Phospholipase A2 Triggered siRNA against Lp-PLA2 and

Concomitant Darapladib Admistration for Treatment of

Atherosclerosis.
Specific Aims:
Despite many therapies available in the market for the treatment of various heart diseases, the number of
patients due to the disease is increasing at an alarming rate. As per CDCs deaths report of 2009, approximately
780,000 people are dying each year due to any form of cardiac dysfunction. Approximately one half of these
people died due to ischemic heart diseases which comprises of Myocardial Infarction (MI), Atherosclerosis, and
Angina Pectoris (AP). Though the number of people who died of atherosclerosis is less, the fact that the
underlying cause for all these forms of death is atherosclerosis. These numbers stress the importance of
developing newer, better therapeutic strategies to cure the disease.
The current study is built on a premise that the blockade of active enzyme (Lp-PLA2) in the clinical trials with
darapladib was not complete and because the enzyme is ubiquitous the turnaround of enzyme was so high that
the inhibition did not turn out into therapeutic success. We will try to target not only the active enzyme with
darapladib, but also we will inhibit the production of enzyme by using a siRNA targeted knock down of LpPLA2 mRNA. By inhibiting the active enzyme as well as inhibiting the production of Lp-PLA2 we hope to
further decrease the effect of Lp-PLA2 and have beneficial effect in the therapy of atherosclerosis. As the
delivery of siRNA into the system is difficult given the issues regarding stability and efficacy of siRNA, we will
use stealth liposomes to coat the siRNA. Concomitant with liposomal administration, we will administer
Darapladib orally in a mouse model of atherosclerosis (ApoE deficient mouse). By making use of this strategy,
we hope we will have better targeting and drug efficacy and thereby better treat atherosclerosis.
Specific aim 1:
(i)
(ii)
(iii)

To develop and optimize siRNA against Lp-PLA2

To develop and characterize stealth liposomes sensitive to phospholipase A2
To titrate oral doses of Darapladib and dose of Lp-PLA2 siRNA

Specific aim 2:
(i)
(ii)

Formulation of siRNA into stealth liposomes sensitive to phospholipase A2

To determine cellular uptake and inhibitory effects of siRNA and Darapladib in in vitro models

Specific aim 3:
(i)
(ii)

Evaluation of efficacy, safetym release parameters and pharmacokinetic properties of liposomes in

ApoE deficient mice model.
Bionalysis of Phospholipase A2 triggered Lp-PLA2 and concomitant Darapladib.

Background and Significance:

In terms of disease pathology, atherosclerosis is a disease in which the vessel wall, especially arterial vessel wall
thickens due to deposition of plaque in the intimal layer of blood vessel. It is characterized by presence of
atheroma, lumps formed on the inside of blood vessel, which restricts the free flow of blood in those blood
vessels. In response to an injury of the blood vessel as a part of defense, the body sends inflammatory mediators
such as white blood cells and other mediators like macrophages to fight the injury/ stimulus. This causes
formation of a clot which will be worsened by the presence of lipid in the blood. Lipids usually travel in the

blood in the form of Low Density Lipoprotein (LDL), High Density Lipoprotein (HDL), Chylomicrons, and
Triglycerides. Low density lipoprotein (LDL), and Chylomicrons specifically is more dangerous in terms of
progression to atherosclerosis, since these are prone to oxidation by free radicals.

Source: CDC (http://www.cdc.gov/heartdisease/images/heart_disease_deaths.png)

Oxidized LDL will further cause damage to blood vessel by promoting diapedesis (transendothelial migration)
of monocytes and activating vascular endothelial cells (17194459). Once inside the blood vessel, these
monocyte transform into macrophages and will try to repair the atheroma. Disparities in this process will cause
build up of macrophages inside the atheroma which will attract immunological mediators (T cells and
macrophages) which will absorb the oxidized LDL leading to formation of characteristic foam cells inside the
atheroma. The continued presence of oxidized LDL leads to build up of these foam cells inside the atheroma
which keeps on adding on size which may ultimately lead to rupture of atheroma. The ruptured atheroma, as it
contains oxidized LDL and other inflammatory mediators will in turn cause more number of atheroma in the
blood vessel. If this process is not interrupted the blood vessel will be constricted leading to decreased blood
supply to the target organ. Based on the target organ, the result could be either myocardial infarction, if the
atheroma appears in coronary arteries, or tissue sepsis.
The role of Lipoprotein associated Phospholipase A2 (Lp-PLA2) in the pathogenesis of atherosclerosis and
coronary heart disease is well established. Lp-PLA2 is an enzyme produced by inflammatory cells which
hydrolyses the oxidized LDL to purported inactive fragments (like (lysophosphatidylcholine and oxidized

nonesterified fatty acids). But newer studies have demonstrated that the hydrolyzed products of LDL are not
inactive, as they were thought to be, but they in turn activate inflammatory cascades. These characteristics of
Lp-PLA2 have interested investigators to inhibit the enzyme and use the inhibitor as a biological target for
treatment of atherosclerosis. Many clinical trials were conducted to estimate the therapeutic efficacy of drugs
inhibiting Lp-PLA2 for treatment of various cardiovascular diseases like Coronary artery disease,
Atherosclerosis, Coronary Heart Disease, (AIM I, II, & III, SOLID-TIMI 52, STABILITY, NCT00695305,
NCT01916720, NCT00269048, NCT01000727, NCT00799903). Though the drug has showed excellent
efficacy and safety in animal models and in vitro analyses, all the trials failed to show any significant protection
from the disease. Though the plasma concentrations of drug after administration and related parameters are
good following oral dosing, the drug failed to deliver therapeutic benefit.
Introduction: Coronary Heart Disease is one of the devastating illness responsible for numerous number of
deaths worldwide. In spite of therapy with beta blockers and other related drugs, the number of deaths keep on
increasing given the role of stress and life style of people. Though many new targets have been employed for
many clinical trials in the recent past and most of them have failed for reasons including lack of specificity, nonspecific targeting, delivery and release of drugs in the system. One such trial is IMMEDIATE in which the
investigators tried to treat the disease by using a new drug called Darapladib which targets Lipoprotein
associated Phospholipase A2 (Lp-PLA2), also known as Platelet activating factor acetylhydroxylase. Lp-PLA2
is an important regulator of lipid metabolism and inflammation which travels in blood with Low Density
Lipoprotein (LDL).
In the process of progression to atherosclerosis, LDL also helps in carrying Lp-PLA2 into the arterial wall and
in the formation of plaques. Inside the arterial wall, Lp-PLA2 mediate macrophages recruitment and foam cell
evolution which ultimately makes plaques vulnerable to progression of atheorosclerosis. Histological
examination of diseased coronary arteries showed higher presence and activity of Lp-PLA2 in atherosclerotic
plaques prone to rupture. Elevated levels of circulating Lp-PLA2 correlate with risk of being affected with
coronary heart disease and atherosclerosis. Lp-PLA2 is an ideal target to treat atherosclerosis as it is a molecule
which mediates events necessary for
progression of atherosclerotic plaque and
subsequent evolution of the plaque to a
vulnerable state of rupture and progression of
the disease to Ischemic Heart Disease and
Myocardial Infarction. This is an enzyme of a
larger family of enzymes which has an ability
to hydrolyze glycerophospholipids to liberate
lysophospholipids and free fatty acids. There
are also reports that show Lp-PLA2 has a dual
functioning role as both atheroprotective and
atherosclerotic.
The content of Lp-PLA2 in LDL increases in
proportion with the status of inflammation of
the arteries and this ensures LDL to further
enter and invade the arterial wall. Once in
subintimal space the tissue oxidases oxidize
the surface phospholipids and these oxidized
phospholipids are substrates for Lp-PLA2,
which degrade these phospholipids to
Lysophosphatiylcholine
(LYSO-PC)
and

Source: Charo et al., doi:10.1038/nrd3444

oxidized fatty acids. LYSO-PC stimulates pro-inflammatory signaling events in vascular wall such as increased
expression of VCAM-1 and MCP-1. These events lead to vascular smooth muscle cell migration and
proliferation of tissue macrophages. Immunostaining studies also showed very high content of Lp-PLA2 in
advanced stage thin cap atheroma lesions which are ready to rupture but not in the early stage or middle stage
atheroma. All these studies put together support an active role for Lp-PLA2 in making the atherosclerotic
plaque more vulnerable to damage and targeting this enzyme would be of therapeutic significance for
atherosclerosis.
Darapladib: Given the role of Lp-PLA2 in the process of progression of atherosclerosis and the benefits
obatined by inhibiting the enzyme, many studies were focused to inhibit the enzyme for treatment of
atherosclerosis and coronary heart disease. The initial studies with a series of pyrimidone substituents showed
good inhibitory activity against Lp-PLA2 in vitro. Darapladib was one of those pyrimidone compounds which
showed good in vitro inhibitory activity against recombinant Lp-PLA2 and excellent lipophilic activity good
cell permeability. Further studies in hyperlipidemic rabbits with oral darapladib at a dose of 10mg/Kg reduced
plasma Lp-PLA2 levels and the inhibition was maintained for 24 hours with peak inhibition at 2 hours. The
same result was also seen in the porcine model of atherosclerosis. These effects are also found in phase 1 and
phase 2 clinical trials with minimal adverse effects. Though the drug was a success in phase 1 and 2 clinical
trials, darapladib did not show any significant therapeutic effect in decreasing major coronary events in patients
with acute coronary artery disease. Though there were no discrepancies in drug disposition, metabolism and
effects in the body, the drug did not see therapeutic success.
Specific aim 1:
To
develop
and
optimize
siRNA
against
Lp-PLA2:
The
target
sequence
(5GCAAGCTGGAATTCTCCTTTG-3) within the murine Lp-PLA2 mRNA will be used to build the siRNA for
the experiment. Based on the published reports and available literature this siRNA has good efficacy in blocking
the mRNA of Lp-PLA2 both in vitro and in vivo. We will follow the protocol followed by Zhang et al., to test
the efficacy and safety of siRNA. This group showed an approximate 84% inhibition of Lp-PLA2 mRNA in

cultured mouse macrophages.

The same group also showed effects of darapladib on atherosclerotic plaque formation and its protective effect
in the disease in a mouse model of atherosclerosis. In an elegant study using ApoE deficient mice, they showed
good reductions in lipid content and pro-inflammatory cytokines and increase in levels of anti-inflammatory
cytokines and collagen content following inhibition of Lp-PLA2 using both darapladib and RNAi using siRNA
for Lp-PLA2. We will follow the same method to estimate the efficacy of darapladib and siRNA.

Source: Zhang et al., DOI: 10.3892/ijmm.2013.1248

Source: Zhang et al., DOI: 10.3892/ijmm.2013.1248

To develop and characterize stealth liposomes sensitive to phospholipase A2: We will use the double
emulsion technique for preparation of stealth liposomes, a method published by Camilla et al., for preparation
of
stealth liposomes sensitive to phospholipase A2. In one study, they used stealth liposomes prepared from
Dipalmitoylphosphatidylglycerol
(DPPG),
Dipalmitoylphosphatidylcholine
(DPPC),
and
Dipalmitoylphosphatidylethanolamine-polyethylene glycol (DPPE-PEG 2000). We will follow the same method
for encapsulating Lp-PLA2 siRNA into the liposomes. We will test different concentrations of siRNA to get the
optimum concentration effective for inhibiting the mRNA. For this purpose we will prepare phospholipase A2
sensitive liposomes with varying concentrations of siRNA and will check for the biological activity of siRNA in
a method similar to one employed by Zhang et al, in which we will use cultured macrophages and induce
activity of Lp-PLA2 in these cells by treatment with oxLDL. These cells will be transfected with the liposomes
sensitive to PLA2 and the concentration of siRNA which shows least toxicity and good efficacy will be chosen
for further studies. Liposomes without any siRNA will be prepared and used as negative controls.

Source: Camilla et al.,doi:10.1016/j.ijpharm.2006.11.010

Specific aim 2:
Characterization of liposomes: Characteristics of liposomes such as size, shape, lipid content, lipophilicity,
encapsulation efficacy, polydispersity index, zeta potential will be estimated. For better efficacy, liposomes
with a size range of 60-90nm, zeta potential values between -20 and -30 mV, siRNA encapsulation efficacy of
7-9%, will be chosen. The above limitations will be applied as per various studies done which have shown
better
availability and
release
characteristics
upon
i.v.
administration
and to avoid
clearance from
RES. We will
also conduct a
study to check
Source: Camilla et al.,doi:10.1016/j.ijpharm.2006.11.010
if the harsh
conditions used in the preparation of liposomes will affect biological activity of siRNA. For this purpose, we
will label Lp-PLA2 siRNA with Cy3 fluorescent dye and will estimate the efficacy of transfection efficiency
using flow cytometry.
We will use another suitable cell line such as HeLa as published by Camilla et al., for estimating the biological
activity of siRNA. We will use blank liposomes without siRNA as negative control and liposomes with siRNA
for estimating the biological activity. We will also have another group without inherent expression of Lp-PLA2,
to show the specificity of siRNA release pattern of liposomes.
Specific aim 3:
We will also use ApoE deficient
atherosclerotic mice model to estimate
of
concomitant
treatment
of
atherosclerotic mice with Darapladib
PLA2 siRNA. For this purpose mice
homozygously deficient for ApoE will
from Jackson laboratories and will be
as per IACUC guidelines in LARC,
Mice will develop atherosclerosis
diet they receive. Hence, mice to be
the drugs will be given a high fat diet
18% hydrogenated cocoa butter,
cholesterol, 7% casein, 7% sucrose,
maltodextrin ad libitum and this diet
continued till 17 weeks starting at 6
age. The number of mice per group
determined based on power calculation
be done after the preliminary studies.

the efficacy
and

Lp-

be obtained
maintained
UMKC.
based on the
tested using
containing
0.15%
and
3%
will
be
weeks
of
will
be
which will
Source: Camilla et al.,doi:10.1016/j.ijpharm.2006.11.010

The mice which were used for the study will be also used to obtain blood for serum lipid analyses. Blood from
mice will be collected from retroorbital plexus and will be initially centrifuged at 1000g at 4C for 3 minutes.
This is done to separate the serum from whole blood and the obtained serum will be stored at -80C until further

analys
es. This serum will be used to analyze the levels of total cholesterol (TC), High Density Lipoprotein (HDL),
Low Density Lipoprotein (LDL), and Triglyceride (TG). We will follow the manufacturer's protocol for
enzymatical analyses of these samples which we will purchase from Wako Inc.

A better estimate of efficacy of siRNA and the drug will be the levels of plasma Lp-PLA2 after treatment of
mice with these drugs. So, we will also estimate the levels of plasma Lp-PLA2 using a method published by
Wang et al. For this purpose we will use the ability of Lp-PLA2 to degrade 2-thio-PAF in serum. Also we will
use staining techniques to estimate the efficacy of treatment with Lp-PLA2 siRNA and darapladib in terms of
reduction of plaque volume. We will perform Sudan IV staining and Immunohistochemical analyses to
determine if the treatment has beneficial effects in terms of reduction of plaque volume. For either of analyses
mice will be sacrificed and dissected to obtain aortas. The obtained aortas will be stored in paraformaldehyde
solution until further analyses. The effect will be best elucidated if we can have a long part of aorta to study
staining, hence we will use a segment of aorta from heart till 3mm distal to iliac bifurcation. The aorta will be
cut open longitudinally and will be fixed on silica gel plate. After initial washing in 70% ethanol, we will stain
the aortas in 1% Sudan IV in 50% acetone and 35% ethanol for 10 minutes. After a final wash with 80% ethanol
for 5 minutes, the sections will be photographed and analyzed using suitable software.

For Immunohistochemical analyses, paraffin sections prepared from aortic sinus will be used. After
deparaffinization and initial treatment with 0.3% H2O2, for 10 minutes, to remove any endogenous peroxide
activity, the sections will be incubated with 5% BSA for 1 hour to avoid non specific binding of antibody. Then
the sections will be incubated overnight at 4C with murine smooth muscle actin antibody or Mac-2 antibody.
After this the sections will be washed and incubated with secondary antibody for 1 hour. The sections will be
visualized using suitable software to check the levels of actin in the smooth muscle of aorta and the number of
activated macrophages in the aorta.

References: