Effects of the selective mineralocorticoid receptor antagonist eplerenone on extracellular adenosine formation in humans in vivo

Despite state-of-the-art reperfusion strategies, mortality and morbidity in
patients with an acute myocardial infarction remain significant. This is
caused, at least in part, by *lethal reperfusion injury*. Therefore, novel
therapeutic options to further limit ischemia-reperfusion (IR) injury are
urgently needed to improve outcome in these patients.
It has been suggested that the mineralocorticoid receptor (MR) antagonists
spironolactone and eplerenone could potentially serve this goal, because these
drugs reduce mortality in patients with heart failure. Indeed, recent studies
in murine models of myocardial infarction have shown that MR antagonists can
directly limit infarct size. In more detail, in vitro studies and studies in
animal models of myocardial infarction have reported that spironolactone,
eplerenone and/or canrenoate (the active metabolite of spironolactone) reduce
myocardial infarction size, and protect against left ventricular remodeling; by
reducing interstitial fibrosis, cardiomyocyte hypertrophy and/or collagen
synthesis, preventing apoptosis, improving cardiac function, and reducing mRNA
synthesis of (pro-)collagen. The underlying mechanisms of these
cardioprotective effects are not yet fully understood, but it has been
suggested that the endogenous purine nucleoside adenosine is crucially
involved. In a recent animal study, canrenoate caused a dose-dependent
reduction in infarction size. This protective effect of canrenoate was
completely abolished in CD73 knock-out and adenosine A2b receptor knock-out
mice. In rats, eplerenone significantly reduced infarction size, and this
beneficial effect was abolished by co-administration of adenosine receptor
antagonists. (2) These findings suggest that extracellular formation of
adenosine is crucial for the protective effect of MR antagonists on IR injury.
Adenosine is an endogenous purine nucleoside, which is formed by intra-, and
extracellular degradation of adenosine monophosphate by the enzyme ecto-5*-
nucleotidase, which is also named CD73. Degradation of adenosine occurs in the
intracellular compartment. As a consequence, facilitated diffusion of adenosine
over the cellular membrane by the equilibrative nucleoside transporter (ENT) is
normally directed inwards. Stimulation of membrane-bound adenosine receptors
induces various effects, including vasodilation, inhibition of inflammation,
and protection against IR-injury. Indeed, endogenous adenosine acts as a key
mediater of the infarct size-limiting effect of several drugs.
Measurement of adenosine is extremely difficult (24), because the half life of
adenosine in blood is approximately one second, due to rapid uptake and
degradation. (25) Indirectly, the effects of substances which resemble the
effects of adenosine, could serve as a surrogate for adenosine formation.
Dipyridamole increases the extracellular adenosine concentration by inhibition
of the ENT transporter (26) and induces local vasodilation. (27) Therefore,
increase in forearm blood flow (FBF) response to dipyridamole acts reflects
adenosine formation.
Dipyridamole increases FBF and this effect is blocked by caffeine. (28) Statins
have been shown to promote adenosine receptor stimulation by activation of
ecto-5*-nucleotidase activity. (29-31) Metformin facilitates adenosine receptor
stimulation, probably by increasing the intracellular formation of adenosine.
(32) Arterial occlusion is an endogenous stimulus to increase adenosine
formation and hereby enhancing blood flow, resulting in so-called *post-
occlusive reactieve hyperemia* (PORH). This effect is also potentiated by
dipyridamole and prevented by co-administration of caffeine. (33)

In this research proposal, we aim to investigate for the first time in humans
in vivo whether eplerenone promotes adenosine receptor stimulation by
activating CD73. The results of these studies will provide a possible
explanation for the positive effects of MR antagonists on IR-injury. To test
our hypotheses, we will use the vasodilator response in the forearm vascular
bed to various stimuli as a well-validated surrogate of adenosine receptor
stimulation, as will be explained in more detail in the next section.

Single center, double blinded, randomized, placebo controlled trial in a
cross-over design

-eplerenone 50mg bid during 8 days and placebo 50mg bid during 8 days (or vice
versa)

Nature and extent of the burden:
-participants will visic our clinic 9 times in total
-before inclusion in the study, volunteers will be screened by history taking,
physical examination, venous blood drawing (1 time) and electrocardiography
-During the study, participants take eplerenone 50mg bid during 8 days,
followed by placebo 50mg bid during 8 days, or vice versa. Venous blood
sampling will be performed 6 times during te study, and a 27 gauge-needle will
be inserted into the brachial artery 4 times (day 7 and 8 during eplerenone
treatment, and day 7 and 8 when on placebo). Participants are not allowed to
take any caffeine or alcohol 24 hours before both experiments.

Risks:
-The risk of an hematoma will be very small, because for the insertion of a
needle into the brachial artery we use a very small needle (27 gauge).
-we will reduce the risk of hyperkalemia by excluding volunteers with a serum
potassium of 4.8 mmol/L or higher. During the first week of treatment we will
monitor serum potassium again. At this time, participants will be excluded from
the study if they reach a serum potassium of 5.1 mmol/L or higher.
-Although we don't expect an effect on blood pressure during eplerenone
treatment in our healthy volunteers, we will exclude male volunteers with a
blood pressure of <100/<60 mmHg.
-we don't expect endocrine side effects to occur in our volunteers, since
eplerenone is a selective mineralocorticoid receptor antagonist. Therefore,
anti-androgenic or progestagene side effects (as seen during spironolactone
treatment) are not likely to occur. Furthermore, treatment is only 8 days.