Download Curcumin Analogs as Anticancer Agents

Targeted Drug Delivery System (TDDS):
Encapsulating Newly Synthesized Anti-cancer
Compounds-conjugated gold nanoparticles
•Professor Dr. khairia Mohammed Youssef, Dept. of
Pharm. Chemistry, Faculty of Pharmaceutical Sciences
and Pharmaceutical Industries, Future University,P.O.
BOX 12311, Cairo, Egypt
•[email protected]
•[email protected]
• 1- Introduction
• 2- Synthetic Methods of Curcumin and Curcumin
analogs.
• 3-In vitro Assessment of Curcumin and Curcumin
•
•
•
Analogs as Anti-Oxidant and Anti-Cancer Agents.
4-Mechanisms of Action of Curcumin and
Curcumin analogs as Anti-oxidant and AntiCancer Agents.
5- Mechanism of Cytotoxic Tubulin Polimerization
Activities.
6- In vivo Assessment of Curcumin as AntiCancer.
– Preclinial Safety Evaluation in Mice and Rats.
– Chemoprotective and Chemopreventive effects
in DMH-Induced Colon Cancer in Albino Rats
Model.
7- Conjugation with gold nanoparticles
Introduction:
Cancer and Curcumin as anticancer
• Cancer is perhaps the most progressive and
•
•
devastating disease posing a threat of mortality to
the entire world despite significant advances in
medical technology for its diagnosis and treatment.
Recently, considerable attention has been focused on
identifying naturally occurring chemopreventives.
Wide arrays of phenolic substances, particularly those
present in dietary and medicinal plants, have been
reported to possess substantial anticarcinogenic and
antimutagenic effects
Curcumin
diferuloylmethane, 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione
the major constituent of turmeric powder extracted from the rhizome of the plant
curcuma longa
Biological Properties of Curcumin and
its Derivatives
•
•
•
•
•
•
•
•
•
1.
2.
3.
4.
5.
6.
7.
8.
9.
Antioxidant
Anti-inflammatory
Antibacterial, Antifungal and Antiparasitic
Antiviral
Antihistaminic
Treatment of Skin Diseases
Chemopreventive Effects
Anticancer
Treatment of Alzeheimer
Synthetic Methods for the Preparation
of Curcumin and Curcumin derivatives
O
RO
CHO
1
NaOH/EtOH, NaOEt/EtOH
2
RO
O
+ O
or c.HCl
OR
1
XXX
HO
O
HO
1
OH
In an attempt to prepare curcumin and curcumin analogs, search of the literature
demonstrated that chalcones, Claisen-Schmidt condensation in general, are known
to be prepared via the classic enolate condensation reaction which is an acyl
addition reaction of a nucleophilic enolate to an electrophilic carbonyl carbon
In 1973, Roughley and Whiting reported the instability of curcumin in alkaline
medium and explained its alkaline degradation as shown in the following equations:
alkaline degradation of Curcumin
O
O
OCH 3
H3CO
i
OH
HO
O
H3CO
COOH
H3CO
+
HO
Feruloylmethane
HO
Ferulic acid
i
iv
H3CO
CHO
CO2 (BaCO3)
+
HO
vanillin
acetone
ii
iii
H3CO
CH3COCH3
COOH
CHI3
iodoform
HO
Vanillic acid
iv
CO2 (BaCO3)
Roughley, P.J. and Whiting, D.A.; J. Chem. Soc. Trans I (1973),
2379-2388.
Pabon Procedure for the Synthesis
of Curcumin and its Analogs
R1O
2
CHO
O
O
(BuO)3B/ n-BuNH2
/ B2O3
+
HO
O
O
R1O
OR1
HO
OH
the acetylacetone-boric anhydride complex method was adopted successfully for the
preparation of Hundreds of Curcumin and Curcumin Derivatives.
•
•
•
•
Pabon, H.J.J; Rec. Tr. Chim. Pays-Bas (1964), 83: 379-386.
Synthesis of Curcumin Analogues as Potential Antioxidant, Cancer Chemopreventive Agents. Khairia M.
Youssef, Magda A. El-Sherbeny, Faiza S. El-Shafie, Hassan A. Farag, Omar A. Al-Deeb, Sit Albanat A.
Awadalla. Arch. Pharm. Pharm. Med. Chem. 2004, 337, 42-54.
Synthesis and Antitumor Activity of Some Curcumin Analogs. Part II. Khairia M. Youssef, Magda A. ElSherbeny; Arch. Pharm. Chem. Life Sci., 338, 181-189, 2005.
Synthesis of bioconjugate analogs of curcumin as potent antitumor agents. Khairia M. Youssef, Omaima A.
Abu-Alwafa, Reem I. Al-Wably. Med Chem Res (2012) 21:874–890.
CH 3
H3C
2
+
O
O
H3C
B2O3
CH 3
O
+
O
BO2-
B
O
O
(A)
H3C
CH 3
OCH 3
4
O
HO
n-BuNH2
H
Cl-
H+O
HC=HC
H3CO
O
O
OCH 3
HO
OH
CH=CH
B
OCH 3
O
(B)
O
HC=HC
OCH 3
O+H
CH=CH
Cl-
HCl
O
2
H3CO
O
OCH 3
(C)
HO
OH
+ H2O
Synthetic Methods for the Preparation
of Curcumin Analogs
O
HO
O
OH
OCH 3
OCH 3
• The Biological Study of Curcumin reported
•
the instability of curcumin at a pH above 6.5
which is attributed to the active methylene
group.
Omitting the active methylene group and one
carbonyl group may lead to potent
antioxidative compounds
Synthesis of Curcumin Analogs
O
O
CHO
+
R'O
N
OCH3
R
N
R'O
OR''
R
OR'
OR''
R = H, Me, Et, Propyl, acetyl
R' = H, Me, Et
R''= H, Me, Et, alkyl derv.
3,5-Bis(substituted
benzylidene)-N-alkyl-4-piperidones
was
obtained
through
condensation of the appropriate 1-alkyl-4-piperidone with the appropriate aldehyde
under acidic condition.
Prepared Compounds
O
O
HO
O
HO
OCH 3
N
OCH3
CH3
OH
O
OCH 3
OH
HO
OCH3
N
OC2H5
CH3
OH
OC2H5
O
O
HO
N
OC2H5
•
•
•
C2H5
OH
OC2H5
HO
N
OCH3
CH3
OH
OCH3
Synthesis of Curcumin Analogues as Potential Antioxidant, Cancer Chemopreventive Agents. Khairia M.
Youssef, Magda A. El-Sherbeny, Faiza S. El-Shafie, Hassan A. Farag, Omar A. Al-Deeb, Sit Albanat A.
Awadalla. Arch. Pharm. Pharm. Med. Chem. 2004, 337, 42-54.
Synthesis and Antitumor Activity of Some Curcumin Analogs. Part II. Khairia M. Youssef, Magda A. ElSherbeny; Arch. Pharm. Chem. Life Sci., 338, 181-189, 2005.
Synthesis and in vitro antioxidant activity of some new fused pyridine analogs. Mohamed A. Al-Omar, Khairia
M. Youssef, Magda A. El-Sherbeny, Sit Albanat A. Awadalla, Hussein, I. El-Subbagh; Arch. Pharm. Pharm.
Chem. Life Sci. 2005, 338, 181-189.
Curcumin as Anti-Oxidant Agents
O
O
H3CO
OH
OCH3
HO
O
H3CO
OH
OCH3
HO
(a)
OH
(b)
(1)
Curcumin and curcumin derivative have a unique conjugated
structures which show a typical radical trapping ability as a chainbreaking antioxidant
Mechanism of Antioxidant Activity
of Curcumin
O
O
H3CO
.
O
OCH3
O
.
AH: is the phenolic antioxidant.
A●: is the antioxidant radical.
X●:
is another radical. species or the same as A●.
During
Metabolism,
Biomolecules
produce reactive peroxyl radicals
during their oxidation, which may act
as X● and couple with the antioxidant
radical A● in the second step of the
antioxidation process
1- In vitro Chromatographic Determination
of Free Radical Scavenging Activity of
Curcumin Analogs
a: Diphenylpicrylhydrazyl Free Radical Test
• Diphenylpicrylhydrazyl absorbs strongly at 515 nm. The
reaction is carried out under pseudo-first-order conditions using
excess of the tested compound compared to the reagent and
monitoring the mixture for the decrease of absorption at 515
nm.
• Form the linear regression line of the absorbency against time,
the pseudo-first-order rate constant (Kapp) and the
corresponding half-life time (t1/2) of the reaction was calculated.
b- Chemiluminescence Measurement Scavenging
Activity of Curcumin Analogs of Free Radical oxygen
Produced by Peripheral multinuclear Neutrophil
Cells (PMNs)
• All of the synthesized compounds were tested for their ability
to scavenge oxygen free radical produced by peripheral
multinuclear neutrophil cells (PMNs) collected from apparently
healthy blood donors.
• Phorpol-12-myristate-13-acetate
(PMA),
in
a
final
concentration of 2ng/ml was added to PMNs (5x105cells/ml) to
stimulate respiratory burst which was magnified by luminol
(10-4M) to be able to be measured by the LKB luminometer.
• 100µg of synthesized drugs were added to detect its effect on
the amount of oxygen radical liberated and the percentage of
inhibition was calculated.
Conclusion
• The Anti-oxidant screening of most of Curcumin Analogs reveals that the
results go hand by hand with the in vitro Anti-cancer effects results which
performed at The National Cancer Institue (NCI).
• The anti-oxidant and anti-cancer effects of these compounds depend
mainly on the stabilization of the formed phenoxy free radical. The phydroxy phenyl moiety is very essential to produce the phenoxy free
radical which is responsible for free radical scavenging effect.
• o-Substitution by electron–donating group like o-methoxy group, increases
stability of phenoxy free radical and hence increasing both free scavenging
and antitumor effects.
• o-Substitution by ethoxy group rather than methoxy, increases stability of
phenoxy free radical and thus increasing both free scavenging and
antitumor effects.
• Increasing the alkyl group chain from methyl to ethyl on the N in the series
of substituted N-alkyl piperidones, increases the activity toward both free
radical scavenging and antitumor effects which may be attributed to
increased positive inductive effect and / or increased lipophilicity of the
new compounds.
• Extension of conjugation, increases stabilization of phenoxy free radical.
Chemopreventive Properties of Curcumin and
Curcumin Analogs
Mechanism of Action
• Stimulation of phase I and phase II detox
•
•
•
•
•
•
systems
Inhibition of COX-1 and COX-2 enzymes
Stimulation of glutathione-S-transferase
Interference with cell growth by inhibition of
protein kinases
Neutralization of carcinogenic free radicals
Curcumin significantly inhibits the activity of the
isoenzymes of cytochrome P-450 involved in the
metabolism of some carcinogens
It inhibits SK-Hep-1 hepatocellular carcinoma cell
invasion in vitro and suppresses matrix
metalloproteinase-9 secretion
• Cytotoxic Tubulin Polymerization
Activities
Invitro Assessment as Cytotoxic Targeting
Tubulin Polymerization Activity
Microtubules represent one of the fiber systems of the eukaryotic
cytoskeleton. They are essential for a wide variety of cellular functions,
notably: 1- cell motility, 2-transport, 3-cell shape, 4-polarity and 5-mitosis.
Microtubules consist of a core cylinder built from heterodimers of α and β
tubulin monomers.
*Mary Ann Jordan & Leslie Wilson, “Microtubules as a target for anticancer drugs”. Nat. Rev. Cancer, (2004), 4:
253-265.
Microtubules binding sites
There are three different target sites on the tubulin
heterodimer:the vinca alkaloid, the colchicine, and
the paclitaxel binding sites.
a. Vinblastine bound to highaffinity
sites
at
the
microtubule plus end suffice
to suppress microtubule
dynamics.
b. Colchicine forms complexes
with tubulin dimers and
copolymerizes
into
the
microtubule
lattice,
suppressing
microtubule
dynamics.
c. A microtubule cut away to
show the interior surface is
shown.
Paclitaxel
binds
along the interior surface of
the
microtubule,
suppressing its dynamics.
Tubulin Inhibition Study of
Curcumin Analogs
tubulin dimer of
P.
falciparum
tubulin
is
represented
as
dimer of alpha
(yellow) and beta
(green) subunit.
curcumin diketo form at the interface of Panel A shows all the predicted
bound poses, mostly at the interface of the dimer.
Panel B shows the most probable binding pose according to Autodock (Rank
1) with the curcumin diketo form at the interface of alpha and beta tubulin
monomers.
Panel C shows binding sites of colchicine (purple), paclitaxel (red) and
vinblastine (brown) on parasite tubulin dimer. Curcumin was proved to have
inhibition activity on tubulin inside P. falciparum cells with IC50 = 5 µM.
*Chakrabarti R, Rawat PS, Cooke BM, Coppel RL, Patankar S, “Cellular Effects of Curcumin on Plasmodium falciparum
Include Disruption of Microtubules”. PLoS ONE, (2013), 8 (3): e57302.
SAR Performed on Novel Curcumin
Analogs
Docking Study Using Discovery
Studio 2.5 and MOE Programs
1, 5-diaryl-3-oxo-1,4pentadienyl pharmacophore
[Reacts at the primary binding
site A of colchicine]
O
Auxiliary
binding site
B
R'
N
R'
O
Groups capable of Van
der Waal bonding
Atoms capable of
hydrogen bonding
The group on N atom could lead to increases in cytotoxic potencies due to either additional binding with cellular
constituents at an auxiliary binding site (site B) or by facilitating the interaction of the cytotoxin at site A.
Newly Designed and Synthesized Curcumin Analogs with in vitro Cytotoxicity and Tubulin Polymerization
Activity. Iten M. Fawzy, Khairia M. Youssef, Nasser S. M. Ismail, Joachim Gullbo and Khaled A. M. Abouzid, Chemical Biology &
Drug Design. Early View, Article first published online: 25 NOV 2014.
European Patent, PCT/EG 2013/000028.
Invivo Assessment as Anticancer Agents
O
HO
O
Curcumin
OCH 3
O
O
OH
OCH 3
HO
OH
OCH 2CH 3
OCH 2CH 3
Ethyl Curcumin
O
HO
CH 3
N
CH 3
OH
CH 3
Curcumin Analog
Curcumin (1) and curcumin analogs (2,3) were synthesized and the preclinical safety
evaluation in mice and rats were done.
Preclinical Safety Evaluation in Mice and
Rats
Acute Toxicity (LD50) of tested
compounds procedure
Pilot experiments were performed by oral administration of increasing doses of tested
compounds into different groups of mice (each consisted of 5 mice). Doses which
produce 0 % and 100 % mortality were determined.
Doses which used in the assessing of
chemopreventive, antitumor effects
Compound
1 (curcumin)
Dose
0.05mg/gm/orally/day
2
3
0.12mg/gm/orally/day 0.16mg/gm/ora
lly/day
Litchfield JT, Wilconsxon F. J. Pharmcol. Exp. Ther. 1949, 96, 99-113.
Blood Pressure and Electrocardigrahic
Recording
Test
Control
Curcumin
2
3
B.P. Mean
109
108.4
109.9
103.9*
± SEM
± 0.573
± 0.57
± 0.575
± 0.546
Three groups of virgin Sprague-Dawley female rats, each consisted of 10 rats: Group A
administered the calculated dose of compounds 1, 2 and 3 daily for two weeks. Group B
administered the calculated dose of compounds 1, 2 and 3 daily for four weeks.
Results of Blood Pressure and
Electrocardigrahic Recording
• These tests show that there is significant
reduction in systolic B.P. only with
pretreatment with compound 3 for 14
days P.O. administration at P , 0.05 while
there is no changes in E.C.G. or heart
rate.
Histopathological Study
• Different organs sections of untreated and
treated animals e.g. mammary gland,
heart, kidney, liver, spleen and colon were
stained with routine hematoxylin and
eosin (HX and E) to be examined under
light microscope (Fig. 2-7)
Histopathological Results
Group 1: Control (untreated)
Fig. (1) Light microscope image of
the Mammary gland stained with
H&E (X20)
Fig. (2) Light microscope image of
the Heart stained with H&E (X10)
Fig. (4) Light microscope image of
the Liver stained with H&E (X20)
Fig. (3) Light microscope image of
the Kidney stained with H&E (X40)
Fig. (5) Light microscope image of
the Spleen stained with H&E (X20)
Fig. (6) Light microscope image of
the Lung stained with H&E (X20)
Fig. (7) Light microscope image
ofthe Colon stained with H&E (X20)
Group 2 : Compound 1 (Curcumin) After Four Weeks
Fig. (8) Light microscope image of
the Mammary gland stained with
H&E (X20)
Fig. (9) Light microscope image of
the Heart stained with H&E (X10)
Fig. (11) Light microscope image of
the Liver stained with H&E (X20)
Fig. (10) Light microscope image of
the Kidney stained with H&E (X40)
Fig. (12) Light microscope image of
the Spleen stained with H&E (X20)
Fig. (13) Light microscope image of
the Lung stained with H&E (X20)
Fig. (14) Light microscope image of the Colon
stained with H&E (X20) showing normal
structure
Group 3: Compound 2 (Ethyl Curcumin)
Fig. (15) Light microscope image of
the Mammary gland stained with
H&E (X20)
Fig. (16) Light microscope image of
the Heart stained with H&E (X10)
Fig. (18) Light microscope image of
the Liver stained with H&E (X20)
Fig. (17) Light microscope image of
the Kidney stained with H&E (X40)
Fig. (19) Light microscope image of
the Spleen stained with H&E (X20)
Fig. (20) Light microscope image of the
Lung stained with H&E(X20) showing
thickening of the interestial space
Fig. (21) Light microscope image of
the Colon stained with H&E (X10)
showing normal structure
Group 4: Compound 3
Fig. (22) Light microscope image
of the Mammary gland stained
with H&E (X20)
Fig. (23) Light microscope image
of the Heart stained with H&E
(X10)
Fig. (25) Light microscope image
of the Liver stained with H&E (X20)
Fig. (24) Light microscope image
of the Kidney stained with H&E
(X40)
Fig. (26) Light microscope image of
the Spleen stained with H&E (X20)
(a) Accumulation of fluids in the alveoli
(b) Accumulation of lymphocytes and RBC
in the alveolar space
Fig. (27) Light microscope image of the Lung stained
with H&E (X20) showing in
(a) X10
(b) X40
Fig. (28) Light microscope image
of the Colon stained with H&E
showing an increase in the goblet
cells in the base of the crypts (arrows)
Conclusion
compound 1 (curcumin) and compound 2 (ethyl curcumin
showed normal structure after 4 weeks treatment identical
of that in 2 weeks .

Compound 3 shows some intraalveolar lung haemorhage
after four weeks.
Chemoprotective Effect of
Curcumin and its Analogs in
DMH-Induced Colon Cancer in
Albino Rats Model.
Bird RP. Role of aberrant crypt foci in understanding the pathogenesis
of colon cancer .Cancer Letters 1995; 93 : 55-71.
Histological evaluation and Aberent
Cript Focy assay
• Group A: animals constituted the normal
•
•
•
•
untreated controls
Group B: Comprised of carcinogen control
animals.
Dimethylhydrazine(DMH) was administered
subcutaneously, at a dose of 20mg/kg-body
weight ,once a week in 0.9%Na Cl solution(pH
7.2) for a total period of 30 weeks .
Chemoprotective treatment part(1) Two weeks
then DMH was administered
Chemoprotective treatment part(2) Four weeks
then DMH was administered
Normal untreated control
Fig. 29: No signs of abnormality
0.3% methylene blue stained colon . X 200
Putative signs of the induction of cancer in colon:
•
•
•
•
•
Increase in No. of ACF near epithelial surface
Increase in size of ACF
ACF are lined with thick, deeply stained epithelial cells
Nuclei are started to show mitotic changes
Increase in pericryptal zones
DMH Carcinogen Control
a X 200
b X 400
0.3% methylene blue stained colon
Fig. 30: Adenocarcenoma induced by DMH
•Invasion of malignant cells to muscle layers
•Tumor consists of crowded irregular malignat acini separated by thin
fibrovascular stroma
Fig. 31: Adenocarcenoma induced by DMH in colon (adenomatous polyps )
0.3% methylene blue stained colon X 200
Curcumin was given orally daily for 2
weeks ,then DMH
a X 200
b X 400
0.3% methylene blue stained colon
Fig. 32: Crowding cells at the surface with enlarged and hyperchromatic
nuclei with scattered aberrant crypt foci (ACF) were seen.
Ethyl Curcumin was given orally daily
for 2 weeks ,then DMH
a X 200
b X 400
0.3% methylene blue stained colon
Fig. 33: Moderate protection was seen. Crowding cells at the surface
with enlarged and hyperchromatic nuclei with scattered aberrant crypt
foci (ACF) were seen.
Compound 3 was given orally daily for
2 weeks ,then DMH
a X 200
b X 400
0.3% methylene blue stained colon
Fig. 34: Few areas of crowding cells at the surface were seen. Few
numbers of aberrant crypt foci (ACF) were seen.
Curcumin was given orally daily for 4
weeks ,then DMH
a X 200
b X 400
0.3% methylene blue stained colon
Fig. 35: Few areas of crowding cells at the surface were seen. Few
numbers of aberrant crypt foci (ACF) were seen.
Ethyl Curcumin was given orally
daily for 4 weeks ,then DMH
a X 200
b X 400
0.3% methylene blue stained colon
Fig. 36: Beter protection was seen. Few numbers of aberrant crypt foci
(ACF) were seen with less sign of neoplastic changes.
Compound 3 was given orally daily for
4 weeks ,then DMH
a X 200
b X 400
Fig. 37: 0.3% methylene blue stained colon
 No aberrant crypt foci (ACF) were seen.
No focal cell crowding were seen.
Normal globlet cells.
Only some inflammatory cellular infiltration.
Aberrant Crypt Foci (ACF) Assay
Chemoprotective efficay of different forms of curcumin and curcumin analogs
administered for 2 weeks orally before DMH-induced Aberrant crypt foci(ACF)
in male wistar rats .
No of ACF/Rat colon
Inhibition%
(mean+S.E)
Group
No. of rats/G
N.control (A)
10
-------
-----
DMHcontrol(B)
10
100.5+2.54
0
DMH+ curcumin
10
54.3 +1.26
45.98
DMH + ethyl curcumin
10
51.5 +1.20
48.5
DMH + C3
10
37.4 +0.95
62.6
N: Normal control received saline s.c. once weekly for 30 weeks.
DMH : was given at a dose of 20mg/kg s.c once weekly for30 weeks.
DMH+C1 :}Curcumin was given at a dose of 53mg/kg orally once daily orally.
DMH +C2 : Ethyl curcumin was given at a dose of 123mg/kg orally once daily .
DMH +C3 : compound 3 was given at a dose of 161 mg/kg orally once daily
C1,C2, C3 were given 2 weeks before induction of cancer colon by DMH.
Chemoprotective efficay of different forms of curcumin and curcumin
analogs administered for 4 weeks orally before DMH-induced Aberrant
crypt foci(ACF) in male wistar rats .
Group
No of rats/G
No of ACF/
Inhibit
Rat colon(mean+S.E) %
N.control(A)
10
-----
----
DMH control(B)
10
100.5+2.54
0
DMH+ curcumin
10
47.7+1.38
52.3
DMH+ ethyl curcumin
10
45.8 + 1.71
54.2
DMH+C3
10
-----
100
N: Normal control received saline s.c once weekly for 30 weeks .
DMH: was given at a dose of 20mg/kg s.c once weekly for 30 weeks.
DMH+ C1: curcumin was given at a dose of 53mg/kg orally daily .
DMH+C2 : ethyl curcumin was given at a dose of 123mg/kg orally daily.
DMH+ C3 : compound 3 was given at a dose of 161mg/kg orally daily.
C1,C2,C3 were given daily for 4 weeks before induction of cancer colon
by DMH.
Chemoprotective efficacy of different forms of curcumin and curcumin analogs
administered for 2 weeks orally before DMH-induced colonic cancer in rats .
No of rats /G
No.of colon tumors 1
N.control(A)
10
-------
DMH control(B)
10
20
DMH+curcumin
10
8
DMH+ethyl curcumin
10
6
DMH+C3
10
2
N: normal control rats received saline s.c. once weekly for 30 weeks.
DMH: was given at a dose of 20mg/kg s.c. once weekly for 30 weeks .
DMH +C1: curcumin was given at a dose of 53mg/kg orally once daily
for 2 weeks.
DMH+C2: ethyl curcumin was given at a dose of 123mg/kg orally once daily
for 2 weeks .
DMH+C3 : compound 3 was given orally at a dose of 161mg/kg once daily
Chemoprotective efficacy of different forms of curcumin and curcumin analogs
administered for 4 weeks orally before DMH –induced colonic cancer in rats
No. of rats /G
No.of colon tumors/G
N.control
10
---
DMH control(B)
10
20
DMH+curcumin
10
4
DMH +ethyl curcumin
10
2
DMH+C3
10
--
N: normal control rats received saline s.c. once weekly for 30 weeks .
DMH : was given at a dose of 20mg/kg s.c. once weekly for 30 weeks.
DMH+C1 : curcumin was given at a dose of 53 mg/kg .
DMH+C2 : ethyl curcumin was given at a dose of 123 mg/kg .
DMH+C3 : compound 3 was given at a dose of 161mg/kg .
Chemopreventive Effects in DMH-Induced Colon Cancer in
Albino Rats Model
Histopathological Examination of Chemopreventive efficacy of curcumin and
curcumin analogs administered for 2 weeks orally after DMH-induced colonic
cancer in rats
No. of rats /G
No.of colon tumors/G
DMH control(B)
10
20
DMH+C1 (C)
10
8
DMH+C2 (D)
10
6
DMH+C3 (E)
10
2
N: normal control rats received saline s.c. once weekly for 15 weeks.
DMH: was given at a dose of 20mg/kg s.c. once weekly for 15 weeks .
DMH +C1: compound 1 was given at a dose of 53mg/kg orally once daily for 2 weeks.
DMH+C2: compound 2 was given at a dose of 123mg/kg orally once daily for 2 weeks
.DMH+C3 : compound 3 was given orally at a dose of 161mg/kg once daily
Chemopreventive efficacy of of curcumin and curcumin analogs
administered for 4 weeks orally after DMH –induced colonic
cancer in rats
No.
No.of
ofrats
rats/G
/G
No.of
No.ofcolon
colontumors/G
tumors/G
N.control
N.control
10
10
10
-----
DMH
DMHcontrol(B)
control(B)
10
10
20
20
DMH+C1(F)
DMH+C1(F)
10
10
44
DMH
DMH+C2(G)
+C2(G)
10
10
22
DMH+C3(H)
DMH+C3(H)
10
10
---
N: normal control rats received saline s.c. once weekly for 15 weeks .
DMH : was given at a dose of 20mg/kg s.c. once weekly for 15 weeks.
DMH+C1: compound 1 was given at a dose of 53mg/kg.
DMH+C2 :compound 2 was given at a dose of 123mg/kg .
DMH+C3 : compound 3 was given at a dose of 161mg/kg .
Conclusion
• In conclusion, the results of this study
suggest that daily supplementation of
Curcumin and Curcumin Analogs has a
positive
beneficial
effect
against
chemically induced colonic preneoplastic
progression in rats induced by DMH,
which provide an effective dietary
chemoprotective and chemopreventive
approach to disease management .
• Best results were given with compound
(3).
Conjugation with gold
nanoparticles
• The result of the previous work inhanced us to use the
Targeted drug delivery system (TDDS), especially gold-based
nanoparticles (AuNPs) to be used as a model system in this
work.
• The 1.4 nm Nanogold® particle is a gold compound: it is not
just adsorbed to proteins, like colloidal gold, but covalently
reacts at specific sites under mild buffer conditions.
• The main objective is that, the specific conjugated drug is
targeted to fit and improve their binding affinity, to the specific
receptor that ultimately will trigger the pharmacological
activity as anticancer.
• The other objective is the dual destructive effect on cancer
cells after releasing of gold nanoparticles from the drug.
• In this work, the highly stable mercapto capped gold
nanoparticles were prepared adopting Scheme 1
Synthesis and Conjugation with Gold
Nanoparticles
1- Synthesis of biocompatible gold nanoparticles (GNPs) with
different sizes and shapes
2- Physiochemical characterization of the synthesized GNPs using
TEM, XRD, DLS, …etc. techniques
3- Conjugation of the intended drugs with different types of GNPs
(different sizes & shapes)
4- Physiochemical characterization of the synthesized Drug/GNPs
nanocomposite using TEM, XRD, DLS, …etc. techniques to confirm
the formation of the conjugate.
5- Studying the optimization condition such as effect of Temp.,
pH, loading concentrations…etc. to achieve the most probable
(stable) formula of the Drug/GNPs nanocomposite.
6- Studying the rate of drugs release at variable conditions
7- Pharmaceutical formulation of the final product
• The superior stability of these particles under the conditions of
varied pH and electrolyte concentrations will be studied.
• In addition to developing synthetic methodology that is free
from reducing agents and elevated temperature, we will prob
the interactions between the gold core and the stabilizer
molecules using Fourier transformed infrared spectroscopy (FTIR) technique (will be done at University of Georgia Technology
Labs).
• Long term stability of newly synthesized anti-cancer compounds
capped GNPs may provide improved shelf life. While the
controlled release and sustained delivery of the anti-cancer
drugs from the gold core is demonstrated with the help of GSH
of varying concentrations.
• In vitro and In vivo anti-cancer activity of released compounds
against different types of cancer cells will be investigated in
detail. Reasons behind enhanced anti-cancer activity will be also
addressed in this work.
Scheme 1
O
RO
H
O
OR
O
N
R'
H
P2 S 5
CS2
R = CH3, CH5
R' = H, COCH3, CONH2
O
RO
H
S
OR
N
R'
S
H
Au nanoparticles
O
RO
nanoparticles Au
S
OR
N
R'
S
Au nanoparticle
Conjugated compounds
with gold nanoparticles
Molecular docking studies of tubulin
inhibitors
• Docking Study was performed using the MOE software.
• Downloading the crystal structure of CDK2 enzyme complexes
with inhibitor was carried out from protein data bank website
(PDB).
• Regularization and optimization for protein and ligand were
performed.
• Determination of the essential amino acids in binding site was
carried out and compared with that present in literature.
• The performance of the docking method was evaluated by redocking crystal ligand into the assigned active site of to
determine the root mean square deviation (RMSD) value.
• Interactive docking was carried out for all the conformers of
each compound of the test set to the selected active site.
• Each docked compound was assigned a score according to its
fit in the ligand binding pocket (LBP) and its binding mode.
ligand podophyllotoxin E Score = -11 K cal/mol
Comp. k1 E Score = -9.8 K cal/mol
Comp. k2 E Score = -10.9 K cal/mol
Comp. k3 E Score = -11.9 K cal/mol
Comp. k4 E Score = -11 K cal/mol
Comp. k5 E Score = -10.5 K cal/mol
Comp. k6 E Score = -10 K cal/mol
Conclusion
Comp. k3 E Score = -11.9 K cal/mol
Patents and Published Papers
1- U.S. Patent No. 60/670,844, filed April 13,
2005.
2- Egyptian Patent No. 1131/2008, filed July 3,
2008.
3- European Patent, PCT/EG 2013/000028.
1.
Synthesis of Curcumin Analogues as Potential Antioxidant, Cancer Chemopreventive Agents. Khairia M.
Youssef, Magda A. El-Sherbeny, Faiza S. El-Shafie, Hassan A. Farag, Omar A. Al-Deeb, Sit Albanat A.
Awadalla. Arch. Pharm. Pharm. Med. Chem. 2004, 337, 42-54.
2. Synthesis and Antitumor Activity of Some Curcumin Analogs. Part II. Khairia M. Youssef, Magda A. ElSherbeny; Arch. Pharm. Chem. Life Sci., 338, 181-189, 2005
3. Synthesis and in vitro antioxidant activity of some new fused pyridine analogs. Mohamed A. Al-Omar, Khairia
. Albanat A. Awadalla, Hussein, I. El-Subbagh; Arch. Pharm. Pharm.
M. Youssef, Magda A. El-Sherbeny, Sit
Chem. Life Sci. 2005, 338, 181-189.
4. Curcumin Analogs as Anticancer Agents: 1) Preclinical Safety Evaluation in Mice and Rats. 2)
Chemopreventive Effects in DMH-Induced Colon Cancer in Albino Rats Model. Khairia M. Youssef, Azza M.
Ezzo, Moushira I. El-Sayed, Amal A. Hazzaa, Azza H. EL-Medany, Maha Arafa. The International Symposium
on Recent Progress in Curcumin Reseach, September 11-12, 2006 at Yogyakarta, Indonesia.
5. PAC, a novel curcumin analogue, has anti-breast cancer properties with higher efficiency on ER-negative cells
Ensaf M. Al-Hujaily, Ameera Gaafar Mohamed, Ibtehaj Al-Sharif, Khairia M. Youssef and Pulicat S.
Manogaran, et al. Breast Cancer Res Treat (2011) 128:97–107
6. Synthesis of curcumin and ethylcurcumin bioconjugatesas potential antitumor agents Reem I. Al-Wabli
Omaima M. AboulWafa Khairia M. Youssef Med Chem Res (2012) 21:874–890
7. Molecular Modeling of Novel Curcumin Analogs with Anticipated Anticancer Activity: FUE International
Conference on Pharmaceutical Technologies (ICPT) 2012.Iten M. Fawzy, Khairia M. Youssef, Nasser S. M.
Ismail, Khaled A. M. Abouzid.
8. Design and Synthesis and Biological evaluation of Novel Curcumin Analogs with anticipated anticancer
activity. Iten M. Fawzy, Khairia M. Youssef, Nasser S. M. Ismail, J. Gullbo and Khaled A. M. Abouzid. Journal
of Future University in Egypt 2014. In Press.
9. Molecular docking and in silico ADME study of Novel N9-substituted Purines targeting CK1 and abl-tyrosine
kinase. Iten M. Fawzy, Khairia M. Youssef, Nasser S. M. Ismail , Deena S. Lasheen and Khaled A. M.
Abouzid2. FIP conference 2014 at Bangkok.
10. Newly Designed and Synthesized Curcumin Analogs with in vitro Cytotoxicity and Tubulin
Polymerization Activity. Iten M. Fawzy, Khairia M. Youssef, Nasser S. M. Ismail, Joachim Gullbo and
Khaled A. M. Abouzid, Chemical Biology & Drug Design. Early View, Article first published online: 25
NOV 2014.
Acknowledgement
• Taher Salah, Professor of Nanotechnology, Manager of
•
•
•
•
•
•
Nanotechnology & Advanced Materials Central Lab.
(NAMCL).Regional Center for Food & Feed (RCFF),
Agricultural Research Center (ARC).
Nasser Saad, Asst. Prof. Department of Pharmaceutical
Chemistry, Future University in Egypt.
Azza Ahmed, Asst. Prof. Department of Pharmaceutical
Technology, Future University in Egypt.
Azza M. Ezzo, Pharmacology Dept., Faculty of Medicine, AlAzhar University, Cairo, Egypt.
Maha Arafa, Histopathology Dept., College of Medicine, King
Saud University, Riyadh, Saudi Arabia.
King Saud University, Saudi Arabia.
Future University In Cairo.