Studies on synthesis, stability, release and pharmacodynamic profile of a novel diacerein-thymol prodrug

Article abstract of DOI:10.1016/j.bmcl.2012.11.016

Involvement of oxidative stress, leading to chondrocyte senescence and cartilage ageing has been implicated in the pathogenesis of osteoarthritis (OA). New efforts to prevent the development and progression of OA include strategies and interventions aimed at reducing oxidative damage in articular cartilage using antioxidants as adjuncts to conservative therapy. Diacerein is an anthraquinone derivative with a marked disease modifying effect on OA owing to IL-1 β inhibition. In the present work an attempt was made at design and development of a co-drug of diacerein with antioxidant thymol. Structural elucidation was carried out by spectral analysis. When release kinetics of prodrug was studied in phosphate buffer (pH 7.4) and small intestinal homogenates of rats, 91% and 94% diacerein was available respectively at the end of 4.5 h. Chemical linkage of thymol with diacerein improved its lipophilicity and hence bioavailability. Screening of prodrug in Freud's adjuvant-induced arthritis and ulcerogenic potential by Rainsford's cold stress model exhibited significant reduction in paw volume, joint diameter and ulcer index with superior anti-inflammatory/anti-arthritic activities than the standards. Results of histopathology of tibio-tarsal joint indicated that animals treated with diacerein exhibited moderate synovitis while thymol and physical mixture-treated animals showed mild synovitis. Interestingly in prodrug-treated animals synovitis was not observed. The results of this study underline the promising potential of co-drug of diacerein and thymol in the management of OA.

Full text of DOI:10.1016/j.bmcl.2012.11.016

Bioorganic & Medicinal Chemistry Letters 23 (2013) 55–61
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Studies on synthesis, stability, release and pharmacodynamic profile of a novel
diacerein-thymol prodrug
Suneela Dhaneshwar a, , Vriha Patel a, Dipmala Patil a, Gourav Meena b
⇑
a Department of Pharmaceutical Chemistry, Bharati Vidyapeeth Deemed University, Poona College of Pharmacy, Pune 411038, Maharashtra, India
b Department of Pharmacology, Bharati Vidyapeeth Deemed University, Poona College of Pharmacy, Pune 411038, Maharashtra, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Involvement of oxidative stress, leading to chondrocyte senescence and cartilage ageing has been impli-
cated in the pathogenesis of osteoarthritis (OA). New efforts to prevent the development and progression
of OA include strategies and interventions aimed at reducing oxidative damage in articular cartilage using
antioxidants as adjuncts to conservative therapy. Diacerein is an anthraquinone derivative with a marked
disease modifying effect on OA owing to IL-1 b inhibition. In the present work an attempt was made at
design and development of a co-drug of diacerein with antioxidant thymol. Structural elucidation was
carried out by spectral analysis. When release kinetics of prodrug was studied in phosphate buffer (pH
7.4) and small intestinal homogenates of rats, 91% and 94% diacerein was available respectively at the
end of 4.5 h. Chemical linkage of thymol with diacerein improved its lipophilicity and hence bioavailabil-
ity. Screening of prodrug in Freud’s adjuvant-induced arthritis and ulcerogenic potential by Rainsford’s
cold stress model exhibited significant reduction in paw volume, joint diameter and ulcer index with
superior anti-inflammatory/anti-arthritic activities than the standards. Results of histopathology of
tibio-tarsal joint indicated that animals treated with diacerein exhibited moderate synovitis while thy-
mol and physical mixture-treated animals showed mild synovitis. Interestingly in prodrug-treated ani-
mals synovitis was not observed. The results of this study underline the promising potential of co-
drug of diacerein and thymol in the management of OA.
Received 13 October 2012
Revised 3 November 2012
Accepted 7 November 2012
Available online 16 November 2012
Keywords:
Co-drug
Diacerein
Antioxidant
IL-1-beta inhibitor
Thymol
Osteoarthritis
Ulcerogenicity
Oxidative stress
Ó 2012 Elsevier Ltd. All rights reserved.
Osteoarthritis (OA) is a degenerative type of arthritis in which
the biomechanical properties of cartilage are altered due to its
break down in synovial joints by local proteases. The involvement
of mechanical and cytokine-mediated pathways has been empha-
sized in cartilage degeneration and pathogenesis of OA.1 Signs
and symptoms of inflammation, joint pain, swelling and stiffness
causing significant functional impairment and disability are the
highlights of OA. A common feature is infiltration of activated B
cells and T lymphocytes and overexpression of proinflammatory
mediators in early and late OA causing synovitis. This contributes
to dysregulation of chondrocyte function that disturbs the balance
between the catabolic and anabolic activities of the chondrocytes
which is normally involved in remodeling the cartilage. There is
a direct co-relationship between release of inflammatory media-
tors like prostaglandins, nitric oxide, IL-1b and tumor necrosis fac-
OA. Inducible nitric oxide synthase (iNOS), cyclooxygenase-2
(COX-2), microsomal prostaglandin E synthase-1 (mPGEs-1), and
matrix metalloproteinases are the important pro-inflammatory
mediators whose synthesis is stimulated by interleukin IL-1b, pro-
moting the release of nitric oxide (NO) and prostaglandin E2
(PGE2).3–6 Anabolic activities in chondrocytes are also retarded
by IL-1 b causing decreased proteoglycan and collagen synthe-
sis.7–9
The treatment is aimed at relieving pain and suffering, mainte-
nance and restoration of function and ultimately prevention of dis-
ease progression.10 The current treatment modalities for OA are
either conservative or surgical. Conservative measures promi-
nently involve pharmacological intervention in the form of nonste-
roidal anti-inflammatory drugs (NSAIDs), intra-articular steroid
injections or relatively new option of injection of hyaluronan
which improves joint lubrication and can decrease pain. All these
current treatments are basically symptomatic and do not retard
destruction of articular cartilage. Therefore, there is an urgent need
for therapeutic modalities to protect or induce regeneration of car-
tilage on a cellular level by restoring its structural integrity and
function. Disease modifying anti-rheumatic drugs (DMARDS) are
drugs that not only manage symptoms but also favorably affect
joint structure changes over long-term treatment periods and thus
tor-a (TNF)-a in OA synovial fluid and joint tissue. The underlying
mechanism is not well documented but involvement of abnormal
mechanical, and oxidative stresses is indicated.2
Cartilage matrix component’s degeneration and excessive pro-
duction of different cytokines are the most prominent features of
⇑
Corresponding author. Tel.: +91 20 25437237/25436898; fax: +91 20 25439383.
E-mail address: suneeladhaneshwar@rediffmail.com (S. Dhaneshwar).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.11.016

56
S. Dhaneshwar et al. / Bioorg. Med. Chem. Lett. 23 (2013) 55–61
slow or arrest disease progression, so also called structure modify-
ing drugs.11 Therefore, out of the critical need to develop alterna-
tive agents that prevent the destruction of cartilage and/or
stimulate its proper repair, DMARDS came into picture.12
the risk of NSAIDs-induced GI toxicity.27 Thymol is reported to
possess anti-oxidant/anti-inflammatory activity.28–31 Thymol is
listed by the US Food and Drug Administration (US-FDA) as a food
additive on the ‘generally recognized as safe’ (GRAS) list therefore
it would be nontoxic. Thymol is hydrophobic in nature30 that
might enhance lipophilicity of diacerein, in turn increasing its
transcellular absorption.
In the present work, carboxylic group of diacerein was masked
in a transient manner with antioxidant thymol. We expected that
this conjugation would increase lipophilicity and absorption of
diacerein, decrease the gastric irritant effect and enhance it is
anti-inflammatory activity by reducing the oxidative stress in OA.
Evaluation and comparison of the efficacy of this novel mutual pro-
drug with individual drugs and their physical mixture was also one
of the objectives of this work.
Thymol was purchased from LOBA Chemie, Mumbai, India
while diacerein was obtained as a gift sample from Glenmark Phar-
maceutical Pvt. Ltd, Mumbai, India. Synthetic procedures were
optimized on Radley’s 6-station parallel synthesizer. Thin layer
chromatography was performed on pre-coated silica gel plates-
60 F264 (Merck) for purity check and monitoring of reactions.
The IR spectrum of the synthesized compound was recorded on
Jasco V-530 FTIR in anhydrous IR grade potassium bromide. The
1H NMR and 13C NMR spectra were recorded on Bruker Avance II
400 NMR at Sophisticated Analytical Instrument Facility (SAIF),
Panjab University, Chandigarh. Chemical shift values are reported
The pathophysiology of OA is complex with interplay of many
converging mechanisms. The results of study performed by Sura-
paneni et al. suggest higher oxygen-free radical production and
oxidative stress in OA.13 Numerous reports have demonstrated
that oxidative damage due to the over-production of nitric oxide
(NO) and other reactive oxygen species (ROS) may be playing a sig-
nificant role in the pathogenesis of OA and also in the formation of
gastric mucosal lesions associated with NSAIDs therapy.14–16
The current, modern OA therapy has its own limitations in pre-
venting joint destruction which is grossly focused on inhibiting
formation of inflammatory mediators such as prostaglandins and
leukotrienes with anti-inflammatory agents. With the understand-
ing of the significant role of antioxidants in arthritis, they are gain-
ing increasing importance as an adjunct therapy as they would
help in protecting the biological tissues below a critical threshold
of reactive oxygen species17,18 by forming a mutually supportive
defense team against ROS in OA. Impaired antioxidant defense
and increased lipid peroxidation suggest that treatment with anti-
oxidants at the initial stages of illness may prevent further oxida-
tive injury and deterioration of associated musculoskeletal deficits
in OA.19 Oxidative stress leads to increased risk for OA but the pre-
cise mechanism remains unclear. The findings of Yudoh et al.
(2005) clearly show that the presence of oxidative stress induces
dysfunction of chondrocytes in OA cartilage, suggesting that oxida-
tive stress, leading to chondrocyte senescence and cartilage ageing,
might be responsible for the development of OA.20 New efforts to
prevent the development and progression of OA may include strat-
egies and interventions aimed at reducing oxidative damage in
articular cartilage.
in ppm downfield on d scale. The absorbance maximum (kmax
)
was determined on Jasco V-530 UV–Visible double-beam spectro-
photometer. Partition coefficient was determined experimentally
in n-octanol/phosphate buffer (pH 7.4) by flask shake method. In
vitro release of diacerein from its prodrug and logP values were
determined using Jasco V-530 UV–Visible double-beam spectro-
photometer. Institutional Animal Ethical Committee’s approved
experimental protocols were followed for pharmacological screen-
ing of synthesized prodrug which was carried out with at the CPC-
SEA-approved animal facilities of Poona College of Pharmacy, Pune,
India. Anti-arthritic activity was evaluated by Freund’s adjuvant-
induced arthritis using Complete Freund’s adjuvant (F-5881, Sig-
ma–Aldrich Corporation, USA). The body weight, joint diameter
and paw volumes (both the paws) were measured using weighing
balance, digital vernier caliper and UGO BASILE Plethysmometer
7140, Italy respectively. The photomicrographs were taken on in-
verted microscope attached with digital olympus model no: E-
PL1 camera with 40ꢀ resolution.
In the last decade scientists have proven that some antioxidants
have anti-inflammatory properties. In addition to scavenging free
radicals, there are antioxidants that actually block inflammation.
The antioxidant effect (the blocking of certain oxidizing proteins)
lowers the activation of inflammatory signals.21
Diacerein is a very recently introduced, symptomatic slow act-
ing disease modifying IL-1b inhibitor, known to possess antiar-
thritic and moderate anti-inflammatory, antipyretic and analgesic
activities.22 The mechanism of action of diacerein differs from
those of NSAIDs or corticosteroids. Neither diacerein nor its active
metabolite rhein, inhibit prostaglandin biosynthesis; indeed, cyclo-
oxygenase/lipoxygenase pathways. This unique feature seems to
be the reason for the excellent gastric safety profile of diacerein
during OA treatment. But according to the theory of Cioli et al.
diacerein might produce local irritant effect on the gastric mucosa
due to its free carboxylic group.23 It is reported that diacerein’s dis-
ease modifying effect is more prominent while its anti-inflamma-
tory effect is mild to moderate with a late onset.22
Dhaneshwar et al. (2009, 2012) have reported mutual prodrugs
of diacerein with aminosugar as well as amino acids possessing
lowered ulcerogenic tendency and more aqueous solubility.
These prodrugs had quicker onset of action as compared to
diacerein.24–26 The present work was inspired by promising results
of our earlier work. The role of increased levels of ROS and oxidative
stress is well documented in the literature. Antioxidants have
been suggested as a secondary therapy aimed at limiting tissue
destruction.18 Therefore, an antioxidant carrier; thymol was chosen
as a carrier for its covalent linkage with –COOH group of diacerein
so as to develop its mutual prodrug with reduced local irritant
effect, improved absorption, prolonged release of drug and
enhanced anti-inflammatory effect. It has been suggested that
co-administration of antioxidants and NSAIDs in formulated dosage
forms may possibly suppress the progression of OA and decrease
Prodrug of diacerein with thymol (DTH) was synthesized by
DCC coupling method32 (Fig. 1) and then characterization of its
physico-chemical properties was performed by spectral analysis.33
In vitro release profile of synthesized prodrug was investigated
in aqueous buffers (HCl buffer pH 1.2 and phosphate buffer pH 7.4)
and tissue homogenates of upper gastro-intestinal tract (GIT). As
the difference between k max of diacerein (258 nm) and DTH
(230 nm) was substantial (28 nm), a new method was developed
for simultaneous estimation of diacerein in presence of its prodrug
DTH by employing UV spectrophotometer. The method was vali-
dated as per ICH guidelines and carried out in triplicate. The K val-
ues from the plots were calculated separately and average K and SD
value was determined.
Release kinetics of the prodrug was studied in aqueous buffers
of varied pH. DTH (10 mg) was introduced in 100 ml of HCl buffer34
taken in different beakers kept in a constant temperature bath at
37 1 °C. The solutions were occasionally stirred and 5 ml aliquot
portions were withdrawn at various time intervals (0–180 min)
each time replenishing with fresh 5 ml HCl buffer (pH 1.2). The ali-
quots were directly estimated on UV spectrophotometer at 232 nm
for the amount of DTH remaining and at 259 nm for released
diacerein. Same procedure as above was followed to study release

S. Dhaneshwar et al. / Bioorg. Med. Chem. Lett. 23 (2013) 55–61
57
B
C
A
B
A
C
D
Figure 1. Scheme of synthesis.
kinetics in phosphate buffer pH 7.4 except that the HCl buffer was
replaced by phosphate buffer pH 7.435 and release of diacerein was
monitored over a period of 4.5 h at 230 nm for the amount of DTH
remaining and at 258 nm for amount of diacerein released.
To study the release kinetics in upper GI homogenate, a male
Wistar rat was anesthetized by ether and midline incision was
made. The stomach and small intestine were separated out,
homogenized with their contents and diluted to half concentration
with isotonic hydrochloric acid buffer (pH 1.2) and phosphate buf-
fer (pH 7.4) respectively. DTH was weighed (10 mg) and dissolved
overall kinetic studies are compiled in Table 1 and depicted graph-
ically in Figure 2.
Anti-arthritic effect of DTH was investigated in Freund’s adju-
vant—induced arthritis as per protocols described by Vogel35 and
our earlier publication24 Male Wistar rats (200–250 g) were di-
vided into 6 groups viz healthy control, arthritic control which re-
ceived FCA injection sub-plantar (day 1) + 0.25% of sodium CMC
suspension p.o. (day 13–28), reference standard diacerein at the
dose 5 mg/kg p.o. (day 13–28), thymol at the dose of 2.04/kg p.o.
(day 13–28), physical mixture of diacerein and thymol at the dose
in HCl buffer (pH 1.2) and volume was made up to 10 ml (1000
ml). 1 ml of this solution was added to 10 ml volumetric flask and
volume was made up to 10 ml with HCl buffer (pH 1.2) (100 g/
lg/
Table 1
l
Kinetics data for release of diacerein from its prodrug DTH at 37 °C
ml). This was considered as the stock solution. To each eppendorf
tube (1 ml); 0.8 ml of the stock solution of prodrug and 0.2 ml of
stomach homogenate was added and kept in incubator at 37 °C.
The Eppendorf tubes were taken out at appropriate time intervals,
centrifuged at 10,000 rpm at 4 °C for 10 min, filtered through
membrane filter and estimated at 232 nm and 259 nm taking HCl
buffer as blank. Concentrations of DTH/diacerein at respective
times were calculated from equations of their calibration curves
in HCl buffer. For release studies in small intestinal homogenates,
same procedure as above was employed for 4.5 h. Absorbance
was observed at wavelength of 230 nm (for DTH) and 258 nm
(for diacerein) taking phosphate buffer as blank. The results of
a
Medium
Order of t1/2
K (minꢁ1
)
% Diacerein
released over
4.5 h
kinetics
(min)
HCl buffer
(pH 1.2)
Stomach
homogenates
Phosphate buffer First
(pH 7.4)
—
—
—
—
—
Stable
Stable
—
193
168
3.59 ꢀ 10ꢁ2 0.0001 90.6
4.12 ꢀ 10ꢁ2 0.0001 93.7
Small intestine
homogenates
First
a
Average of three readings.

58
S. Dhaneshwar et al. / Bioorg. Med. Chem. Lett. 23 (2013) 55–61
subsequently stained with haematoxylin and eosin for evaluation
under light microscope. Sections were examined for the presence
of hyperplasia of synovium, pannus formation and destruction of
joint space. The photomicrographs were taken on inverted micro-
scope attached with digital olympus model no: E-PL1 camera with
40ꢀ resolution. The photomicrographs of histopathological studies
of rat ankle joint are depicted in Figure 7.
Orally administered diacerein induces moderate ulceration in
the upper GIT due to its carboxylic group, temporary masking of
which might lower its propensity for producing local ulcers. So
to test our hypothesis, DTH was screened for its gastro-protective
effect on GIT mucosa at ten times higher dose by Rainsford’s cold
stress method36 as per protocols described by Dhaneshwar
et al.24 Suspensions of diacerein and test compounds in 0.25%
CMC were administered orally. Male Wistar rats (200–250 g) were
fasted overnight and divided into different groups (n = 6). Animals
were treated with diacerein (50 mg/kg, p.o.) and equimolar dose of
DTH (68 mg/kg) and physical mixture of diacerein + thymol
(50 + 20.4 mg/kg). Control group was treated with an equal volume
of 0.5% CMC solution. Images of stomach dissected from the sacri-
ficed rats were captured using a Crystal Clear Display (CCD) Scan-
ner (UMAX ASTRA 5600) at magnification of 2400 dpi. Images were
analyzed using adobe photoshop and image software to calculate
ulcer area and total area of each stomach. Ulcers were scored as
per Cioli’s scoring method.24,37 Average of six readings was calcu-
lated and all data was expressed as mean SEM (Table 2). Statistical
differences between the groups were calculated by One-Way ANO-
VA followed by Dunnett’s post hoc test. Differences were consid-
ered at a p value of <0.01.
The logP (n-octanol: phosphate buffer pH-7.4) of DTH was
found to be higher (0.565) than diacerein (0.46) which would en-
sure better absorption and increased bioavailability in the body be-
cause of enhanced lipophilicity. The IR spectrum of the synthesized
compound showed absorption band at 1780 cmꢁ1 characteristic of
C@O stretching of ester. Absence of phenolic OH stretching at
3219 cmꢁ1 (thymol) and carboxylic OH stretching at 2553 cmꢁ1
(diacerein) further confirmed the formation of an ester. The 1H
NMR spectrum of DTH showed chemical shifts for protons of iso-
propyl and methyl groups (thymol) as well as aromatic protons
and protons of methyl ester (diacerein) which were characteristic
of the anticipated structure of the prodrug. Chemical shifts for pro-
ton of –OH of –COOH of diacerein at 11 ppm and phenolic –OH of
thymol at 5 ppm were totally absent in 1H NMR of DTH that
Figure 2. Release kinetics of DTH prodrug in phosphate buffer (pH 7.4) and small
intestinal homogenates at 37 °C.
of 7.04 (5 + 2.04) mg/kg p.o. (day 13–28) and DTH at the dose of
6.8 mg/kg p.o. (day 13–28). The dose of prodrug was calculated
on an equimolar basis to diacerein. Parameters studied under this
model were change in body weight, joint diameter, paw volume
and percent inhibition of edema which were measured on 0th,
3rd, 7th, 12th, 15th, 19th, 21st and 28th days using weighing bal-
ance, digital vernier caliper and UGO BASILE Plethysmometer 7140,
Italy, respectively. Percent inhibition of edema was determined by
the following formula:
%Inhibition of edema ¼ 1 ꢁ ðVt=V Þ ꢀ 100
c
where Vt is the mean relative change in paw volume in test/
standard group and Vc is the mean relative change in paw volume
in arthritic control group. Average of six readings was calculated
and all data were expressed as mean SEM. Statistical differences
between the groups were analyzed using Graphpad Prism 5.0 soft-
ware by two way ANOVA followed by BonFerroni’s post test. The
results of various parameters of anti-arthritic activity are summa-
rized in Figures. 3–6.
All the animals were sacrificed on 28th day and samples of an-
kle joints were sent for histological studies. Ankle joints were sep-
arated from the hind paw, weighed and immersed in 10% buffered
formalin for 24 h followed by decalcification in 5% formic acid,
processed for paraffin embedding sectioned at 5
l thickness and
Figure 3. Effect of prodrug on improvement of body weight (g) of rats in FCA-induced arthritis. Values are expressed as mean SEM of six readings, ⁄p <0.05, ⁄⁄p <0.01, ⁄⁄⁄
<0.001 when compared to arthritic control and ###p <0.001 when compared to healthy control.
p

S. Dhaneshwar et al. / Bioorg. Med. Chem. Lett. 23 (2013) 55–61
59
Figure 4. Effect of prodrug on reduction of left joint diameter of rats in FCA-induced arthritis. Values are expressed as mean SEM of six readings, ⁄p <0.05, ⁄⁄p <0.01,
⁄⁄⁄p <0.001 when compared to arthritic control and ###p <0.001 when compared to healthy control.
Figure 5. Effect of prodrug on change in left paw volume of rats in FCA-induced arthritis. Values are expressed as mean SEM of six readings, ⁄p <0.05, ⁄⁄p <0.01, ⁄⁄⁄p <0.001
when compared to arthritic control and ###p <0.001 when compared to healthy control.
Figure 6. Graph plot showing percent inhibition of edema in FCA induced arthritis model.

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S. Dhaneshwar et al. / Bioorg. Med. Chem. Lett. 23 (2013) 55–61
Figure 7. Photomicrographs of histopathological studies of rat ankle joint. Healthy control (HC): No cellular infiltration, no bone necrosis, no connective tissue proliferation,
no edema no adjacent tissue involvement. Arthritic control (AC): Cellular infiltration, necrosis of bone, connective tissue proliferation, edema, adjacent tissue involvement.
Diacerein (D): Cellular infiltration, necrosis of bone, connective tissue proliferation, edema, adjacent tissue involvement. Thymol (T): Cellular infiltration, necrosis of bone,
connective tissue proliferation, edema, adjacent tissue involvement. Physical mixture (D+T): No cellular infiltration, no bone necrosis, no connective tissue proliferation, no
edema, no adjacent tissue involvement. DTH: No cellular infiltration, no bone necrosis, no connective tissue proliferation, no edema, no adjacent tissue involvement.
Table 2
Results of ulcerogenic activity
with the similar behavior in HCl buffer (pH 1.2). However 94.3%
release of diacerein was furnished in small intestinal homogenate
Compounds
Dose (mg/kg)a
Ulcer index SD**
at the end of 4.5 h which supports hydrolysis of DTH in phosphate
buffer (pH 7.4). The rate of hydrolysis of DTH in small intestinal
homogenate was more as compared to phosphate buffer (pH
7.4). These results indicate that hydrolysis of ester (DTH) was pH
(weakly basic) as well as enzyme (esterase)—mediated.
Statistically significant changes in various parameters were ob-
served thirteen days after administration of 0.1 ml FCA injection
into the sub-plantar region of left hind paw. During the course of
28 days, weight in healthy control animals increased owing to reg-
ular feed and normal growth while in arthritis control the weight
decreased due to full blown development of arthritis. Diacerein,
physical mixture (D + T) and DTH increased the body weight near
to normal (baseline value) while thymol had a mild effect on body
weight.
In arthritic control, joint diameter increased slowly from 3rd
day onwards reaching a maximum on 28th day indicating severity
of inflammation in joint. Diacerein-treated group showed a gross
reduction in joint diameter from 15th day onwards reaching a min-
imum on 28th, indicating significant anti-inflammatory activity.
DTH brought joint diameter almost near the baseline value. Effect
of DTH was more pronounced than diacerein, thymol and physical
mixture.
Healthy control
Diacerein
Thymol
Physical mixture
DTH
—
50
20.4
50 + 20.4
68
1.78 0.60
6.03 0.15
1.98 0.70
5.90 0.12
4.17 1.03
a
Dose 10 times the equimolar dose.
**
Average of six readings; p <0.01.
confirmed formation of ester linkage. The 13C NMR of DTH exhib-
ited characteristic chemical shifts for aromatic, isopropyl and
methyl carbons which also confirmed the structure of DTH.
Stability of DTH was studied in HCl buffer (pH 1.2) and phos-
phate buffer (pH 7.4) simulating the environment in stomach
and small intestine respectively. DTH was stable in HCl buffer
(pH 1.2) till 3 h and acid resistant. In phosphate buffer (pH 7.4),
hydrolysis of DTH started at around 30 min after introduction with
total 90.6% release of diacerein at the end of 4.5 h following first or-
der kinetics. To simulate the enzymatic environment of GIT, release
of diacerein from DTH was also studied in stomach and intestinal
homogenates of rat that inherently contain esterases which are in-
volved in the hydrolysis of ester linkage. Negligible hydrolysis of
DTH was observed in stomach homogenate which is in accordance
Change in paw volume was calculated as a difference between
paw volume of each day and paw volume of 0th day. Therefore,

S. Dhaneshwar et al. / Bioorg. Med. Chem. Lett. 23 (2013) 55–61
61
the group showing minimum change indicates maximum
Acknowledgements
anti-inflammatory activity because the minimum change implies
that the inflamed/swollen paw was almost brought near to the
normal condition. In this respect, DTH exhibited maximum protec-
tion against inflammation. Physical mixture, diacerein and thymol
also had significant anti-inflammatory effect which was less than
DTH. On 28th day, diacerein offered 57% inhibition of inflammation
while plain thymol offered 29.4% inhibition, physical mixture
offered more protection against inflammation (68%) while the pro-
drug exhibited highest protective effect in terms of 79% inhibition
of edema.
After 28 days study, all animals were sacrificed and samples of
ankle joints were sent for histopathological studies. The histopa-
thological evaluation of tibio-tarsal joint of healthy control animal
showed unremarkable synovial tissue without any evidence of
inflammation or granuloma formation. The section exhibited nor-
mal architecture characterized by normal muscle, subcutaneous
and synovial tissue. Sections of joints of arthritic control group
indicated inflammation and granuloma formation along with
inflammatory cell infiltration in the subcutaneous tissue and
chronic mononuclear inflammatory cell infiltration in synovial tis-
sue indicating chronic synovitis with subcutaneous inflammation.
The impressions observed for diacerein and thymol-treated groups
were almost similar with respect to subcutaneous inflammation.
However, the former showed moderate synovitis and the latter
showed mild synovitis. Animals treated with physical mixture of
diacerein and thymol indicated inflammatory cell infiltration in
sub-cutaneous tissue and chronic mononuclear inflammatory cell
infiltration in synovial tissue. The overall impression was of mild
synovitis with subcutaneous inflammation. Animals treated with
DTH exhibited no inflammatory cell infiltration in the sub-cutane-
ous tissue with mild mononuclear cell infiltration in the synovial
tissue. The impression was absence of synovitis with subcutaneous
inflammation.
Authors thank the UGC for financial assistance and Glenmark
Pharmaceutical Pvt. Ltd., Mumbai, India, for the gift sample of
diacerein.
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The results of ulcerogenic activity (Table 2) revealed that diace-
rein when directly administered orally in plain form, showed high-
er ulcer index (6.03 0.15), whereas DTH exhibited lower
ulcerogenic potential (ulcer index: 4.17 1.03) which may be as-
cribed to temporary masking of carboxylic group and stability of
prodrug at the acidic pH of stomach.
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In conclusion, conjugation of diacerein with thymol increased
its lipophilicity, which might be responsible for its better
absorption and enhanced bioavailability. The prodrug was stable
in acidic pH and stomach homogenates while almost complete
release of diacerein was observed (91–94%) in phosphate buffer
(pH 7.4) and small intestinal homogenates. Selection of thymol
as an antioxidant carrier in mutual prodrug design was justified
as the prodrug brought about gross reduction in joint diameter,
paw volume and ulcer index and exhibited better anti-inflam-
matory/anti-arthritic effects than standard drugs and their
physical mixture which could be ascribed to positive contribu-
tion of thymol. The chronic synovitis induced by FCA in rats
was significantly brought back to normal by marked anti-
arthritic effect of DTH in comparison to moderate effects of
diacerein and physical mixture while thymol showed mild
anti-arthritic effect. It can be concluded that combination ther-
apy of disease modifying agent with antioxidant in the form of
co-drug could be a promising approach in the effective manage-
ment of RA.
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33. DTH: mp 256 °C (d; uncorrected), Rf 0.73 (Toluene: methanol; 7:1 v/v) yield
73.54%, LogP 0.565, IR (ˆ, cmꢁ1, KBr): 2918, 2851 (aromatic C–H stretching),
1780 (C@O stretching conjugated ester), 1745(C@O stretching non-conjugated
ester), 1701, 1672 (C@O cyclic ketone of anthraquinone), 1311, 1269, 1244 (C–
O stretching ester), 1450, 1381(CH3 bending), 1047–640 (aromatic C–H out of
plane bending). 1H NMR (d, ppm, DMSO-d6): 1.18–1.29; 2 ꢀ CH3; isopropyl
group [d; 6H], 1.71–1.77; 2 ꢀ CH3; acetyloxy group [s; 6H], 2.48; CH3; benzillic
methyl [s, 3H], 3.23–3.35; CH; benzillic methine [m; 1H], 7.35–7.97 ring
protons [m, 8H]. 13C NMR (d, ppm, DMSO-d6): 122.09–137.36; 12 ꢀ C aromatic
rings A & C; 191.77, 191.94; 2 ꢀ ester carbonyls [non-conjugated], 161.30,
161.60, 161.92; 2 ꢀ ketone carbonyls [conjugated] and 1 ꢀ ester carbonyl
[conjugated], 20.74–25.51; 5 ꢀ CH3, 30.14; CH methine, 115.48–118.27; 6 ꢀ C
aromatic ring D.
34. Indian Pharmacopoiea 2007; Vol. I, Chapter 4, p 241.
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38.