12 - Chapter 5 Syn

Chapter: Five
Biological Activity of Non-steroidal Anti-inflammatory & Anti-

Cholesterol Prodrugs
Section 1: Biological Activity of Non-steroidal Anti
inflammatory Prodrugs
1E7
5.1.0 Introduction to inflammation
Inflammation is part of the complex biological response of vascular tissues to

harmful stimuli, such as pathogens, damaged cells, or irritants. It is characterized by
blood flow to the tissue causing, increased temperature, redness, swelling and pain. It is a
protective attempt by the organism to remove the injurious stimuli as well as initiate the
healing process. Inflammation is classified as acute inflammation and chronic
inflammation. In this process body’s white blood cells and chemicals protect us from
infection and foreign substances such as bacteria and viruses. In some diseases however
the body defense system (immune system) inappropriately triggers an inflammatory
response when there are no foreign substances to fight off. In these diseases called
autoimmune diseases, the body’s normally protective immune system causes damage to
its own tissues. The body responds as if normal tissues are infected or somehow abnormal
e.g. asthma, rheumatoid arthritis, multiple sclerosis, chronic obstructive pulmonary
disease, transplant rejection etc. Inflammatory diseases are dignosed after careful
evaluation of complete medical history and physical examination, the location of painful
joints, joint stiffness in the morning, evaluation of other symptons and results of X- ray.
There are number of treatment options for inflammatory diseases including medication,
rest and exercise and surgery to correct the joint damage. The type of treatment
prescribed will depend on several factors including the type of disease, the patient’s age,
type of medication, overall health, medical history and severity of symptoms. The goal of
treatment as to avoid or modify activities that aggravate, relieve pain through analgesics
and anti-inflammatory medications. There are many medications are available for
inflammatory diseases to reduce joint pain, swelling and inflammation and hopefully
prevent or minimize the progression of the diseases. The medications include:
1) Non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin, ibuprofen,

diclofenac sodium, ketorolac tromethamine, flurbiprofen, ketoprofen etc.
2) Corticosteroids such as glucocortisone, prednisone.
3) Other medications include methrotrexate, sulfasalazine and anti-TNF medications.
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5.2.0 Test methods
5.2.1 Carrageenan-induced Paw Edema in rat (Anti-inflammatory assay)
Carrageenan are a complex group of polysaccharides made up of repeating galactose
related monomers and are three main types i.e. lambda, kappa, and iota. Each has their
own characteristics which are all thermally reversible. The lambda form does not get gel
strongly at room temperature and is injectable to induce an inflammatory response.
Inflammation induced by carrageenan, originally described by Winter,1 is acute,
nonimmune, well researched, and highly reproducible.

Carrageenan induced paw edema is a standard and most commonly used technique to
screen the anti-inflammatory activity. It expected that after tissue injury an animal will
display spontaneous pain behaviour. This peripheral hypersensitivity or pain perception
can be explained on the basis of local release various inflammatory mediators i.e.
bradykinin, prostaglandins or cytokines which can activate and sensitize the peripheral
nerve endings. This phenomenon can be evaluated in laboratory by carrageenan induced
paw edema. The inflammatory response is usually quantified by increase in paw size
(edema) which is maximal around 5 h post carrageenan injection and modulated by
inhibitors of specific molecules within the inflammatory cascade.
Paw swelling, or footpad edema, is a convenient method for assessing inflammatory
responses to antigenic challenges and irritants. The protocol described here uses
carrageenan as the irritant to induce paw edema. Typically, test compounds are assessed
for acute anti-inflammatory activity by examining their ability to reduce or prevent the
development of carrageenan-induced paw swelling. This model has long been used to
assess the anti-inflammatory properties of agents such as nonsteroidal anti-inflammatory
drugs (NSAIDs) that inhibit prostaglandin production.
5.2.2 Metabolic stability
Metabolic stability is an important property of drug candidate since it affects parameters
such as clearance, half life and bioavailability. Thus, knowledge of metabolic stability is
essential both in drug discovery and development program. By using species specific
liver microsomes or plasma (i.e. rat, human), stability assays give essential information
on a compound’s potential pharmacodynamic properties. Metabolic stability study
indicates, how long drug compound remain circulating in plasma within body. A
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successful prodrug candidate is expected to undergo rapid, complete conversion to parent
compound in the plasma or microsomes within 1-3 h.
Microsomal stability
Drugs are most often eliminated by biotransformation and/or excretion into urine or bile.
The liver is the major organ for xenobiotic biotransformation and is thereby important in
characterizing the metabolism stability, toxicology, and drug-drug interaction properties
of drugs. Drug metabolism is achieved via two major enzyme reactions within the liver,
Phase I and Phase II reactions.
Phase I enzymes include the cytochrome P450 (CYP) family of enzymes which are
located in the smooth endoplasmic reticulum. The basic processes in phase I reaction are
oxidation, reduction and/or hydrolysis many of which are catalysed by the CYP system
and require NADPH as a cofactor. Phase II enzymes are located in the cytoplasm and
endoplasmic reticulum and characteristic of conjugation reactions including glucaronic
acid, glutathione, sulfate, and glutamine conjugations. Phase II reactions generally
inactivate the drug if it is not alrerady therapeutically inactive following Phase I
metabolism, and make the drug more water soluble to facilitate its elimination. Some
drugs are metabolized by Phase I and Phase II enzyme alone whereas others are
metabolized by both Phase I and Phase II enzymes.
Microsome assay uses subcellular fractions of liver called microsomes to investigate
the metabolic fate of compounds. Liver microsomes consist mainly of endoplasmic
reticulum, and contain many drug metabolozing enzymes including cytochrome P 450s
(CYPs), flavin monoxygenases (FMO), carboxylesterases, and epoxide hydrolase.
Compounds (IpM final concentration) are incubated with rat liver microsomes in
potassium phosphate buffer. The microsomal protein concentration in the assay is 1
mg/mL and the final percent DMSO is 0.2%. Reaction is started by the addition of
NADPH and stopped either immediately or after 15 min for screening assay or at 5, 15,
30 and 60 min for a more precise estimate of clearance. The corresponding loss of
compound is deterimed by HPLC.
Data analysis: percentage remaining of compound is calculated compared to the initial
quantity at time zero.
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Plasma stability
The quantitative analysis of drags in plasma stability assay is a well accepted surrogate
marker for drag discovery and develop groups. Assay addresses the stability of drag in
plasma. The stability of drags in plasma is an important parameter, which not only affects
in vivo results, but also the bioanalytical assay strategy and design. Normally plasma
stability of new drags is performed early in the discovery process in order to assess
potential degradation and/or protein binding issues.
Method
A solution of test compound in plasma is prepared and incubated for a predetermined
time period. Aliquots are removed at predefined time points and analysed by HPLC. The
peak area for die drag compound is compared to the time zero sample in order to assess
the amount of compound still available. To determine plasma stability, test compounds
are incubated in rat plasma at 37 °C. Aliqots of samples are rmoved at various time points
followed by proteins precipitation. Percentage remaining of compound is determined by
HPLC in respect to the amount of compound at time zero.
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5.3.0 Biological evaluation of mdomethacin prodrugs
Carrageenan-induced Paw Edema in rat
Test method: The test compounds (2,5,6, 8) as well as the reference drug indomethacin
(1) were evaluated by using the in-vivo rat carrageenan-induced foot paw edema model
reported previously.1
Chemicals: Carrageenan was purchased from Sigma (St. Louis, MO, USA).
Indomethacin (EP-grade) is gifted by R & D division of Orchid Chemicals and
pharmaceutical Ltd and prodrugs were synthesized in the laboratory.
Animals: Male Wistar rats weighing 150-200 g were used for the study. All the
experiments were approved by the Institutional Animal Ethics Committee (IAEC) and
complied with the NIH guidelines on handling of experimental animals. The animals
were housed in a group of 3 rats per cage under well-controlled conditions of temperature
(22 ± 2°C), humidity (55 ± 5%) and 12 h / 12 h light-dark cycle. Animals had free access
to diet purchased from vet care and water ad libitum.
Paw edema was induced with carrageenan 1% (0.1 mL) administered as a single
subplantar injection under light ether anesthesia in left paw. Animals were divided in
groups, namely normal control, carrageenan and carrageenan treated with indomethacin
and prodrug (100 mg/kg, p.o.) 1 h prior to carrageenan administration. Paw volume was
measured using the plethysmograph 3 h after the carrageenan administration. Percentage
of inhibition was calculated by following equation.
Carrageenan - Test compound
% Inhibition = ------------------------------------------- x 100
Carrageenan - Normal
Results and discussion

The anti-inflammatory activity of prodrugs 2, 5, 6, and 8 was evaluated by using the in-
vivo rat carrageenan induced paw edema method when administered orally to rats.
Prodrugs 2, 5 and 6 were found potent anti-inflammatory compounds and produced a
significant anti-inflammatory effect (Table 1).
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Table 1: Anti-inflammatory activities of prodrugs (2,5,6,8) and indomethacin (1)
groups dose (mg/kg) paw volume (mL) % inhibition at 180 min

normal — 1.88 ±0.04 -
carrageenan
— 3.12±0.04a -
(1%) 0.1 mL
1 100 2.52 ± 0.06b 48.39%
2 100 2.52 ± 0.08c 48.39%
5 100 2.56±0.18c 45.16%
6 100 2.58 ± 0.09° 44.35%
8 100 2.96 ±0.08 12.10%
On ^ r\f\ i _____ 7 tn .
aP < 0.001, compared to normal control; bP < 0.001, compared to carrageenan control;
CP < 0.01, compared to carrageenan control.
Acute ulcerogenic assay

Test method
The test compounds (2, 5, 6, 8) as well as the reference drug indomethacin (1) were
evaluated for ulcerogenic side effect by using distension ulcer model reported
previously,2
Animals (n= 6 / group) were kept for fasting for 18 h with free access to water before
administration of test compounds. Ulcerogenic activity was evaluated after 100 mg/kg
single dose oral administration of indomethacin, and prodrugs 2, 5, 6, and 8. Animals
were sacrificed under ether anesthesia after 6 h dosing drug compounds, stomach was
removed, opened along the greater curvature, washed and mounted on thermostat sheet
and examined for ulcers. Ulcerative lesions were scored as follows.
• Length of all lesions was measured using Vernier caliper.
• The ulcers are classified as level I, ulcer area < 1 mm diameter; level -II, ulcer area
1-3 mm diameter; level-III, ulcer area > 3 mm diameter
• Ulcerative Lesion Index (UI): as 1 * (number of ulcer level I) + 2 x (number of ulcer
level II) + 3 x (number of ulcer level III). Mean value of six animal readings was
reported as ulcer index (UI).
Prolonged administration of NSAIDs exhibit several undesired side effects. The most
important side effects are gastro-intestinal irritation and ulceration which represent still
an unsolved therapeutic problem. With the aim of minimizing the ulcerogenicity, a series
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of indomethacin ester prodrugs (2, 5, 6, 8) were synthesized and evaluated. The potential
ulcerogenic effects of 2, 5, 6, and 8 were determined after single dose administration in
rats (100 mg/kg dose) and compared to those produced by the parent compound
indomethacin (1) with identical conditions. Lesser degree of ulcers was observed in
animals treated with prodrugs 2, 5, 6, 8 compared to parent compound indomethacin
(Table 1).
Table 1: Ulcerogenic effect of indomethacin (1) and prodrugs (2,5,6,8) in rats
compound number of level I level II level III

ulcers® (< 1 mm) (1-3 mm) (> 3 mm)
1 41 37 (90.2 %) 2 (4.9%) 2(4.9%)
2 19 19 (100.0 %) 0 0
5 51 44 (86.3 %) 3 (5.9 %) 4 (7.8 %)
6 0 0 0 0
8 0 0 0 0
The results were obtained with an average ofsix animals analyzedper group (n = 6)
Animals in each group (n = 6) was treated with 1, developed an average of 41

ulcerogenic lesions with single dose administration. Around 4.9 % of the lesions were
considered to be large (higher than 3 mm diameter). This allowed to the classification of
lesions, which was scored depending upon the severity of mucosal damage. Prodrug 2 on
single dose administration, led to the development of only 19 small lesions (< 1 mm
diameter) and 5 on single dose administration, led to the development of around 51
lesions with level I (86.3 %), level II (5.9 %) and level HI (7.8 %). However 6 and 8
developed no ulcerogenic lesions on identical dose. These findings proved that masking
of carboxylic function of indomethacin in prodrugs 2, 6 and 8 successfully decreases the
gastro-ulcerogenicity. All the prodrugs (except 5) showed an improved safety profile than
the parent reference compound (Table 2).
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Table 2: Ulcer index for compounds (2,5,6,8) and indomethacin (1)
groups dose (mg/kg) ulcer index (UI)a

Normal — NIL
1 100 47
2 100 19
5 100 62
6 100 0
8 100 0
“Ulcer Index (UI) is calculated based on the lesions developed in stomach on single dose
administration in rats in each group (n-6). Data are presented as mean ± SEMat 6 h
after oral administration ofthe test compound.
The prodrug 5 (UI = 62) produced more detectable lesions on the gastric mucosa in
the group of animals examined, 2 (UI = 19) produced less detectable lesions, and 6, 8
showed no visible lesions. This represents the prodrugs 2, 6, and 8 were found
significantly less irritating compounds to gastric mucosa than 1 (UI = 47).
Metabolic stability of indomethacin and indomethacin prodrugs (In-vitro)
Indomethacin ester prodrugs were synthesized with aim of obtaining enzymatically labile.
Metabolic stability is an important property of drug candidate since it affects parameters
such as clearance, half life and bioavailability. A successful prodrug candidate is
expected to undergo rapid, complete conversion to parent compound in the plasma or
microsomes within 1-3 h. Based on in-vivo study in rats, prodrugs 2 and 6 were selected
for metabolic stability study in rat liver microsomes and rat plasma.
Procedure for metabolic stability in rat plasma
Rat plasma was harvested from in-house rats. Fresh blood was collected from the male
rat using retro orbital bleeding method in the tube containing heparin (100 lU/mL
blood). After the collection of blood, plasma was separated from the blood by
centrifugation at 9,000 rpm for 5 min. The supernatant plasma was separated and utilized
for the further experiment.
Procedure: The test compound solution (5 pL of 5 mM) was dissolved in rat plasma (495
jiL). Immediately after addition (0 min), aliquots (100 pL) was removed and added to
ice-cold acetonitrile (100 pL) and mixed well by vortexing for 2 min. The mixture was
centrifuged at 14000 rpm for 10 min. The supernatant was diluted with acetonitrile, and
analyzed by HPLC. After 0 min sample, remaining sample was incubated at 37 °C for 60
min. After 30 and 60 min, sample (100 pL) was treated with ice cold acetonitrile (100
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|llL) and centrifuged at 14,000 rpm for 10 min. The supernatant was diluted with
acetonitrile and injected in HPLC. The percentage of prodrug remaining was calculated
as per following equation.
Peak area at respective time (min)
Percentage remaining = ------------------------------------------- * 100
Peak area at 0 min
5.3.1 Procedure for metabolic stability of indomethacin prodrugs in RLM

Rat liver microsomes (RLM) were prepared in-house by known method and used
immediately in experiments. Protein concentrations were determined by the Biorad
protein Assay (Bio-Rad, Hercules).
Material used
NADPH (Sigma, Lot No. N 6674); acetonitrile (HPLC grade, Merck, India); Tris HCI (s.
d. fine chem, India), Rat liver chromosomes (in-house), prodrugs and indomethacin
reference compound.
Requirement
Rat liver microsomes (10 mg/mL protein concentration), NADPH (10 mM solution), Tris
HCI buffer (pH 7.4), Eppendorf tubes, test compounds solution (concentration 5 mM).
Study conditions
Incubation period, 60 min; Incubation condition, 37°C with 60 rpm shaking; Protein
precipitation solvent, acetonitrile.
Assay procedure
Tris HCI buffer (395pL), 10 mM NADPH solution (50 pL) and 50 pL of rat liver
microsomes were mixed and vortexed for 10 sec. To this mixture, 5 mM drug solution (5
pL) was injected and vortexed well. Sample (75 pL) was immediately taken out (0 min)
and transferred to the centrifuge tube containing ice cold acetonitrile (75 pL) vortexed
and centrifuged at 14,000 rpm for 10 min, aliquots of the supernatant were separated and
used for analysis by HPLC. Then the assay mixture was incubated in water bath at 37°C
for 60 min and at specific time points (30 min, 60 min) assay mixture (75 pL) was taken
out and added to the centrifuge tube containing equal volume of cold acetonitrile. Then
tubes were vortexed and centrifuged at 14,000 rpm for 10 min, aliquots of the supernatant
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were separated and used for analysis by HPLC. The percentage of prodrug remaining was
calculated as per following equation.
Peak area at 0 min
Results and discusion
Prodrugs (2 and 6) and reference parent drug 1 were subjected to metabolic stability in
the presence of rat liver microsomes and rat plasma. The parent drug 1 was stable up to
60 min in both rat liver microsomes and rat plasma, however prodrugs were highly
metabolized (Table 1).
Table 1: Metabolic stability of 1 and prodrugs (2 and 6) in RIM and RP
metabolic stability in RLM metabolic stability in RP

Time—► Omin 30 min 60 min Time—► Omin 60 min
compd % remaining in RLMa compd % remaining in RPa
1 100 90.3 80.2 1 100 97.1
2 100 0 0 2 100 0
6 100 1.14 0 6 100 0
aThe percentage ofprodrug remaining after metabolism at respective time points was
calculated by ratio ofpeak area at respective time (min) to peak areafound at 0 min
multiply by 100.
% Remaining =------------------------------------------- x 100
Peak area at 0 min
Formation of parent compound indomethacin (1) from prodrugs (2, 6) in RLM and
RP
The experimental findings depicted in table 1 and table 2 proved that prodrugs (2 and 6)
were enzymatically labile and converted rapidly to parent compound (1). Indomethacin
active metabolite was observed after biotransformation in both RLM and RP. Release of
parent drug from these ester prodrugs upon hydrolysis was confirmed by HPLC. Prodrugs
peak in HPLC (retention time: 2 ~14.1 min; 6-11.1 min) was disappeared in 30 - 60 min
and peak area of parent compound 1 (retention time: - 9.15 min) was increased (Table 1
and 2)
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Tablet: Formation of parent compound 1 from prodrugs (2 and 6) in RLM and RP
formation of indomethacin parent compound formation of indomethacin parent

from prodrugs in RLM compound from prodrugs in RP
Time----- ► 0 min 30 min 60 min Time—► Omin 60 min
Compd indomethacin peak area in Compd indomethacin peak area
HPLC analysis in HPLC analysis
2 0 1435186 1801169 2 0 101220
6 0 739541 738838 6 0 855753
Table 2: Disappearance of prodrugs by metabolic transformation
prodrug peak area in RLM prodrug peak area in RP

time----- ► 0 min 30 min 60 min time —► Omin 60 min
compd prodrug peak area in HPLC compd prodrug peak area in HPLC
2 1157146 0 0 2 491862 0
6 794700 9082 0 6 748633 0
5.3.2 Physicochemical properties of indomethacin prodrugs

a) Aqueous solubility
Test method
The solubility of indomethacin prodrugs (2, 5,6, and 8) was determined at 40°C in 0.1M
boric acid buffer at pH 9,0.2 M phosphate buffer at pH 7.4,0.1 M phosphate buffer at pH
5.2, 0.1 M citric acid buffer at pH 3.0 and 0.2 M hydrochloric acid buffer at pH 1.0. Test
compound (5 mg each) in buffer solution (10 mL) was incubated at 40°C for 4 h at 400
rpm on mechanical shaker. The solution was filtered through 0.20 pm membrane filter,
and the filtrate was analyzed quantitatively by HPLC at wave length 254 nm for its
solubility and hydrolysis.
The orally administered drug should be stable at various pH environments encountered in
the gastrointestinal tract to deliver the intact prodrug to the systemic circulation. Four
prodrug candidates (2,5,6, and 8) were evaluated for solubility and stability in a series of
buffer solutions ranging from pH 1 to 9. All the prodrug compounds were found stable
and practically insoluble in buffer solutions (below detection limit). Aqueous solubility of
(2, 5, 6, and 8) was lower than that of parent drug indomethacin (1), which is related to
the increased lipophilicity of prodrugs. Limit of detection (LOD) of prodrugs (2,5,6, and
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8) was estimated by HPLC (LOD: 2, 0.084 pg/mL, 5, 0.119 pg/mL, 6, 0.111 pg/mL, 8,
0.0638 pg/mL). The aqueous solubility of indomethacin at 40 °C was 0.03 pg/mL and 24
pg/mL, 1500 pg/mL, and 1600 pg/mL at pH 1.0, 3.0, 5.2, 7.4, 9.0 respectively as against
reported in literature (3.882 pg/mL and 767.5 pg/mL at pH 1.2 and pH 7.2). Experimental
solubility value of 1 was slightly different than the reported earlier could be because of
difference in solubility study parameters. Solubility of indomethacin was pH dependent
and increases with increase in pH. Insignificant hydrolysis of prodrug compounds to
parent drag was observed in acidic to neutral pH buffer solutions (Table 1) at 40°C,
indicated that prodrags 2, 5, 6, and 8 were resistant towards hydrolysis at pH of stomach
acidic environment, pH of the small intestinal mucosa and at physiological pH.
Table 1: Aqueous solubility and stability of indomethacin (1) and prodrags ((2,5,6, and
8) in buffer solutions at 40 °C for 4 h
buffer hydrolysis to parent drag in pg/mL solubility in pg/mL
pH 2 5 6 8 2 5 6 8
1.0 0.0210 0.010 0.0103 0.0191 BDL BDL BDL BDL
3.0 0.0075 0.00539 0.0092 0.0134 BDL BDL BDL BDL
5.2 0.1525 0.01379 0.0197 0.0244 BDL BDL BDL BDL
7.4 0.2940 0.01332 0.0093 0.0101 BDL BDL BDL BDL
9.0 2.187 0.24614 0.369 0.5106 BDL BDL BDL BDL
BDL, below detection limit. Limit of detection (LOD) are estimated by HPLC method, 2
(0.084 yg/mL), 5 (0.119 yg/mL), 6 (0.111 yg/mL), 8 (0.0638 yg/mL).
b) Determination of partition coefficient (log P)

Test method
The partition coefficients of indomethacin ester prodrag (2, 5, 6, 8) were determined in
octanol-buffer system by HPLC method at 25 °C. The aqueous phase was 0.2 M
phosphate buffer of pH 7.4 and 0.1 M citric acid buffer of pH 3.0. Before use, the 1-
octanol and buffer solution were mutually saturated for 24 h at 400 rpm on mechanical
shaker at 25 °C, A known concentration of compounds in 1-octanol (5 mL) and buffer
solution (5 mL) was shaken on mechanical shaker for 60 min at 400 rpm at 25 °C,
centrifuged at 3500 rpm for 5 min. The concentration of the compound (solute) in both
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phases was analyzed quantitatively by HPLC at 254 nm. Each experiment was repeated
in triplicate. The partition coefficient was calculated by following equation.
log fl-v— - log <)

The partition coefficient (log P) of parent compound indomethacin (1) and prodrugs (2,5,
6, and 8) were determined at room temperature in n-octanol - phosphate buffer at pH 7,4,
and 1-octanol-citric acid buffer at pH 3.0. Partition coefficient of all prodrugs at pH 7.4
was higher than 1, indicated that prodrugs are more lipophilic than the parent drug.
Partition coefficient of indomethacin and prodrugs are summarized in Table 1 and results
of indomethacin are in line with the literature value.
Table 1: Partition coefficient of indomethacin (1) and prodrugs (2,5,6, and 8) in buffer
solution
compound partition coefficient (log Pf
pH 7.4 pH 3.0
1 2.1 4.2
2 4.7 4.6
5 5.0 5.1
6 3.5 4.2
8 2.9 4.5
aResults are reported an average ofthree experiments (n =3)
c) Solid state morphology

The solid state morphology of an active pharmaceutical ingredient (API) is a key-
parameter for their further utilization when several forms can co-exist either as crystalline
or amorphous, and/or as different polymorphs (allotropes). This is particularly important
for API as their morphology can have a significant impact on their bio-availability and
stability. Hence it was necessary to know the solid state morphology of prodrugs (2,5,6,
and 8). Prodrugs (2, 5, 6, and 8) were crystalline when screened in Bruker AXS D8
advance diffractometer. Powder XRD profiles of prodrugs are shown in Figure 1,2,3 and
4.
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Figure 1: XRD diffractogram for prodrug 2
2-Theta-Scale
2-IheJa-Seale
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i i » t i ■i"i... i'—t...r"'T-r —| i------*----- 1----- 1 | » i------1 r- | i i i
» »
2-TheW-Scale
Figure 4: XRD diffractogram for prodrag 8
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5.4.0 Biological evaluation of diclofenac prodrugs
a) Carrageenan-induced paw edema in rat
Test method
Anti inflammatory assay of the test compounds (10,13,14) and reference drug diclofenac
sodium la was evaluated by using in-vivo rat carrageenan foot paw edema model
reported previously.1
Chemicals
Carrageenan was purchased from Sigma (St. Louis, MO, USA). Diclofenac sodium (EP)
is procured from local bulk drug manufacturer.
Animals
All the experiments were approved by the Institutional Animal Ethics Committee (IAEC)
and complied with the NIH guidelines on handling of experimental animals. Male Wistar
rats weighing 150-200 g were used for the study (n = 6). The animals were housed in a
group of 3 rats per cage under well-controlled conditions of temperature (22 ± 2 °C),
humidity (55 ± 5%) and 12 h/12 h light-dark cycle. Animals had free access to diet
purchased from vet care and water ad libitum.
Paw edema was induced with carrageenan 1% (0,1 mL) administered as a single
subplantar injection under light ether anesthesia in left paw. Animals were divided in
groups, namely normal control, carrageenan and carrageenan treated with diclofenac
prodrug (100 mg / kg) as well as parent drug diclofenac sodium at dose of 50 mg/kg, 1 h
prior to carrageenan administration. Paw volume was measured using the plethysmograph
after 3 h carrageenan administration. Percentage inhibition was calculated by using
following equation
Carrageenan - Test compound
% Inhibition = ------------------------------------------- x 100
Carrageenan - Normal
Ressults and discusion

Anti-inflammatory assay
The anti-inflammatory activity of prodrugs was evaluated by using the in-vivo rat
carrageenan induced paw edema method1. All diclofenac produgs (10,13,14) produced a
significant anti-inflammatory effect when administered orally in rats. Prodrug 10 emerges
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as better anti-inflammatory drug than the prodrug (13 and 14). Inhibition of paw volume
in carrageenan-induced paw edema is shown in Table 1.
Table 1; Anti-inflammatory activity of prodrugs (10,13,14) and parent compound (la)
in carrageenan paw edema in rats (data represented as Mean ± SEM, n = 6).
dose paw volume % inhibition at 180
groups
(mg/kg) (pmol/kg) (mL) min
normal - - 1.95 ±0.05 -
carrageenan (1%)
- ###3.77 ± 0.2 la -
0.1 mL -
la 50 157 ***2.88± 0.12B 49.17

10 100 244 ***2.74 ±0.076 56.33
13 100 273 **3.03 ± 0.14° 40.88
14 100 261 ***2.90 ± 0.07B 47.51
aP < 0.001, compared to normal control; bP < 0.001, compared to carrageenan control;
CP < 0.01, compared to carrageenan control. dInhibitory activity in a carrageenan-
induced raw paw edema assay in the group ofanimals (n = 6) was calculated by using
following equation.
Carragennan - Test compound
% Inhibition = ------------------------------------------- x 100
Carragennan - Normal
b) Acute ulcerogenic assay

The most common side effects associated with the long-term administration of NSAIDs
are gastric erosions, ulcer formation, and sometimes severe bleeding. Therefore, the
potential ulcerogenic side effects of prodrugs were determined in rats and compared to
those produced by the parent compounds after single dose oral administration.
Test method
The ability to produce gastric damage was evaluated according to reported procedure.2
Animals (n = 6 / group) were kept for fasting for 18 h with free access to water. Prodrugs
at (100 mg/kg) while diclofenac sodium at (50 mg/kg) were administered orally and
evaluated for ulcer potential. Animals were sacrificed under ether anesthesia after 6 h
dosing drug compounds, stomach was removed, opened along the greater curvature,
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washed and mounted on thermostat sheet and examined for ulcers. Ulcerative lesions
were scored as follows.
• Length of all lesions were measured using Vernier caliper.
• The ulcers are classified as level I, ulcer area < 1 mm diameter; level -II, ulcer area
1-3 mm diameter; level-III, ulcer area > 3 mm diameter
• Ulcerative Lesion Index (UI): as 1 x (number of ulcer level I) + 2 x (number of ulcer
level II) -i- 3 x (number of ulcer level III). Sum of six animal ulcer readings was
reported as ulcer index (UI).
The ulcerogenicity assay was performed with single dose diclofenac sodium (50 mg/kg)
and prodrug (100 mg/kg) in rats. Lesser degree of ulcers was observed in rats which were
treated with prodrugs (10,13,14) compared to animals treated with diclofenac sodium la
(Table 1).
Table 1: Ulcerogenic effect of diclofenac sodium (la) and prodrugs (10,13,14) in rats.
compd number of level I level II level IU
ulcers (< 1 mm) (1-3 mm) (> 3 mm)
la 50 40 (80.0 %) 7 (14 %) 3 (6.0 %)
10 6 6 (100.0 %) 0 0
13 35 32(91.4%) 1 (2.86 %) 2 (5.71 %)
14 8 8 (100.0 %) 0 0
Animals treated with diclofenac sodium developed an average of 50 ulcerogenic

lesions with only single dose administration and around 6.0% of the lesions were
considered to be large (higher than 3 mm diameter). This allowed to the classification of
lesions, which was scored depending upon the severity of mucosal damage. Prodrug 10
on 100 mg/kg single dose administration, led to the development of only six small lesions
(< 1 mm diameter). Prodrug 13 on 100 mg/kg single dose administration, led to the
development of around 35 lesions with level I, 91.42 %; level II, 2.86 % and level III,
5.71 %, and prodrug 14 developed only eight ulcerogenic lesions of level I (< 1 mm
diameter) on similar oral dose concentration. The results were obtained with an average
205
of six animals analyzed per group (n = 6). These findings suggest that masking of
carboxylic function of diclofenac successfully decreased gastro-ulcerogenicity.
Ulcer Index (UI) is calculated based on the lesions developed on single dose
administration in rats. All the three prodmgs showed an improved safety profile than the
parent reference compound even at higher dose (Table 2).
Table 2: Ulcer index for prodrugs (10,13,14) and diclofenac sodium (la)
groups dose ulcer index8 (UI)
mg/kg (pmol/kg)
normal __ 0
la 50 157 63
10 100 244 6
13 100 273 40
14 100 261 8
aResults are expressed as a mean ± SEM(n = 6).
Unlike parent compound la (UI = 63), prodrug 10 (UI = 6) and prodrug 14 (UI = 8)
produced very few detectable lesions on the gastric mucosa in the group of animals
examined and prodrug 13 (UI = 40) produced comparatively more detectable lesions.
This represents a remarkable improvement considering that of diclofenac sodium (UI =
63) was the most irritant compound on the molar basis.
5.4.1 Metabolic stability of diclofenac sodium and diclofenac prodrngs (in-vitro)
Diclofenac ester prodrugs (10, 13, 14) were synthesized with aim of obtaining
enzymatically labile and with less ulceration drugs than the parent drag diclofenac
sodium (la). The prodrags were evaluated in vitro and in vivo for their potential use as
prodrags for oral delivery,
a) Rat plasma (RP)
Rut plasma was harvested from in-house rats. Fresh blood was collected from the male
rat using retro orbital bleeding method in the tube containing heparin (100 lU/mL
blood). After the collection of blood, plasma was separated from the blood by
centrifugation at 9000 rpm for five min. The supernatant plasma was separated and
utilized for the further experiment.
206
In-vitro physiological stability of prodrugs (10,13,14) and diclofenac sodium (la) in
rat plasma
The test compound solution (5 pL of 5 mM) was dissolved in rat plasma (495 pL).
Immediately after addition (0 min), aliquots (75 pL) were removed and added to ice-cold
acetonitrile (75 pL) and mixed well by vortexing for 2 min. The mixture was centrifuged
at 14000 rpm for 10 min and the supernatant was diluted with acetonitrile, and analyzed
by HPLC. After 0 min sample, remaining sample was incubated at 37 °C for 60 min and
120 min. After 60 min and 120 min, sample (75 pL) was treated with ice cold acetonitrile
(75 pL) and centrifuged at 14000 rpm for 10 min. The supernatant was diluted with
acetonitrile and injected in HPLC. The percentage of prodrug remaining was calculated
as per following equation.
Peak area at 0 min
b) Metabolic stability in rat liver microsomes (RLM)

Rat liver microsomes were prepared in-house by previously published method26 and used
immediately in the experiments. Protein concentrations were determined by the Biorad

protein Assay (Bio-Rad, Hercules).
Material
NADPH (Sigma, Lot No. N 6674); acetonitrile (HPLC grade, Merck, India); Tris HC1 (s.
d. fine chem, India), Rat liver chromosomes (in-house). Test compounds.
Requirement: Rat liver microsomes (10 mg/mL protein concentration), NADPH (10
mM solution), Tris HC1 buffs- (pH 7.4), Eppendorf tubes, test compounds solution
(concentration 5 mM).
Study conditions
Incubation period, 60 min; Incubation condition, 37°C with 60 rpm shaking; Protein
precipitation solvent, acetonitrile.
Assay procedure
Tris HC1 buffer (395pL), 10 mM NADPH solution (50 pL) and 50 pL of rat liver
microsomes were mixed and vortexed for 10 seconds. To this mixture.
207
5 mM drug solution (5 pL) was injected and vortexed well. Sample (75 pL) was
immediately taken out (0 min) and transferred to the centrifuge tube containing ice cold
acetonitrile (75 pL). The assay mixture was incubated in water bath at 37 °C for 60 min
and at specific time points (30 min, 60 min) assay mixture (75 pL) was taken out and
added to the centrifuge tube containing equal volume of cold acetonitrile. Then all tubes
were vortexed and centrifuged at 14,000 g for 10 min, aliquots of the supernatant were
separated and used for analysis by HPLC. The percentage of prodrug remaining was
calculated as per following equation.
Percentage remaining = ------------------------------------------- x 100
Peak area at 0 min

Prodrugs (10,13, and 14) and parent diclofenac sodium (la) were subjected to metabolic
stability in the presence of rat liver microsomes and rat plasma. Diclofenac sodium (la)
was fairly stable in rat liver microsomes and rat plasma, however prodrugs 10,13, and 14
were highly metabolized, and converted to desirable parent compound la in 30-60 min
(Table 1). Chemical degradation was not observed during metabolic stability study. The
corresponding loss of pro drug compounds and formation of parent drug 1 was determined
by HPLC.
Table 1: Metabolic stability of diclofenac sodium (la) and prodrugs (10,13,14) in rat
liver microsomes (RLM) and rat plasma (RP)
metabolic stability in rat liver microsomes metabolic stability in rat plasma
time —► Omin 30 min 60 min time —► Omin 60 min 120 min
compound % remaining in RLMa compound % remaining in RPa

la 100 84.4 88.8 la 100 86.2 69.4
10 100 2.5 0.0 10 100 0.0 0.0
13 100 2.2 1.2 13 100 0.0 0.0
14 100 0.0 0.0 14 100 0.0 0.0
aPercentage ofprodrug remaining after metabolism was calculated by ratio ofpeak
area at respective time (min) to peak areafound at 0 min multiply by 100.
% Remaining =------------------------------------------- x 100
Peak area at 0 min
208
Formation of parent compound from prodrugs in rat liver microsomes (RLM) and
rat plasma (RP)
The experimental findings proved that all prodrugs were enzymatically labile and
converted to parent compound diclofenac as metabolite by enzymatic hydrolysis of ester
group. The peak area of prodrugs in HPLC analysis (Retention time: prodrug 10, ~18.7
min; prodrug 13, -11.97 min; prodrug 14, -7.91 min) observed below detection level
(BDL) after 60 min and correspondingly the parent drug diclofenac peak area (Retention
time, 4.4 min) was increased (Table 2 and 3).
Table 2: Formation of diclofenac parent compound (la) from prodrugs (10,13,14)
formation of parent compound from prodrugs formation of parent compd from prodrugs in
in rat liver microsomes rat plasma
time----- ► 0 min 30 min 60 min Time—► 0 min 30 min 60 min
compd diclofenac peak area in HPLC compd diclofenac peak area in HPLC
10 0 250711 280652 10 0 100565 101220
13 0 396209 452071 13 0 46076 36528
14 0 256817 295451 14 0 105658 109889
Table 3: Disappearance of prodrugs (10,13,14) by enzymatic biotransformation in RLM

and RP
peak area of prodrugs in presence of RLM peak area of prodrugs in presence of RP
time----- ► Omin 30 min 60 min Time—► 0 min 30 min 60 min
compound prodrug peak area in HPLC compound prodrug peak area in HPLC
10 311622 7636 0 10 101079 0 0
13 569902 12271 0 13 46076 0 0
14 310206 0 0 14 117720 0 0
209
5.4.2 Physicochemical properties of diclofenac sodium and prodrugs
a) Aqueous solubility
Test method
Diclofenac sodium and prodrugs solubility was determined in various pH buffers at pH
1.0, pH 3.0, pH 5.2, pH 7.4 and pH 9.0 at 25 °C. An excess compound was added to
buffer solutions and the suspension was shaken for 5 h at 25 °C on mechanical shaker at
350 rpm. The solution was filtered through a Millipore filter (0.22 pm) and analyzed
quantitatively by HPLC.
Aqueous solubility of diclofenac sodium (la) and prodrugs (10,13,14) was determined
in buffer solutions at various pH 1- 9. The solubility of la was depends on the pH of the
dissolution medium at 25 °C and increased with increase in pH. Experimental values are
in line with literature value. Limit of detection for diclofenac sodium la (0.008 pg/mL)
was determined by HPLC at wavelength 254 nm. The aqueous solubility of all prodrugs
was independent of pH and was practically insoluble in acidic, neutral and basic aqueous
solution (Table 1). The limit of detection (LOD) of prodrugs (10,13,14) was determined
by HPLC (10, 0.0837 pg/mL; 13, 0.166 pg/mL; 14, 0.0636 pg/mL) at wavelength 254
nm. Aqueous solubility of prodrugs (10, 13, and 14) was lower than that of parent
diclofenac sodium (la), which is related to the increased lipophilicity of esters. Increased
molecular size by introducing a bulky group through ester linkage, as well as masking the
hydrophilic carboxylic group of diclofenac can lead to poor solubility of prodrugs.
Table 1: Solubility of diclofenac sodium (la) and prodrugs (10,13,14) in buffer solution
pH Medium® Solubility pg/mL

cla °la °10 D13 D14
1.0 0.2 MHC1 buffer - 1.27 BDL BDL BDL
1.2 0.1MHC1 1.20 - - - -
3.0 0.1 M Citric acid buffer - 1.785 BDL BDL BDL

5.2 0.1 M Phosphate buffer - 43.7 - - -
5.5 Acetate buffer 36.0 - BDL BDL BDL

7.4 0.2 M Phosphate buffer 5150 5280 BDL BDL BDL
9.0 0.1 M Alkaline borate buffer 15180 13960 BDL BDL BDL
aBuffer solutions were prepared as per USP 32. bExperimentally determined, results are
reported as mean ±SD (n = 2). cSolubiIity are reported in literaturature, BDL, Below
detection limit.
210
b) Determination of partition coefficient (log P)
Test method
The partition coefficient of Diclofenac sodium and prodrugs were determined by HPLC
method. Before a partition coefficient, 1-octanol and buffer solution were mutually
saturated for 24 h by stirring vigorously on mechanical shaker at 25 °C, and allowed the
phases to separate for 24 h at 25 °C. A stock solution of test compound was prepared at
25 °C, filtered through 0.22 pm membrane filter and determined the percentage of test
compound by HPLC. Three tests were carried out in duplicate with various volume ratios
of octanol (stock solution) to pre-saturated buffer i.e. 1:1, 1:2, 2:1. Die test vials were
shaken on mechanical stirrer at 350 rpm for 60 min, centrifuged for 5 min at 3500 rpm at
room temperature. The concentration of the test compounds in both phases was
determined precisely by HPLC at wavelength 254 nm. The partition coefficient (log F)
was determined by the logarithm of the ratio of concentrations of un-ionized compound
(solute) in octanol to aqueous solution.
The partition coefficients (log P) of diclofenac sodium (la) and prodrugs (10, 13, 14)
were determined by HPLC method in octanol-buffer system. Partition coefficient values
for all prodrugs were found to be higher than diclofenac sodium at pH 7.4. Results of
log P at pH 3.0 and pH 7.4 are summarized in Table 1. Higher log P values of all
prodrugs at pH 7.4, indicated that prodrugs are more lipophilic than the parent drug.
Partition coefficient results of diclofenac sodium are in line with the value reported in
literature.3'6
Table 1: Partition coefficient of diclofenac sodium (la) and prodrugs (10,13,14)

compd Partition coefficient (log Pf
phosphate buffer pH 7.4 citric acid buffer pH 3.0
(0.2 M) (0.1 M)
°la 0.853 & 1.1 -
la 0.91 3.99
10 3.72 4.08
13 2.46 3.90
14 4.57 4.33
On ^ An. ...... j- t-.- ...__ 3-6
aResults were expressed as mean ofsix tests (n = 6). bReported in literature3-6
211
c) Powder X ray diffraction of prodrugs (PXRD)
The solid state morphology of active pharmaceutical ingredient (API) is a key-parameter
for their further utilization when several forms can co-exist either as crystalline and
amorphous, and/or as different polymorphs (allotropes). Solid state morphology is
particularly important for API, as their morphology can have a significant impact on their
bio-availability and stability. Hence it is necessary to know solid state morphology of
these prodrugs (10,13, 14) in which biological studies were performed. The diffraction
spectrum of prodrugs (10,13,14) showed the drugs are highly crystalline powder when
screened in Bruker AXS D8 Advance diffractometer. The prodrug 10 possesses sharp
peaks at 20 equal to 8.27,” 9.54,° 9.96,° 11.29,° 15.91,° 17.15,° 22.63.° The prodrug 13
possesses sharp peaks at 20 equal to 8.06,°,9.01,° 11.41,° 12.4,° 14.25,° 16.13,° 16.66,°
16.87,° 17.2,° 17.63,° 18.03,° 18.6,° 19.8,° 20.2,° 20.7,° 21.99,° 22.57,° 23.09,° 23.4,°
23.74,° 23.9,° 24.29,° 24.66,° 25.61,° 26.58,° The prodrug 14 possesses sharp peaks at 20
equal to 113,° 12.89,° 13.19,° 15.05,° 22.66,° 25.47.° XRD patterns for prodrugs (10,13,
14) are displayed in Figure 1,2 and 3.

tSSstiUBSC
2-Ttwta-ScaIa
212
“pp" iwtm :OCEUA»DO«ra

LumoMniMakii fi Broker AXSDSAdvaac* ________ Ref, No. XRDH09H
L m w m p*
CaH»,«e:
.
213
5.5.0 Metabolic stability of ibuprofen (1), and ibuprofen prodrug compound No. 18
in rat plasma (RP)
Stability of Ibuprofen in rat plasma

Time (min) Peak Area of i in HPLC
0 829
15 955
30 857
60 751
120 911
Stability of compound No.18 in rat plasma

Time (min) Peak Area of Ibuprofen (1) Peak area of Compd No. 18
0 3844 9500
15 27096 6798
30 53098 10437
60 83535 4161
120 98579 971

Ibuprofen and ibuprofen prodrug compound No. 18 subjcted for metabolic stability in rat
plasma (in-vitro) by known method. All processed samples were analyzed by HPLC.
Ibuprofen was found stable in rat plasma, however prodrug was enzymatically labile and
converted to ibuprofen within 2 h, which was as expected.
214
5.6.0 Metabolic stability of flurbiprofen and prodrugs (26 and 32) in rat plasma
(RP)
Stability of flurbiprofen in rat plasma

Time (min) Peak Area in HPLC
0 3B403
15 38474
30 42408
60 42706
120 42059
Stability of prodrug No. 26 in rat plasma

Peak Area of Flurbiprofen in Peak area of Compd No. 26 in
Time (min)
HPLC HPLC
0 18281 19074
15 73133 265
30 78199 45
60 81243 141
120 93735 70

Time Peak Area of Flurbiprofen in Peak area of Compd No. 32 in
(min) HPLC HPLC
0 27514 3008
15 52278 2015
30 52402 2748
60 52953 2435
120 52619 2477

Flurbiprofen and prodrug No. 26 and 32 were subjected for metabolic stability in rat
plasma (in-vitro) by known method. All processed samples were analyzed by HPLC.
Flurbiprofen was found stable in rat plasma, however prodrug compounds were found
enzymatically labile and converted to flurbiprofen within 2 h, which was as expected.
215
5.7.0 Metabolic stability of ketorolac tromethamine (1) and prodrug No. 46 (in-
vitro)
Stability of ketorolac tromethamine (1) in RLM
Peak Area of. %ofl %ofl Mean%
Time Peak Area
1 remaining remaining
00 of 1 (Set-II) remaining
(Set-I) (Set-I) (Set-II)
0 1653504 1336778 100.0 100.0 100.0
0.5 1710246 1326156 103.4 99.2 100.0
1 1513881 1694820 91.6 126.8 100.0
Stability of ketorolac tromethamine (1) in rat plasma

%ofl %ofl
Peak Area Peak Area Mean% ofl
Time (h) remaining remaining
of 1 (Set-II) of 1 (Set-II) remaining
(Set-I) (Set-II)
0 1536206 1344770 100.0 100.0 100.0
1 1454205 1545511 94.7 114.9 100.0
2 1413090 1551573 92.0 115.4 100.0
Stability of prodrug No. 46 in RLM

% of prodrug
Time (h) Peak Area of prodrug in HPLC
remaining
0 404365 100.0
0.5 5668 1.4
1 0 0.0

Time (h) Peak Area of prodrug in HPLC % of prodrug remaining
0 83926 100.0
1 0 0.0
2 0 0.0

Metabolic stability of ketorolac tromethamine (1) and ketorolac prodrg No. 46 was
performed in microsomes (RLM) and rat plasma (RP). Ketorolac tromethamine was
found stable in both RLM and RP. However ketorolac prodrug was enzymatically labile
and transformed to ketorolac active metabolite, which was confirmed by HPLC.
216
5.8.0 Testing procedures (HPLC)
a) In-process analysis, chromatographic purity, and partition coefficient
In-process analysis, chromatographic purity and partition coefficient of prodrugs (10,13,
14) were performed by HPLC (Waters alliance), pump 2695 and UV detector 2487 with
following chromatographic parameters.
Wavelength 254 nra. Column: YMC-Pack Cg, 100 mm x 4.6 mm, 3pm, injection volume
20pL, runtime 20 min. Separation was performed isocratic elution.
Mode of operation: Isocratic
Mobile phase
Solution A: Mix 2.0 mL glacial acetic acid in 1000 mL of water (pH 3.0).
Solution B: Acetonitrile (filtered and degassed).
Mobile phase mix ratio: 30 volumes of solution A and 70 volumes of solution B.
Flow rate: 0.8 mL/min at 25 °C.
b) Metabolic stability analysis by HPLC
Analysis was performed by using HPLC (Waters alliance), pump 2695 and PDA detector
2996 with following chromatographic parameters. Wavelength: 275 nm. Column, Inertsil
Cig. 3V, 250 x 4.6 mm, 5pm, injection volume 20 pL, run time 20 min. Mode of
operation: Isocratic.
Solution A: Mix 2.0 mL glacial acetic acid in 1000 mL of water (pH 3.0)
Solution B: Acetonitrile (filtered and degassed)
Mobile phase: Mixture of 30 volumes of solution A and 70 volumes of solution B.
Flow rate 1.0 mL/min at 25 °C.
217
References
(1) Winter, C. A.; Risley, E. A.; Nuss, G. W. Carrageenan-induced edema in hind
paw of the rat as an assay for anti-inflammatory drugs. Proc. Soc. Exp. Biol. Med.
1962, 111, 544-547.
(2) Szelenyi, L; Thiemer K, Distension ulcer as a model for testing of drugs for
ulcerogenic side effects. Arch Toxico. 1978,41, 99-105.
(3) Wills, J. V.; Kendall, M. J.; Flinn, R. M.; Thornhill, D. P.; Welling, P. G. J Clin.
Pharmacol. 1979,16,405-410.
(4) Khazaeinia, T.; Jamali, F, J. Pharm. Pharm. Sci. 2003,6,352-359.
(5) Hendriksen, B. A.; Sanchez Felix, M. V.; Bolger, M. B. J. Pharm. Sci. 2002, 91
(9), 2076- 2089.
(6) Arora, P.; Mukherjee, B. J. Pharm. Sci. 2002,91 (9), 2076-2089.
218
Section II; Biological activity of anti-cholesterol prodrugs
(fibrates)
219
6.0.0 Introduction and literature review of hypolipidemic drugs
Cardiovascular diseases remain by far the number one cause of death for both
men and women of all ethnic back ground. Although many creative factors of these
diseases are recognized high serum LDL-C and elevated total cholesterol levels are the
most prevalent indicators for susceptibility to atherosclerotic heart diseases'
Atherosclerosis is a disorder of the arterial wall characterized by accumulation of
cholesterol esterin ceils derived from the monocyte-macrophage line, smooth muscle cell
proliferation and fibrosis, and the results in narrowing the blood vessel.3 There are many
classes of lipid lowering agents available, these drugs have different mechanisms of
action and variable efficacy depending on the lipid profile of an individual. Inspite of
their lipid lowering effect, these drugs have many side effects. Thus research is still
pursuing to find out novel agents that are more effective and safe.
Fibrates have been used for the treatment of hypertriglyceridemia or mixed

hyperlipidemia for more than 30 years. Their main action is to lower plasma triglyceride
levels, but they also reduce total and LDL cholesterol concentrations and induce a
moderate increase in HDL cholesterol. It is now known that fibrates act by stimulating
the activity of peroxisome proiiferator-activated receptor (PPAR)-o, a member of the
PPAR subfamily of nuclear receptors.4 PPAR-a is mainly expressed in liver, kidney,
heart, and muscles,5,6 as well as in cells of the arterial wall, monocytes, macrophages7,
and lymphocytes.8 It controls the transcription of regulatory genes of fatty acids and
cholesterol metabolism. The decrease in plasma triglycerides induced by fibrates has

been attributed to an inhibition of the synthesis and secretion of triglycerides by the liver
and a stimulation of the degradation of triglyceride-rich lipoproteins.9 This increased
clearance of triglycerides results from a stimulation of the expression of lipoprotein lipase

(LPL)10,11 and a decreased expression and concentration of apolipoprotein CIII,12 an
inhibitor of LPL activity. The mechanisms of the action of fibrates on cholesterol

metabolism remained unclear until it appeared that PPAR-a activation modified the
expression of several key genes controlling HDL cholesterol metabolism.10 It was first
recognized that PPAR-a stimulated the expression of human apolipoprotein A-I and A-II
genes.13 More recently, Chinetti and colleagues14,15 demonstrated that PPAR-a activation
220
stimulated the expression in differentiated human macrophages of CD36 and LImPII
analogous 1 (CLA-l)/scavenger receptor class B type I (SR-BI) and ABCAI, which both
play key roles in the reverse transport of cholesterol16,17 SR-BI is a cell surface receptor
that binds HDL with high affinity and mediates the selective uptake by liver and
steroidogenic tissues of cholesterol esters from HDL. SR-BI could also play a role in the
cellular efflux of cholesterol. ABCAI exports unesterified cholesterol and phospholipids
from cells to nascent HDL and therefore has a key role in the control of the first step of
reverse cholesterol transport. However, most of these data on the mechanisms of action of
fibrates were obtained in vitro or in rodent animals. There are differences in the tissular
expression and activity of PPAR-a between rodents and humans, as well as large
differences in the regulation of lipoproteins metabolism, particularly HDL cholesterol.
Hamsters are considered a more appropriate model of lipoprotein metabolism but also
have some divergent responses to fibrates, such as decreases in HDL cholesterol
concentrations and apoprotein-AI expression instead of the increases seen in humans.18
Data on the mechanism of action of fenofibrate in human subjects, particularly in

hyperlipidemic patients, are scarce and limited, to our knowledge, to measurements of the
concentrations and kinetics of apoprotein B100 and apoprotein A.18"19
The present work aims at investigating the antihyperlipidemic activities of

beazafibrate ester prodrugs in rats. Bezafibrate was adopted in this study as reference
hypolipidemic drug for data comparison.
221
References
1. Habib, N. S.; Ismail, K. A.; El-Tombary, A. A.; Abd El-Aziem, T. Pharmazie
2000,55,495-499.
2. Fuster, V.; Alexander, P. W.; O’Rourke, R. A.; King, S. B.; Wellens, H. J.
Anthrogenesis ands its determinants. In Hurst’s the heart Volume 3, 10th edition,
New York, Me Graw-Hill 2001,1065.

3. Devlin, T. Textbook of biochemistry with clinical coorelations 3rd edition. New
York, Wiley-Liss Inc. 1995,376-381.

4. Gervois, P.; Torra, I.; Fruchart, J.; Staels, B.; Clin. Chem. Lab Med 2000, 38, 3-
11.
5. Auboeuf, D.; Rieusset, J,; Fajas, L.; Vallier, P.; Frering, V.; Riou, J. P.; Staels, B.;
Auwerx, J.; Laville, M.; Vidal, H. Diabetes. 1997,46,1319-1327.
6. Su, J.; Simmons, C.; Wisely, B.; Ellis, B.; Winegar, D. Hybridoma 1998,17, 47-
53.
7. Gbaguidi, F.; Chinetti, G.; Milosavljevic, D.; Teissier, E.; Chapman, J.;
Olivecrona, G.; Fruchart, J. C.; Griglio, S.; Fruchart-Najib, J.; Staels, B. FEES
Lett. 2002,512,85-90.
8. Jones, D.; Ding, X.; Daynes, R. J. Biol. Chem. 2002,277,6838-6845.
9. Staels, B.; Dallongeville, J.; Auwerx, J.; Schoonjans, K.; Leitersdorf, E.; Fruchart,
J. Circulation 1998,98,2088-2093.
10. Heller, F.; Harvengt, C. Eur.J Clin. Pharmacol. 1983,23,57-63.
11. Zhang, Y.; Repa, J.; Gauthier, K.; Mangelsdorf, D. J. Biol. Chem. 2001, 276,
43018-43024.
12. Malmendier, C.; Lontie, J.; Delcroix, C.; Dubois, D.; Magot, T.; De Roy, L.
Atherosclerosis 1989, 77,139-149.
13. Fruchart, J. Am. J. Cardiol. 2001,88 (Suppl.), 24N-29N.
14. Chinetti, G.; Gbaguidi, F.; Griglio, S.; Mallat, Z.; Antonucci, M.; Poumlain, P.;
Chapman, J.; Fruchart, J. C.; Tedgui, A.; Najib-Fruchart, J.; Staels, B.
Circulation 2000,101, 2411-2417.
222
15. Chinetti, G.; Lestavel, S.; Bocher, V.; Remaley, A. T.; Neve, B.; Torra, I. P.;
Teissier, E.; Minnich, A.; Jaye, ML; Duverger, N.; Brewer, H. B.; Fruchart, J. C.;
Clavei, V.; Staels, B. Nat Med. 2001, 7, 53-58.
16. Trigatti, B.; Rigotti, A.; Braun, A. Biochim BiophysActa 2000,1529,276-286.
17. Young, S.; Fielding, C. Nat Genet. 1999,22,316-318.
18. Guo, Q.; Wang, P.; Milot, D.; Ippolito, M.; Hernandez, M.; Burton, C. A.;
Wright, S. D.; Chao, Y. Biochim BiophysActa 2001,1533,220-232.
19. Faix, D.; Neese, R.; Kletke, C.; Wolden, S.; Cesar, S.; Countlangus, M.;
Shackleton, M.; Hellerstein, M. L Lipid Res. 1993,34,2063-2075.
223
7.0.0 Pharmacoligical evaluation of bezafibrate prodrugs
a) Introduction
Bezafibrate is 2-(4-(2-[(4-chIorobenzoyl)amino]ethyl}phenoxy)-2-methylpropanoic acid
and it is used to lower levels of lipids such as cholesterol and other fats in the blood.
Lipids are made naturally in the body and are also absorbed from the food we eat. If
levels of lipids are too high in the blood, they are deposited on the walls of blood vessels.
Eventually this leads to a narrowing of the blood vessels and can even block them
completely. High levels of lipids do not make people feel ill but can cause problems if
left untreated. By lowering the levels of cholesterol and other fats in the blood,
bezafibrate helps prevent any long-term effects caused by fat building up in blood
vessels, such as heart disease.
Bezafibrate is a fibrate drug used for the treatment of hyperlipidemia. It helps to
lower LD cholesterol and triglyceride in the blood, and increase HDL. Bezafibrate was
first introduced by Boehringer Mannheim in 1977. Bezafibrate is a fibrate drug used for
the treatment of hyperlipidemia. It helps to lower LD cholesterol and triglyceride in the
blood, and increase HDL. Bezafibrate improves markers of combined hyperlipidemia,
effectively reducing LDL and triglycerides and improving HDL levels.1 The main effect
on cardiovascular morbidity is in patients with the metabolic syndrome, the features of
which are attenuated by bezafibrate.2 Studies show that in patients with impaired glucose
tolerance, bezafibrate may delay progress to diabetes3, and in those with insulin
resistance it slowed progress in the HOMA severity marker.4
224
b) Hypolipidemic effect of bezafibrate (la) and prodrugs (1-3, and 5- 8) in SAM
Materials & method
Animals: Male Swiss albino mice weighing 21-30 g were obtained from in-house animal
facility of Orchid Research Laboratories and were maintained at a controlled temperature
(23±1 °C) under 12:12 h light and dark cycle. All animals were given standard laboratory
chow and water ad libitum.
Materials: The following chemicals were purchased and used for the study.
Carboxy methyl cellulose (CMC)-Sigraa Aldrich Chemicals (St Louis, U.S.A.), Tween
80- Sigma Aldrich Chemicals, Triglyceride kit- ERBA diagnostics
Method: Animals were grouped into 9 groups (n = 10) based on their body weight Then
animals were treated with NCEs/vehicle for 8 days as mentioned below. After 8 days of
treatment animals were fasted for 12 hours. On day 9th day after giving respective
treatment blood samples were collected one hour post administration of the drug. Plasma
was separated for the estimation of triglyceride and cholesterol.
Group I: Normal Control (Vehicle)
Group II: Bezafibrate: 50 mg/kg
Group HI: Prodrug No. 1:50 mg/kg
Group IV: Prodrug No. 2:50 mg/kg
Group V: Prodrug No. 3: 50 mg/kg
Group VI: Prodrug No. 5: 50 mg/kg
Group VII: Prodrug No. 6: 50 mg/kg
Group VIE: Prodrug No.7:50 mg/kg
Group IX: Prodrug No. 8:50 mg/kg
Formulation: All the compounds were suspended in vehicle (Tween 80- 5pL/mL in 0.5%
CMC, 10 ml/kg).
Biochemical analysis: Plasma triglyceride and cholesterol were measured
spectrophotometrically using commercially available ERBA kit.
Statistical analysis: All the data were expressed as Mean±SE. Statistical analysis was
done by one-way analysis of variance (ANOVA), followed by Dunnett’s test and P< 0.05
was considered as statistically significant.
225
c) Results and discusion
Bezafibrate (la) and prodrags (1-3, and 5- 8) were treated at 50 mg/kg p.o. for 9 days in
Swiss albino mice (SAM). Triglyceride and cholesterol levels were measured on the 9th
day in 12 h fasted animals. Bezafibrate ester prodrags (1-3, and 5- 8) were evaluated in
vivo for lipid lowering activity in male Swiss Albino mice (SAM), a moderate
hypertriglyceridemic model by known techniques. The mice were treated with prodrugs
((1-3 and 5- 8)) and Bezafibrate (la) by oral gavage at a dose of 50 mg/kg body weight
for 9 days. The in vivo profile of compounds ((1-3, and 5- 8)) was compared with a
reference drag la for hypolipidemic activity. The daily oral administration of prodrags
(1-3,5- 8) lowered the plasma triglyceride level (TG) by 3.89%, 18.80%, 17.24%, 3.02%,
9.63%, 11.78%, and 30.18% respectively, where as la lowered TG by 11.38% in an
identical conditions (Table 1 and Figure 1). Prodrags 6 and 7 showed equipotent
triglyceride lowering activity as bezafibrate and comapratively less potent than the
prodrugs (2, 3, 8). Total cholesterol did not show significant changes in bezafibrate
prodrags (1, 2, 3, 5, 6, and 7) treated animals. Interestingly, prodrags 8 appears to be
more potent in triglyceride as well as total cholesterol lowering activity (30.18% and 13.0
%) relative to la (11.38% and 10.42%) (Table 2 and Figure 2).
Table 1: Effect of bezafibrate (la) and prodrags (1- 4 and 5 - 8) on triglyceride level in
Swiss Albino mice
Normal
Group la 1 2 3 5 6 7 8
Control
178 189 183 135 142 197 183 192 98
168 159 155 118 122 189 163 120 110
191 126 196 148 181 129 134 134 121
217 172 117 138 105 135 203 117 200
190 113 145 94 159 165 159 161 153
151 120 213 152 168 167 119 152 82
164 137 156 162 129 145 173 162 107
163 123 133 165 156 175 139 172 101
124 189 134 146 145 165 156 181 90
177 199 224 141 119 204 128 129 141
Mean 172.30 152.70 165.60 139.90 142.60 167.10 155.70 152.00 120.30
SE 7.94 10.37 11.52 6.65 7.57 8.01 9.29 8.25 11.25
% 11.38 3.89 18.80 17.24 3.02 9.63 11.78 30.18
Reduction
226
Figure 1: Effect of bezafibrate (la) and bezafibrate prodrugs (1- 3 and 5 - 8) on
triglyceride level in Swiss Albino mice
EM' Normal control

200 wwbM 19
<
-<
Tripyeoide (mg/dl)
m in
100
id h*
w
0
**P < 0.01 one-way ANOVA followed by Dunnett’s test
Table 2: Effect of bezafibrate (la) and prodrugs (1- 3 and 5 - 8) on total cholesterol level
in Swiss Albino mice
Normal
Group la 1 2 3 5 6 7 8
Control
122 137 • 105 111 125 127 114 129 104
129 120 132 112 112 129 113 128 92
126 110 128 117 112 126 121 115 93
136 104 124 124 118 121 131 122 120
121 113 126 126 113 126 106 115 121
110 89 156 132 no 120 133 108 109
133 102 140 115 130 90 124 131 106
126 100 132 112 136 118 118 107 115
106 118 143 126 127 123 139 118 111
129 116 121 121 102 121 109 128 106
Mean 123.80 110.90 130.70 119.60 118.50 120.10 120.80 120.10 107.70
SE 3.01 4.18 4.36 2.30 3.35 3.52 3.85 2.79 3.12
% 10.42 -5.57 3.39 4.28 2.99 2.42 2.99 13.00
Reduction
227
Figure 2: Effect of Bezafibrate la and Bezafibrate Prodrugs (1- 3 and 5 - 8) on Total
Cholesterol Level in Swiss Albino Mice
HI Normal control
la
1
E=g 2
imrim 3
5
6
7
Q
rk
**P < 0.01 one-way ANOVA followed by Dunnett’s test
d) Conclusion
After 9 days of treatment of Bezafibrate and Bezafibrate prodrugs were tested for
triglyceride and total cholesterol reduction. Bezafibrate prodrugs 2, 3, and 8 were found
more potent in triglyceride reducing activity (18.8%, 17.24%, 30.18% respectively) than
the reference drug bezafibrate (11.38%), which is currently prescribe as hypolipidemic
agents. Prodrugs 6 and 7 are equipotent in lowering triglyceride (9.63% and 11.78%), and
prodrugs 1 and 5 are less potent (3.89%, and 3.02%) than bezafibrate (11.38%). Prodrugs
(1-3, 5- 8) were also evaluated for total cholesterol reduction activity in Swiss Albino
mice, prodrug 8 is only showed equipotent cholesterol reducing activity as bezafibrate.
Prodrug 8 lowered total cholesterol by 13.0%, where as bezafibrate lowered total
cholesterol by 10.42%. Hence prodrugs 2, 3, 7 and 8 were potent drugs for loweing
triglyceride and prodrug 8 is the most active drug amongst other prodrugs and
bezafibrate, which was showed significant reduction (P < 0.01) in triglyceride and
cholesterol level as compared to Normal control.
228
7.1.0 Physicochemical properties of bezafibrate and prodrugs
a) Particle size distribution (PSD)
Bioavailability of an active pharmaceutical ingredient (API) administered orally is
depends upon its particle size. The pharmacological profiles of prodrugs (1- 3, and 5- 8)
indicate that prodrugs 2, 3, and 8 were more potent than reference drug bezafibrate la,
prodrugs 6 and 7 showed equipotent in hyperlipidemic activity and prodrugs 1 and 5 were
less potent than reference drug bezafibrate la even at almost identical particle size
distribution. Hence it can be concluded that bioavailability of prodrugs is not depends on
particle size distribution (Table 1).
Table 1: Particle size distribution of bezafibrate (la) and prodrugs (1,2,3,5,6,7,8)
compd particle size distribution
< 10% < 50% < 75% <90% < 100%
la 4.6 pm 36.1 pm 92.0 pm 170.4 pm 385.8 pm
1 14.6 pm 79.1 pm 126.0 pm 177.9 pm 314.1 pm
2 6.2 pm 92.2 pm 145.1 pm 209.1 pm 795.0 pm
3 26.0 pm 115.9 pm 175.5 pm 231.1 pm 385.8 pm
5 1.2 pm 48.0 pm 98.2 pm 151.7 pm 314.1 pm
6 1.8 pm 42.3 pm 112.5 pm 183.6 pm 350.1 pm
7 1.3 pm 11.9 pm 89.5 pm 167.7 pm 348.1 pm
8 0.9 pm 41.4 pm 104.0 pm 167.6 pm 348.1 pm
b) Aqueous solubility
Aqueous solubility is an important physicochemical parameter of an active
pharmaceutical ingredient (API), and its knowledge has fundamental importance in a
wide range of applications and research areas. The orally administered drug should be
stable at various pH environments encountered in the gastrointestinal tract to deliver the
intact prodrug to the systemic circulation. Five bezafibrate ester prodrags (2, 3, 6, 7, 8)
evaluated for aqueous solubility at pH 1- 9. Excess quantity of test compounds was
stirred in buffer solution for 4 h at 40 °C on mechanical shaker at 350 rpm, filtered
through 0.22 pm filters and the filtrate solution was analyzed quantitively by HPLC.
Prodrags (2, 3, 6, 7, 8) and the reference drag bezafibrate (la) were found stable and
229
practically insoluble in aqueous solution at pH 1-9. The bezafibrate solubility result was
in line with the reported earlier. Limit of detection (LOD) and limit of qualification
(LOQ) of la and prodrugs (2,3,6,7, 8) were estimated by HPLC at a wavelength of 254
nm. The experimental results are depicted in Table 5. Aqueous solubility of prodrugs was
lower than that of parent drug bezafibrate (la), which is related to the increased
lipophilicity of prodrugs. Limit of detection (LOD) of prodrugs was estimated by HPLC
at wave length 254 nm. Prodrug 7 solubility decreased with increase in pH, mainly due to
morpholine moiety (Table 1). All the ester derivatives were found to have sufficient
chemical stability in buffers (pH 1- 9), ensuring them to be absorbed as intact moieties
from the gastrointestinal tract (Table 2).
Table 1: Aqueous solubility, Limit of detection, Limit of qualification of bezafibrate la
and prodrugs (2,3,6,7,8) in buffer solutions at 40 °C for 4 h
a
compd aLOD aLOQ aqueous solubility (pg/mL)
(pg/mL) (pg/mL) pH 1.0 pH 3.0 pH 5.2 pH 7.4 pH 9.0
la 0.031 0.093 0.042 0.050 390 4500 8600
2 0.133 0.401 BDL BDL BDL BDL BDL
3 0.04 0.12 0.28 1.2 1.2 1.3 0.86
6 0.17 0.51 1.6 3.3 3.5 2.9 3.0
7 0.043 0.13 1500 940 37 0.44 0.57
8 0.095 0.29 0.12 0.30 0.29 0.18 BDL
*Buffer solutions HCl buffer pH 1.0 (0.2 M), citric acid buffer pH 3.0 (0.1 M), phosphate
buffer pH 5.0 (0.1M), phosphate hiffer pH 7.4(0.2 M), alkaline borate buffer pH 9.0 (0.1
M) were prepared as per USP 32.
aExperimentally determined. Results are reported as mean (n = 3). BDL, below detection
limit. LOD, limit ofdetection; LOQ, limit ofqualification.
c) Chemical stability
Chemical hydrolysis of bezafibrate ester prodrugs was performed to evaluate stability of
molecule so that they can be formulated in a stable dosage form. Buffers employed in
chemical hydrolysis study covered pH of microclimate of stomach (pH 1-2), duodenum
230
(pH 4.0-5.5), jejunum (pH 5.5-7.0), and systemic circulation (pH ~7.4). Results of
hydrolysis of prodrugs 2, 3, 6, 7, and 8 in aqueous buffer at pH 1-9 are summarized in
table 1. All prodrugs were highly stable at pH 1.0 and 3.0 and marginal hydrolysis was
observed at pH 5.2, 7.4 and 9.0. All the ester prodrugs have sufficient chemical stability
in buffers (pH 1- 9), ensuring them to be absorbed as intact moieties from the
gastrointestinal tract (Table 1).
Table 1: Aqueous stability of prodrugs (2,3,6,7,8) in buffer solutions at 40 °C for 4 h

compd formation of bezafibrate in aqueous solution (pg/mL)
pH 1.0 pH 3.0 pH 5.2 pH 7.4 pH 9.0

2 BDL BDL 1.7 2.2 0.79
3 BDL BDL 0.21 0.88 6.6
6 BDL BDL 0.7 1.8 2.5
7 BDL BDL 6.3 6.2 8.8
8 BDL BDL 0.24 0.50 2.6
Results are reported as mean (n = 3). BDL, below detection limit.
231
References
(1) Secondary prevention by raising HDL cholesterol and reducing triglycerides in
patients with coronary artery disease: the Bezafibrate Infarction Prevention (BIP)
study". Circulation 2000,102 (1), 21-27.
(2) Tenenbaum, A.; Metro, M.; Fisman, E. Z.; Tame, D.; Boyko, V.; Behar, S;
"Bezafibrate for the secondary prevention of myocardial infarction in patients
with metabolic syndrome". Archives of internal medicine 2005,165 (10), 1154—
1160.
(3) Tenenbaum, A.; Motro, M.; Fisman, E. Z.; Schwammenthal, E.; Adler, Y.;
Goldenberg, L; Leor, J.; Boyko, V. et al. "Peroxisome proliferator-activated
receptor ligand bezafibrate for prevention of type 2 diabetes mellitus in patients
with coronary artery disease". Circulation 2004,109(18), 2197-2202.
(4) Tenenbaum, A.; Fisman, E. Z.; Boyko, V.; Benderly, M.; Tame, D.; Haim, M.;
Matas, Z.; Motro, M. et al. "Attenuation of progression of insulin resistance in
patients with coronary artery disease by bezafibrate" Archives of internal
medicine 2006,166 (7). 737-744.
232
8.0.0 Fharmacoligical evaluation of fenofibric acid prodrugs
a) Materials & method
Animals: Male Swiss albino mice weighing 22-35 g were obtained from in-house animal
facility of Orchid Research Laboratories and were maintained at a controlled temperature
(23+1 °C) under 12 h light and 12 h dark cycle. All animals were given standard
laboratory chow and water ad libitum.
Materials: The following chemicals were purchased and used for the study.
Carboxy methyl cellulose (CMC)-Sigma Aldrich Chemicals (St Louis, U.S.A.).
Tween 80- Sigma Aldrich Chemicals
Triglyceride kit- ERBA diagnostics
Fenofibrate (API) was gifted by D. K. Pharmachem Pvt Ltd., India.
Instrument Name: Clinical chemistry analyzer
Model No. Erba XL 300
Diagnostic Kits; ERBA diagnostics kits
Experimental Design: In the experiment, hypolipidimic effect of fenofibric acid ester
prodrugs (9, 10, 11, 13, 15, 16 ) and fenofibrate la was carried out in normal animals
(Male Swiss albino mice), Male Swiss albino mice were divided into 8 groups (n = 8-10)
based on their body weight.
Group 1: Normal control untreated SAM received 0.5% CMC (control vehicle)
Group 2: Normal SAM administered fenofibrate (la) orally (50 mg/kg body weight) and
considered as reference drug.
Group 3: Normal SAM administered prodrug No. 9 orally (50 mg/kg body weight)
Hypolopidemic Study: Male Swiss albino mice (SAM) were grouped into 8 groups (n =
8-10) based on their body weight. Animals were dosed orally with fenofibrate, prodrugs
and vehicle for 8 days. After 8 days of treatment animals were fasted for 12 h. On day 9th
after giving respective treatment, blood samples were collected one hour post
233
administration of the drugs. Plasma was separated for the estimation of triglyceride and
cholesterol
Formulation: All the compounds were suspended in vehicle (Tween 80 5pL/mL in 0.5%
CMC, lOmL/kg).
Biochemical analysis: Plasma triglyceride and cholesterol were measured
spectrophotometrically using commercially available ERBA kit.
Statistical analysis: All the data were expressed as Mean + SE. Statistical analysis was
performed using one-way analysis of variance (ANOVA), followed by Dunnett’s test.
Results were expressed as mean ± SE from 8 -10 animals in each group. P- values < 0.05
were considered significant,
b) Results and discussion
Fenofibrate (la) is usually orally administered with maximum dose 120 mg per day for
patient suffering from hypertriglyceridemia. It is poorly and variably absorbed when
taken with food and about 60% of the dose of the conventional form is effectively
absorbed and found in the blood as fenofibric acid. Bioavailability of la is very low when
administered without micronization. With the aim of improving hypolipidemic activity
without micronization/ or special kind of formulation, a series of fenofibric acid ester
prodrugs (9, 10, 11, 13, 15, 16) were synthesized from the fenofibric acid (lb) and
evaluated for lipid lowering potential in male Swiss Albino mice (SAM), a moderate
hypertriglyceridemic model.1'2 The mice were treated with prodrugs (9, 10, 11,13, 15,
Id) and la by oral gavage at a dose of 50 mg/kg body weight for 8 days. The in vivo
profile of compounds (9,10,11,13,15,16) was compared with a reference drag la for
hypolipidemic activity. The daily oral administration of prodrags (9,10,11,13,15,16)
lowered significantly the plasma triglyceride level (TG) by 47.03%, 45.03%, 33.15%,
33.15%, 27.62%, and 27.22% respectively, where as la lowered TG by 48.65% in an
identical conditions (Table 1 and Figure 1). Experimental value of triglyceride reduction
of la are in line with reported earlier.2'3 Prodrags 9 and 10 showed equipotent triglyceride
lowering activity as fenofibrate la and were more potent than rest of the prodrags (11,
13,15,16). Total cholesterol did not show significant changes in the fenofibrate treated
animals, and these values are in line with the literature.2"4 Interestingly, prodrags 9, 10,
234
r
13, and 16 appears to be more potent in total cholesterol lowering activity (7.71%, 9.02%,
7.25%, I U8% ) relative to la (5.47%).
Table 1: Effect of fenofibrate la and prodrugs (9,10,11,13,15,16) on triglyceride level
In Swiss Albino mice (SAM)
Normal
Animal la 9 10 11 13 IS 16
Control
1 125 60 74 53 111 75 84 127
2 173 67 48 80 73 102 65 94
3 109 65 65 46 70 65 76 95
4 160 73 74 68 99 79 73 77
5 129 71 68 53 65 125 1?0 95
6 142 71 71 75 79 76 149 85
7 124 71 73 90 97 86 83
8 133 72 68 75 43 100 116 117
9 93 56 77 83 93 79 83
10 109 60 69 90 137 80 68 88
Mean 129.70 66.60 68.7 71.3 86.70 86.70 93.88 94.40
SE 7.61 1.92 2.56 5.01 838 5.54 9.69 5.02
% Reduction - 48.65 47.03 45.03 33.15 33.15 27.62 27.22
Figure 1: Plasma triglyceride levels In Swiss Albino mice treated via oral gavage for 8 days
with fenofibrate la and prodrugs (9,10,11,13,15,16)
140
16
** P< 0.01 one-way ANOVA followed by Dunnett’s test

Bar represents mean ± SE (n = 8-10 mice per group; **p < 0.05 relative to vehicle
control)
235
Table 2: Effect of Fenofibrate la and Prodrugs (9,10, IX, 13,15,10) on Cholesterol
Level in SAM
Normal
S.No. la 9 10 11 13 15 16
Control
1 127 117 128 101 154 96 122 130
2 131 119 128 133 144 124 120 130
3 126 113 117 114 124 131 120 114
4 137 129 125 108 116 105 99 107
5 111 118 121 111 116 118 133 104
6 124 136 120 125 157 107 134 98
7 125 137 114 132 129 140 98
8 150 120 110 117 136 125 140 128
9 114 122 118 115 122 126 _ 116
10 123 115 116 124 145 131 145 127
Mean 126.80 122.6 119.7 118.0 134.3 120.3 126.63 115.2
SE 3.50 2.69 1.88 3.28 4.80 4.34 4.60 4.12
% Reduction - 5.47 7.71 9.02 -3.55 7.25 237 11.18
Figure 2. Decrease in plasma total cholesterol level in Swiss Albino mice treated via oral
gavage for 8 days with fenofibrate and prodrugs (9,10,11,13,15,16)
TewChMiniel (moMI)
20'
Control la 10 11 13 15 16
Compounds
Bars represent percent total cholesterol changefrom vehicle control and represents the mean ±
SE (n = 8-10 mice per group)
236
c) Metabolic stability of fenofibrate (la), prodrng compound No. 10 and 15 in rat
plasma (RP)
Stability of fenofibrate (la) in Rat P asma
Time (h) Peak Area of la in HPLC % remaining
0 498683 100.0
1 61298 12.3
2 0 0.0
St ability of prodrug No. 10 in rat plasma

Time (h) Peak Area of prodrug in HPLC % remaining
0 65022 100.0
1 53421 82.2
2 0 0.0
SIability of prodrug No. 15 in rat plasma

Time (h) Peak Area of prodrug in HPLC % remaining
0 484143 100.0
1 75724 15.6
2 46790 9.7
Fenofibric acid metabolite area after enzymatic hydrolysis in RP

Time (h) Fenofibrate Prodrug No. 10 Prodrug No. 15
0 13907 158612 64660
1 453443 376157 877230
2 576824 409218 985174

Fenofibrate is a prodrug. It is isopropyl ester of fenofibric acid. We have synthesized
novel esters of fenofibric acid, and evaluated for hypolipidemic activity in comparison
with reference drug fenofibrate. Prodrugs are expected to hydrolyzes in rat plasma and
release fenofibric acid as an active metabolite in 1-3 h. Hence we have performed the
metabolic stability of fenofibrate, and novel prodmg No.10 and 15. It was found that
fenofibrate and prodrugs No.10 and 15 were hydrolyzed to fenofibric acid as active
metabolite within 2 h and fenofibric acid peak area was found increasing with respect to
time, when analyzed by HPLC. Hence these prodrugs are enzymatically labile. Based on
this information these prodrugs were selected to study its hypolipedimic activity in mice.
237
8.1.0 Physicochemical properies of fenofibrate prodrugs
a) Particle size distribution of prodrug compounds
Bioavailability of an active pharmaceutical ingredient (API) administered orally is
depends upon its particle size. Fenofibrate (la) is currently micronized before
formulation to get sufficient bioavailability (30-50%).6'8 The pharmacological profiles of
prodrugs (9, 10, 11, 13, 15, 16) indicate that prodrugs 9 and 10, showed equipotent in
hyperlipidemic activity as reference drug la, even with higher particle size distribution.
Hence it can be concluded that bioavailability of prodrugs 9 and 10 was better than la
even at higher particle size and has significant bioavailability without special kind of
pharmacological formulation like micronization/or spray drying to improve its efficacy
(Table 1).
Table 1: Particle size distribution of prodrags 9,10,11,13,15,16 and fenofibrate (la)
compd particle size distribution (in pm)

< 10% < 50% < 75% <90% < 100%
la 0.5 7.8 9.0 18.4 49
9 1.0 20.6 67.1 303 645
10 0.4 3.4 10.6 34 91
11 0.3 4.4 21.5 45 428
13 2.0 293 473 645 879
15 0.8 6.8 9.4 31 61
16 2.0 28.8 37.1 84 200
b) Partition coefficient (log P)

To examine the fundamental process of the transfer of drag to the biological membrane,
the partition coefficient of prodrugs (9, 10, 11, 13, 15, 16) and the reference drag
fenofibrate (la) was determined by HPLC method in octanol-buffer system at 25 °C.
Partition coefficient of la is in line with the value reported in the literature.4 Prodrags (9,
10, 11,13, and 16) were found lipophilic similar to la, and partition coefficient values
were identical to the reference drag la. Prodrag 15 was found less lipophilic than la.
Ionization of prodrags was not observed during determination of partition coefficient.
Results of log P at pH 7.4 are summarized in Table 1.
238
Table 1: Partition coefficient of fenofibrate la and prodrugs (9,10,11,13,15, and 16) in
aqueous solution
compd partition coefficient (log P)
0.2 M phosphate buffer pH 7.4
la 4.78
5 24tt
la
9 5.43
10 4.70
11 4.65
13 4.91
15 3.50
16 4.35
TResults are expressed as the mean of three tests (n = 3).
^Partition coefficient reported in the literature.5
c) Aqueous solubility of prodrugs

Aqueous solubility is an important physicochemical parameter of an active
pharmaceutical ingredient (API), and its knowledge has fundamental importance in a
wide range of applications and research areas. The orally administered drug should be
stable at various pH environments encountered in the gastrointestinal tract to deliver the
intact prodrug to the systemic circulation. Six fenofibric acid ester pro drugs evaluated
for aqueous solubility at pH 1- 9. Excess quantity of test compounds was stirred in buffer
solution for 4 h at 40 °C on mechanical shaker at 350 rpm, filtered through 0.22 pm filter
and the filtrate solution was analyzed quantitively by HPLC. Prodrugs (9,10,11,13,15,
16) and the reference drug fenofibrate (la) were found stable and practically insoluble in
aqueous solution at pH 1-9. The fenofibrate solubility result was in line with the reported
ealrlier.5 Limit of detection (LOD) and limit of qualification (LOQ) of la and prodrugs
(9, 10, 11, 13, 15, 16) were estimated by HPLC at a wavelength of 254 nm. The
experimental results are depicted in Table. Aqueous solubility of ester prodrug 15 was
comparatively more which could be related to the lower lipophilicity (Table 1).
239
Table 1: Aqueous solubility, limit of detection, limit of qualification of fenofibrate la
and prodrugs (9,10,11,13,15,16) in buffer solutions at 40 °C for 4 h
compd aLOD aLOQ aaqueous solubility (pg/mL)
(pg/mL) (pg/mL) pH 1.0 pH 3.0 pH 5.2 pH 7.4 pH 9.0

la 0.036 0.108 BDL BDL 0.075 0.13 0.24
15 0.28 0.084 BDL BDL 38 1.9 4.8
16 0.0094 0.0284 0.23 BDL BDL BDL BDL
“Experimentally determined. Results are reported as mean (n = 3). BDL, below detection
limit. LOD, limit ofdetection; LOQ, limit ofqualification.
d) Solid state morphology

The existence of multiple crystal forms with differences in the solid state properties can
translate into significant effects on the bioavailability and the shelf life of active
pharmaceutical ingredient (API). Hence it is necessary to know the solid state
morphology of prodrug compounds. Compounds (9, 10, 11, 13, 15, 16) were found
crystalline when-screened in a Broker AXS D8 advance diffractometer. Powder XRD
profile of prodrags is shown in Figure 1,2,3,4,5, and 6.
240
Figure 1. XRD diffractogram for prodrug 9
Un(Counts)

H
8
2-Theta - Scale
241
1
I
ml ^--- /
2-Theta-Scale
242
Figure 5. XRD difiractogram for prodrug 15
2-Tteta - Scale
Figure 6. XRD difiractogram for prodrug 16
afr«4aiB5'
hs
-
6
2-Theta - Scale
243
References
(1) Reddy, K. A.; Lohray, B. B.; Bhushan, V.; Reddy, A. S.; Rao Mamidi, N. V.;
Reddy, P. P.; Saibaba, V.; Reddy, N. J.; Suryaprakash, A.; Misra, P.;
Vikramadithyan, R. K.; Rajagopalan, R. J. Med. Chem. 1999,42,3265- 3278.
(2) Dhalwal, K.; Shinde, V. M.; Singh, B.; Mahadik, K. R. International Journal of
Phytomedicine 2010,2,160-165.
(3) Dawn, A. B.; Garret, J. E.; Christopher, J. R.; Antony, J. S.; Samual, J. D.; Aaln,
M. W.; Robert, A.; Janies, R. P.; John, T.; Donald, S. K.; Raymond, F. K.; Carol, L.
B.; Brian, A. O.; Chahzrad, M. R.; Leonard, L. W.; Margaret, M. F.; James, R. M.
J. Med. Chem. 2001,44,2061-2064.
(4) Srivastava, R. A. K. Eur. J. Pharmacol, 2009,607,258-263.
(5) Gidwani, S. K.; Singnurkar, P. S. U.S. Patent 6828334,2004.
(6) Keating, G. M.; Ormrod, D. Drug 2002,62,1-36.
(7) Adkins, J. C.; Faulds, D. Drugs 1997,54,615-633.
(8) Munoz, A.; Guichard, J. P.; Reginault, P. H. Atherosclerosis 1999,110, S45-S48.
244
List of publications
(1) Synthesis and Biological Evaluation of Orally Active Prodrugs of Indomethacin
Babasaheb P. Bandgar* Rajendra Janardan Sarangdhar, Santosh Viswakarma and

Fakradeen AH Ahmad. J. Med. Chem., 2011,54 (5), 1191-1201.
(2) Synthesis, Characterization, and Biological Evaluation of Novel Diclofenac

Prodrugs
Babasaheb P. Bandgar,* Rajendra Janardan Sarangdhar, Fakrudeen Ali Ahamed and
Santosh Viswakarma. J. Med Chem., 2011,54 (5), 1202-1210.
(3) Synthesis and Biological Evaluation of Orally active Hypolipidemic agents
Babasaheb P. Bandgar,* Rajendra Janardan Sarangdhar, Jeyamurugan Mookkan,

Pranesha Shetty, Gajendra Singh, and Khan Fruthous. J. Med. Chem. (Communicated)
(4) Synthesis and Biological Evaluation of Orally Active Prodrugs of Bezafibrate
Babasaheb P. Bandgar,* Rajendra Janardan Sarangdhar, Khan Fruthous,

Jeyamurugan Mookkan, and Pranesha Shetty. J. Med. Chem. (Communicated)
(5) Synthesis and Biological Evaluation of Orally Active Prodrugs of Flurbiprofen
Babasaheb P. Bandgar* Rajendra Janardan Sarangdhar, and Santosh Vlshwakarma.

J. Med. Chem. (Communicated)
245
List of presentations
1) Chemistry and Biology of Novel and Potent Anti-inflammatory and Anti

cholesterol prodrugs useful for Mankind. International Conference on Chemistry
for Mankind: Innovative Ideas in Life Sciences. 9® to 11* February 2011(ICCM-
2011). Rashtrasant Tukadoji Maharaj, Nagpur University, Nagpur, India.
2) Chemistry and Biology of Novel and Potent Anti-inflammatory prodrugs. First

zonal meeting of chemical research society of India, for celebration of international
year of chemistry-2011, National Chemical Laboratory (NCL), Pune. 13* -14* May,
2011. Page No. 19.
246

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Chapter: Five

Biological Activity of Non-steroidal Anti-inflammatory & Anti-

Section 1: Biological Activity of Non-steroidal Anti­

Inflammation is part of the complex biological response of vascular tissues to

1) Non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin, ibuprofen,

nonimmune, well researched, and highly reproducible.

Results and discussion

groups dose (mg/kg) paw volume (mL) % inhibition at 180 min

Acute ulcerogenic assay

compound number of level I level II level III

Animals in each group (n = 6) was treated with 1, developed an average of 41

groups dose (mg/kg) ulcer index (UI)a

5.3.1 Procedure for metabolic stability of indomethacin prodrugs in RLM

metabolic stability in RLM metabolic stability in RP

formation of indomethacin parent compound formation of indomethacin parent

Table 2: Disappearance of prodrugs by metabolic transformation

prodrug peak area in RLM prodrug peak area in RP

5.3.2 Physicochemical properties of indomethacin prodrugs

b) Determination of partition coefficient (log P)

log fl-v— - log <)

Results and discusion

c) Solid state morphology

Figure 2: XRD diffractogram for prodrug 5

i i » t i ■i"i... i'—t...r"'T-r —| i------*----- 1----- 1 | » i------1 r- | i i i

Figure 4: XRD diffractogram for prodrag 8

a) Carrageenan-induced paw edema in rat

Ressults and discusion

la 50 157 ***2.88± 0.12B 49.17

b) Acute ulcerogenic assay

Animals treated with diclofenac sodium developed an average of 50 ulcerogenic

aResults are expressed as a mean ± SEM(n = 6).

b) Metabolic stability in rat liver microsomes (RLM)

immediately in the experiments. Protein concentrations were determined by the Biorad

Results and discusion

compound % remaining in RLMa compound % remaining in RPa

Table 3: Disappearance of prodrugs (10,13,14) by enzymatic biotransformation in RLM

pH Medium® Solubility pg/mL

3.0 0.1 M Citric acid buffer - 1.785 BDL BDL BDL

5.5 Acetate buffer 36.0 - BDL BDL BDL

Table 1: Partition coefficient of diclofenac sodium (la) and prodrugs (10,13,14)

Figure 1: XRD diffractogram for prodrug 10

Figure 3: XRD diffractogram for prodrag 14

“pp" iwtm :OCEUA»DO«ra

Stability of Ibuprofen in rat plasma

Stability of compound No.18 in rat plasma

Results and discusion

Stability of flurbiprofen in rat plasma

Stability of prodrug No. 26 in rat plasma

Stability of prodrug No. 32 in rat plasma

Results and discusion

0.5 1710246 1326156 103.4 99.2 100.0

1 1513881 1694820 91.6 126.8 100.0

Stability of ketorolac tromethamine (1) in rat plasma

Stability of prodrug No. 46 in RLM

Stability of prodrug No. 46 in rat plasma

Results and discussion

Fibrates have been used for the treatment of hypertriglyceridemia or mixed

cholesterol metabolism. The decrease in plasma triglycerides induced by fibrates has

clearance of triglycerides results from a stimulation of the expression of lipoprotein lipase

Section 1: Biological Activity of Non-steroidal Anti

1) Chemistry and Biology of Novel and Potent Anti-inflammatory and Anti