Final Paper

Recombinant Apolipoprotein AImilano and its
Potential as an Atherosclerosis Treatment: A
Review
Aaron Banks
May 7, 2015
1 Abstract
In the small Italian town of Limone Sul Garda, a genetic mutation has devel-
oped that provides significant protection against atherosclerosis and its related
vascular diseases, which make up the most common cause of death in the United
States. The mutation causes a arginine to cysteine replacement at position 173
on apolipoprotein A-I (apo A-I), the primary component of high-density lipopro-
tein (HDL), producing a variant referred to as apo A-Imilano. Individuals with
this mutation have significantly lowered levels of HDL, which would typically
indicate an elevated risk of atherosclerosis. Yet, these individuals exhibit a lower
risk, indicating the milano mutation may cause the production of an HDL that
is more efficient at removing lipids from tissue. Clinical trials have begun, which
attempt to determine the efficacy of a drug produced from recombinant apo A-
Imilano taken through weekly injections or a single injection. In this review, I
will show that such a drug can indeed be effective at reducing arterial plaque.
1

Figure 1: Image showing the
effects of the early stages of
atherosclerosis. Note the de-
crease in lumen size as the
plaque grows larger4
.
2 Introduction
Atherosclerosis is a vascular disease in which plaque, composed of fat, choles-
terol, calcium and other substances found in the blood, forms inside an in-
dividual’s arteries. Beginning during adolescence and continuing for decades,
plaque builds up, narrowing arteries, and thereby reducing the body’s ability
to transport oxygen through blood, as is seen in Figure 11
. Diseases related
to atherosclerosis include coronary heart disease, carotid artery disease and
chronic kidney disease; differences in these diseases are dependent upon where
the arterial plaque forms. While deaths from these diseases have decreased sig-
nificantly over the past forty years, cardiovascular diseases are still the leading
cause of death in the United States, accounting for more than 30% of deaths
in 20102
. Treatment generally focuses on lifestyle changes, along with medica-
tions (statins) that inhibit cholesterol synthesis and decrease the expression of
low density lipoproteins (LDLs), resulting in lower concentrations of both total
cholesterol and plasma concentrations of LDL3
.
High density lipoproteins (HDLs) have been shown to have an inverse cor-
2

Figure 2: A diagram illustrating the process of reverse cholesterol transport.
HDL, along with lipid-poor apoA-I transports cholesterol out of peripheral cells
and into the liver, often with the help of apoB lipoprotein. After cholesterol
reaches the liver, it is then excreted in either feces or bile6
.
relation with risk for atherosclerosis5
. This protection has been suggested to
be due, in part, to the role of HDL in reverse cholesterol transport (RCT), il-
lustrated in Figure 26
. This role in RCT is especially important because most
peripheral cells lack the ability to catabolize cholesterol, relying instead on the
cholesterol eﬄux capabilities of HDL to transport cholesterol to the liver, where
it can be excreted.
Apolipoprotein A-I (apoA-I) and Apolipoprotein A-II (apoA-II) are the pri-
mary components of HDL, constituting 60% and 20% of the mass of HDL,
respectively7
. ApoA-I has been shown to guide HDL formation, maintain its
structure, and contribute to the athero-protective eﬀects of HDL8
. ApoA-I,
along with HDL, has been shown to be inversely associated with the risk of
atherosclerosis, while the ratio of apoA-I:apoA-II has been shown to be inversely
proportional to the risk of atherosclerosis9
.
HDL, as a combination of proteins and lipids, can exist in several forms,
3

Figure 3: A diagram illustrating some of the forms that HDL can occupy11
.
Note the double belt formation of apoA-I. Discoidal HDL is shown in A, with
antiparallel bands of apoA-I circling the cylindrical structure. B shows a pro-
posed form of mature, wild type HDL. Note the tetrafoil formation; it is pro-
posed that the apoA-I belts are bent around flexible Gly-containing hinges. C
shows a proposed model for HDLmilano; the apoA-Imilano bands form two par-
allel rings, with this form likely brought about by a dimer caused by the extra
Cys residue.
depending on the conditions in which it is in. It begins as discoidal HDL,
complexed with two bands of Apo A-I, unesterified cholesterol and phospho-
lipids, and appearing in a short cylindrical form, as can be seen in Figure 3A.
In this form, HDL is an excellent substrate for lecithin cholesterol acyltrans-
ferase (LCAT), which esterifies cholesterol on the surface of HDL. The esterified
cholesterol molecules are now extremely hydrophobic and are able to move to
the center of the HDL molecule, making it larger and more spherical in the
process10
. In this spherical form, illustrated in Figure 3B, the HDL transports
cholesterol to the liver, so it can be excreted.
ApoA-Imilano is a mutant of apoA-I, with a cysteine inserted in place of
arginine in position 17312
. The mutation was first discovered in a family in
Limone sul Garda, Italy; they showed markedly reduced amounts of HDL and
apoA-I, along with hypertriglyceridemia, yet they did not show symptoms of
atherosclerosis13
. In the initial study, the cholesterol and lipoproteins of four
members of the family were studied: a father, his son and two daughters; the
results of which can be seen in Table 1.
4

Table 1: A table containing the results of a study on a family with the apoA-
Imilano mutation. The 13 year old daughter was found not to have the mutation.
The father, son and 12 year old daughter can be seen to express the phenotype
of signiﬁcantly lowered HDL and apoA-I levels along with elevated triglyceride
levels. Yet, they did not have premature coronary heart disease, as would be
expected from such symptoms13
.
In risk assessment studies, individuals are said to be a high risk for cardio-
vascular disease with an LDL:HDL cholesterol ratio of 4.90 or greater, and to
be a low risk with a LDL:HDL ratio of 3.50 or less14
. The three members of this
family who had the mutation all had LDL:HDL ratios greater than 8.75. After
the initial study, further research was performed on rates of coronary heart dis-
ease for the entire town of Limone sul Garda. It was found, that from 1972-1981,
Limone sul Garda experienced 51% fewer deaths from coronary heart disease
than would have been expected in an Italian population15
.
Twenty years later, a subsequent study was performed on carriers of apoA-
5

Table 2: A table showing the results of a 2001 study on the cholesterol levels of
apoA-Imilano carriers16
. According to these results, the carrier group should be
considered high risk for cardiovascular disease, as they had an average LDL:HDL
ratio of 6.65, while the control group had an average ratio of 3.1214
. Yet, both
groups had carotid artery thicknesses that were not significantly different.
Imilano, a kindred control group and two series of subjects with hypoalphalipopro-
teinemia (HA), significantly low levels of Apo A-I, and no history of coronary
heart disease. The carriers of apoA-Imilano once again expressed the phenotype
of lowered HDL levels and hypertriglyceridemia, as is shown in Table 2. The
study also examined the thickness of the carotid artery intima media thickness
(IMT) for each group and found that the arteries of the HA groups had signifi-
cantly larger IMT than did either the control or carrier group16
. This indicates
that, despite the lowered HDL levels and elevated triglyceride levels, the carrier
group did not have an elevated risk of carotid artery disease.
The mechanism behind HDLmilano’s increased ather-protective properties is
6

Figure 4: Proposed models of the double belt configuration of wild type and
milano forms of apoA-I derived from x-ray crystallography; figures are drawn
to scale. Left panels show both apoA-I molecules on top of one another, while
the right panels show the differences between milano and wild type in molecule
1. The location of milano mutations are noted by a yellow diamond, and the
location of the flexible Gly hinges are noted by gray and white circles11
.
not fully known; however, it has been shown that the apoA-Imilano mutation
results in HA due to incomplete LCAT activation17
. The cysteine replacement
at position 173 has been shown to provide a loss in overall stability, yet the loss
in stability is not as great as other insertion mutations in apoA-Imilano
18
. The
slight loss in stability is likely due to the absence of a salt bridge formed by
arginine at position 173 with glutamic acid at position 169 in wild type apoA-I,
while it is mitigated by a disulfide bond connecting the two apoA-Imilano bands,
formed by the inserted cysteines at position 17311
.
These findings indicate that HDLmilano functions much more efficiently than
wild type HDL, suggesting that a recombinant form of apoA-Imilano may be
effective as a treatment for atherosclerosis. In this paper, I will show that, while
7

they are not yet ready for commercial use, drugs incorporating recombinant
apoA-I show great promise in reducing arterial plaque in individuals with acute
cardiovascular disease.
3 Methods
3.1 ETC-216
Two random, double-blind clinical trials (in 2003 and 2006) were performed
to determine the effects of ETC-216, a drug designed to mimic nascent HDL
composed of recombinant apo A-Imilano and a naturally occurring phospho-
lipid. In both studies, roughly fifty individuals with acute coronary syndromes
(ACS) were placed in three treatment groups. One group received a placebo (an
injection of 0.9% saline solution), while the other two groups received weekly
infusions of ETC-216 (15 mg/kg or 45 mg/kg) over the course of five weeks19
.
The atheroma volume of each patient was measured using intravenous ultra-
sound (IVUS). Prior to the study, least and greatest diseased arterial segments
were chosen in each patient, and a baseline was taken. After the five week
course, another IVUS was performed to compare with the baseline20
.
3.2 MDCO-216
In 2014, a study was performed on a different formulation named, MDCO-216.
Composed of recombinant apo A-Imilano and the same phospholipid as ETC-
216, but in a different ratio, the drug is designed to be given in a single injection,
as opposed to five. The study focused on the cholesterol efflux ability of the
drug, whether an individual’s immune system would mount an antibody defense,
and the ideal dosage22
.
The study had two groups of patients with acute coronary artery disease,
8

those receiving a placebo (0.9% saline solution), and those receiving one of four
ascending doses (10, 20, 30 or 40 mg/kg). Cholesterol efflux ability was deter-
mined ex vivo using radio-labeled cholesterol, and blood analysis was performed
to determine if an anti-drug antibody was present. Participants were monitored
for thirty days after injection for any adverse effects22
.
4 Results
4.1 ETC-216
After receiving five weekly injections of a placebo (serving as a negative control)
or two different doses (15 or 45 mg/kg) of a drug composed of recombinant apo
AImilano and a naturally occurring phospholipid (ETC-216), median atheroma
volume decreased in the combined ETC-216 group by 0.81%, and it increased
in the placebo group by 0.03%, as is shown in Table 3. The group that received
the lower dose of ETC-216 (15 mg/kg) exhibited a median decrease of -1.14%,
while the median decrease of atheroma volume of the 45 mg/kg ETC-216 group
was substantially less, at 0.34%19
.
The results of the infusions on mean maximum atheroma thickness in all
three groups can be seen in Table 4; the combined median difference for the
ETC-216 groups was -0.035 mm, and the difference for the placebo groups was
-0.008 mm, a roughly 5-fold difference in atheroma reduction. The median
difference for the high dose ETC-216 group was -0.029 mm, while the median
difference in maximum atheroma thickness for the low dose group was -0.045
mm.
9

Table 3: Table comparing percent atheroma in the target coronary artery of
placebo and ETC-216 groups from baseline testing to a follow-up test five weeks
later. The mean is given with standard deviation in parentheses and median
values are given with interquartile ranges19
.
Table 4: A table showing the median and mean maximum atheroma volume of
the subjects of the placebo and the ETC-216 groups19
.
The difference in atheroma volume for the 10 mm artery segment that con-
tained the greatest atheroma burden is shown in Table 5. The placebo group
showed a median decrease of 0.2 mm3
, while the combined ETC-216 groups
exhibited a decrease of 4.4 mm3
. Once again, the group that received a lower
dose of ETC-216 showed a larger decrease in atheroma size (4.7 mm3
, com-
pared to 2.9 mm3
for the 45 kg/mg group). Figure 5 provides an example of a
cross-sectional intravascular ultrasound (IVUS) of a subject’s artery; 4016 such
images were used to determine the changes in atheroma volume displayed in
Tables 3-5. Of particular importance is the lack of significant change in lumen
size, despite a 34% change in atheroma volume19
.
Table 5: Data on the effects of ETC-216 on the most severely diseased segment
of each subject’s target coronary artery19
.
10

Figure 5: Sample baseline (A) and follow-up (B) IVUS images from a pa-
tient who received the high dose of ETC-216 (45 mg/kg). Note the change
in atheroma volume, yet lack of change in lumen size19
.
Table 6: Data showing the effect of infusion of placebo and two doses of ETC-
216 over the course of five weeks. EEM and lumen volume is obtained from
both baseline and follow-up testing20
.
The Nicholls study, performed three years after the original trial, showed
many of the same results, and the phenomena of the EEM shrinking along with
plaque volume was studied in greater detail. The effects of placebo and ETC-
216 infusions on the volume of the external elastic membrane (EEM) and the
lumen of test subjects from Nicholls et al. can be found in Table 6. The placebo
group showed an 8 mm3
decrease in lumen volume and a 1 mm3
decrease in
EEM volume. The combined ETC-216 groups displayed a median increase of 8
mm3
in lumen volume, yet a 16 mm3
decrease in EEM volume.
Table 7 shows the change in atheroma volume of the most and least diseased
artery segments for both groups given different doses (15 and 45 mg/kg) of ETC-
216 in five weekly infusions. The most diseased segments showed a substantial
reduction in median atheroma volume, while the least diseased segment showed
11

Table 7: Table comparing the eﬀects of ETC-216 on the atheroma, EEM and
lumen volume of the most and least diseased 10 mm segments of the target
coronary artery for both ETC-216 groups20
.
an increase in median volume (a 15.2 mm3
decrease compared to a 1.8 mm3
increase). In both segments, however, the lumen size did not change signiﬁcantly
(1.2 mm3
decrease for the greatest diseased and a 0.5 mm3
decrease in the least
diseased).
Figure 6 demonstrates the relationships between change in plaque volume
and both change in lumen volume and EEM volume. Change in lumen volume
and plaque volume showed little to no correlation, with an r value of 0.13 and a
p value of 0.45. Change in plaque volume did however show a relatively strong
direct correlation with change in EEM volume, with an r value of 0.62 and a p
value< 0.0001.
The lack of change in lumen size is illustrated in Figure 8, with six cross-
sectional representations of target arteries; the left panels show an artery at
baseline and the right panels show an artery at a follow-up examination. The
top two panels are simply illustrations showing a static lumen width despite a
shrinking atheroma. The middle two panels present a cross section that under-
went a 2.87 mm2
reduction in atheroma area (31% of baseline area), yet lumen
area only increased by 0.36 mm2
(4.3% of baseline area). This phenomena is
12

Figure 6: Graph illustrating the corre-
lation between changes in lumen and
atheroma volume, as well as the rela-
tionship between changes in EEM and
atheroma volume. The changes were
studied in the segments containing the
greatest atheroma burden at baseline of
the target arteries of all subjects who
received ETC-21620
.
due to an EEM that decreased by 2.51 mm2
, or 14.2% of baseline area. The
bottom two panels depict a less diseased cross-section that underwent a much
smaller change in atheroma area (0.23 mm2
, or 3.4% of baseline). Despite this
smaller change, EEM area still decreased a similar amount to atheroma area
(0.29 mm2
, or 2.5% of baseline), resulting in a 0.06 mm2
(0.8%) reduction in
lumen area.
4.2 MDCO-216
The trial performed on MDCO-216 showed that a single dose of MDCO-216
is capable of significantly increasing cholesterol efflux, which is consistent with
earlier findings. The four individuals given the 10 mg/kg dose showed the least
increase, with an average of 8.4%, while those receiving the 20 mg/kg dose saw
the greatest increase (13.1%). Those in the 30 and 40 mg/kg groups saw a slight
decline in efflux increase, with 13.0% and 12.4%, respectively22
.
13

Figure 7: Examples of cross-sectional
IVUS images of arterial segments at
baseline (left) and follow-up (right).
The top and middle panels depict the
effects of a change in plaque volume
on both EEM and lumen volume. The
bottom panel shows the results when
plaque volume remains unchanged20
.
With this information, a pharmacodynamic (PD) model was created to pre-
dict the most effective dosage of MDCO-216, which can be found, along with
the average efflux increase for each dose, in Figure 8. The model predicted
that a dose of 16 mg/kg would provide the most effective treatment22
, which is
consistent with both studies on ETC-21620
.
5 Discussion
In both trials of ETC-216, a five week course of injections was shown to bring
about a highly substantial change in atheroma volume. The reduction over the
course of five weeks was orders of magnitude greater than the reduction seen by
either Pravastatin or Atorvastatin in a clinical trial performed over the course
of 18 months23
. The lack of change in lumen size was an unexpected result, one
which suggests an underlying mechanism alters the size of the EEM to maintain
14

Figure 8: Graph depicting the average cholesterol efflux of each ascending dose,
along with the PD model created from that information22
.
a constant lumen size20
. This phenomena must be studied further before such
a drug can be deemed safe to be sold commercially.
More studies are needed, as only 88 subjects received a formulation of recom-
binant apo A-I; consequently, the standard deviation of volume measurements
often eclipsed 50% of baseline and follow-up values20
. In addition, more infor-
mation on adverse effects of the drug are needed if it is to be sold commercially.
Figure 9 lists the adverse effects exhibited in the intent-to-treat populations of
all three study groups of the Nissen study. Several troubling adverse effects,
such as stroke, cholelithiasis and abnormal liver function, were reported by one
or more subjects19
, raising the question of whether ETC-216 or MDCO-216 can
contribute to these events in some patients.
In addition, longer studies, such as those with mortality end points, are
needed to determine if this drug will increase the life expectancy of patients, as
all three studies tracked patients between 30-35 days22
. And while injections
are generally not preferred by patients, were this drug able to greatly reduce
plaque with a single injection, as the MDCO-216 study attempted to prove, it
likely would prove to be commercially viable.
15

Table 8: Table on the prevalence and type of adverse effects found in the intent-
to-treat populations of all three study groups of the study performed by Nissen
et. al.
6 Conclusion
The current models of mature HDLmilano indicate that apo A-Imilano is arranged
in a parallel, double-belt formation, as opposed to the tetrafoil configuration of
wild type HDL. It is thought that this allows HDLmilano to sequester lipids
in smaller amounts but at a faster pace than wild type HDL11
, which may be
the cause of the milano mutation’s atheroprotective effects. High resolution
structural analysis of both wild type and milano HDL is needed to determine
whether the proposed models are correct.
All three clinical studies indicate that a drug formulated from apo A-Imilano
can reduce the amount of arterial plaque in a diseased patient at a much greater
rate than current treatments19
. Although more research is needed, it can be
said with a high degree of certainty that a drug developed from recombinant
apo A-Imilano can be used to reduce the amount of arterial plaque in patients
with acute coronary diseases.
16

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19

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