiro, L.; Silva, N.; Hey, J.; Rautio, J.; Jarvinene, T.; Mota-Filipe, H.;
Moreira, R.; Mendes, E. Arch. Pharm. 2007,340,32-40.
(39) Halen, P.; Kuldeep, K.; Chagti, K. K.; Giridhar, R.; Yadav, M. R. Chem. Biodiv.
2006,3, 1238-1248.
(40) Wallace, J. L. Can. J. Physiol. Pharmacol. 1994, 72,1493-1498.

15
(41) Kim, H.; Jeon, H.; Kong, H.; Yang, Y.; Choi, B.; Kim, Y. M.; Neckers, L,;
Jung, Y. Mol. Pharmacol. 2006, 69, 1405-1412.
(42) Gairola, N.; Nagpal, D.; Dhaneshwar, S. S.; Dhaneshwar, S. R.; Chaturvedi, S.
C. Indian J Pharm. Sci. 2005,67,369-373.
(43) Loeb, D. S.; Talley, N.J.; Ahlquist, D. A. Gastroenterology 1992,102 (6),
1899-1905.
(44) Zeidler, H. J. Rheumato. 1991, (Suppl 28), 2-5.
(45) Geis, G. S.; Stead, H.; Wallemark, C. B. J Rheumatol 1991, (Suppl 28). 11-14.
(46) Cummings, D. M.; Amadio, P. Jr. Am. Fam. Physician 1994,49(5), 1197-1202.
(47) Alegret, M.; Ferrando, R.; Vazquez, M.; Adzet, T.; Merlos, M.; Laguna J, C. Br.
J. Pharmaco. 1994,112, 551-556.
(48) Haubenwallner, S.; Essenburg, A. D.; Barnett, B. J.; Newton, R. S.; Leff, T.;
Bisgaier, C. L. J. Lipid Res. 1995,36,2541-2551.
(49) Miller, D. B.; Spence, J. D. Clin. PharmacoUnet. 1998,34,155-162.

16