A Proof of Principle Study Investigating the Influence of Diclofenac on Perfusion Changes During Cutaneous Negative Pressure Wound Therapy

Negative Pressure Wound Therapy (NPWT) is a common treatment for acute,
chronic, and recently also postoperative surgical wounds.2-6 In the first NPWT
basic research paper, augmented blood flow is put forward as one of its
mechanisms of action.5 Although a multitude of studies currently reports that
NPWT improves blood flow5,7-22, the underlying mechanism remains elusive.
Moreover, an increasing amount of studies that report a negative effect of NPWT
on perfusion and oxygenation has been published in recent years.23-31 This has
resulted in a debate whether NPWT is truly able to increase perfusion
immediately after initiation of therapy23-31, and some investigators currently
advise clinicians to use NPWT cautiously on tissues at risk of ischemia.27,30
Because adequate perfusion is of such vital importance in wound healing32,33,
elucidation of this controversy seems warranted.

The Pressure-induced Vasodilation Phenomenon

Outside of the field of NPWT, several researchers have demonstrated that tissue
blood flow increases during moderate amounts of mechanical deformation in
healthy individuals, for example during tissue compression.34-36 This
phenomenon is commonly referred to as *pressure-induced vasodilation* (PIV,
where pressure stands for the exertion of a compressive mechanical
force).37,38 PIV has been shown to act as a protective mechanism against the
ulcerations from mechanical stress.37,38 Several investigators have elucidated
the physiology behind PIV: mechanical deformation as a result from cutaneous
pressure exertion leads to activation of the mechanosensor acid-sensing ion
channel 3 (ASIC3), an ion channel located in sensory nerve endings (i), which
triggers the release of the neuropeptide calcitonin gene-related peptide (CGRP)
(ii), a potent vasodilator39, thus resulting in vasodilation and ultimately, an
increase in perfusion.38 CGRP is able to induce vasodilation in both the
microcirculation (through release of nitric oxide by the endothelium), or
independently through direct action on vascular smooth muscle cells (e.g.
resistance arteries), depending on the vascular bed involved.39 CGRP is stated
to be the most potent vasodilator currently known to exist in the human body39,
having a potency 10-100 times higher than the most potent prostaglandins or
other vasodilators such as acetylcholine and substance P39, and has also been
shown to increase angiogenesis and wound healing.39-41 Administration of ASIC3-
or CGRP-blockers, as well as ASIC3 knock-out (KO), was shown to eliminate PIV
in response to mechanical deformation in mice, and ASIC3-blockers have been
confirmed to prevent PIV from occurring in humans as well.38 Additionally,
ASIC3-KO was associated with elimination of reactive pressure-induced hyperemia
after compression of skin, and abolishment of ASIC3-mediated CGRP release
resulted in earlier onset and increased severity of pressure-induced
necrosis.38,42 Other researchers have demonstrated that ASIC3 is activated not
only by compression but also by stretch43, implicating that PIV is not only
involved in tissue subject to compression but also in tissues subject to
stretch. Moreover, the main tissues involved in this protective mechanism,
afferent sensory nerves and vasculature, can be found virtually everywhere in
the human body outside of the central nervous system, and PIV already has been
demonstrated to exist in muscular tissue as well.44 The same applies to ASIC3,
which is present in many of these sensory afferent nerves, and CGRP, which is
also present in many sensory afferent nerves, especially those innervating
resistance arteries.39,45 This implies that PIV is a relevant part of
mechanobiology everywhere outside of the central nervous system.

PIV involvement in NPWT?

NPWT has been shown to result in both mechanical deformation of
tissue25,26,46,47 as well as increased blood flow.8,9,48,49 Although these
findings may seem contradictory, they can be explained by involvement of PIV.
Therefore we propose that perfusion increases during NPWT are mediated (at
least in part) by the same physiology involved in PIV (see Figure 1).Thus, in
the articles denouncing the ability of NPWT to quickly increase perfusion
because of concerns regarding negative effects of mechanical deformation on
blood flow23-27,29,30,47, the link has not been made to the release of
vasodilatory PIV mediators during NPWT, which simply has not been previously
investigated in the context of blood flow during NPWT. Yet, NPWT already has
been shown to result in an increase of the PIV mediators CGRP and NO,
contributorily suggesting involvement of PIV (although these authors did not
link CGRP to the vasodilation seen during NPWT in their papers).50-52

















Figure 1. Proposed NPWT-vasodilation pathway. NPWT, Negative-pressure wound
therapy; ASIC3, acid-sensing ion channel 3; CGRP, calcitonin gene-related
peptide.

Impairment of PIV

The research group of Fromy et al have demonstrated that several drugs, such as
Diclofenac and Amiloride (ASIC3 antagonists45) eliminate PIV.38,53,54 Although
NSAIDs such as Diclofenac also inhibit the cyclooxygenase-mediated production
of vasodilatory arachidonic acid metabolites such as prostacyclin (PGI2)55,
abolishment of PIV was attributed to their ASIC3-antagonism45 because
COX-inhibition with the NSAID indomethacin did not completely eliminate PIV,
while ASIC3-antagonists did.56 Because many patients receive both Diclofenac
and NPWT during their treatment, this may have substantial implications for
pain management during NPWT.57

Measurement techniques of perfusion
One of the criticisms brought forth against LDF as a measurement technique in
NPWT perfusion research has been that *the diameter of the underlying vessels
is not factored into the calculation of the laser Doppler device, which records
an effect of increased velocity only*27, *which is erroneously interpreted as
increased perfusion*.27 For this reason, this study will not only perform
perfusion measurements using LDF, but also with single fiber reflectance
spectroscopy (SFRS). SFRS is a validated optic technique frequently used for
providing tissue oxygenation levels and total hemoglobin.58-60 Moreover,
because blood in tissue is strong absorber of certain light frequencies, and
concentrated in blood vessels, this results in a measurable difference in
absorption of light between vessels with a different vessel diameter. Based on
this principle, Van Veen et al developed an approach where SFRS can be used to
calculate a mean vessel diameter, thus allowing for the monitoring of dynamic
processes such as vasodilation of the tissue under investigation.61,62 Although
widely used in other fields, SFRS has not been used before in NPWT perfusion
research, and will provide us with the additional parameters, allowing us to
study these parameters in relation to each other, the LDF measurements, as well
as between study groups. The combination of LDF with SFRS will provide us with
a thorough assessment of the vascular tissue response to the NPWT.

Cutaneous NPWT application has been shown to result in an increase of
LDF-measured blood flow in healthy volunteers.7 Additionally, the experiment of
Fromy et al illustrating elimination of PIV with Diclofenac has been performed
in healthy volunteers as well. Moreover, patients need to be without
neurovascular disease in order for PIV to take place.38 This implies that young
healthy volunteers are the ideal study population for this study.

*
Stepwise summary of background and study rationale

1. Increased perfusion is one of the main mechanisms of action suggested for
Negative Pressure Wound Therapy (NPWT).
2. Acid-sensing ion channel 3 (ASIC3), a mechanosensor located in sensory
nerves, initiates a vasodilatory pathway during mechanical deformation of
tissue, often referred as *pressure-induced vasodilation* (PIV).
3. Because NPWT results in mechanical deformation, this suggests that the
increased perfusion during NPWT may be explained by involvement of PIV.
4. Because Diclofenac is an ASIC3-antagonist that eliminates PIV, this implies
that Diclofenac may eliminate the increase of perfusion normally seen during
cutaneous NPWT.
5. Diclofenac can therefore be used as proof of principle for the hypothesis
that increased perfusion during NPWT is due to ASIC3-mediated PIV.

By potentially revealing the underlying physiology behind the blood flow
changes seen during NPWT, this study will contribute to the scientific
knowledge regarding the mechanisms of action of NPWT This may provide windows
of opportunity for improvement of clinical NPWT treatment.

This study is not a study to investigate clinical application of Diclofenac.
Diclofenac is only used as a challenge agent to induct a physiological blockade
of the normal physiology we hypothesize to lead to increased blood flow during
NPWT. Diclofenac formulations will only be provided once to every participant.
This study should therefore not to be seen as a clinical drug trial.

The primary objective of this study is to investigate the effects of a
Diclofenac challenge on perfusion changes during NPWT as a proof of principle
for the involvement of ASIC3*mediated PIV physiology in the blood flow changes
seen during NPWT.

Secondary Objectives

Secondary objectives of this study are:

* To investigate potential differences between an orally and topically
administered Diclofenac challenge
* To investigate the relationship between LDF and SFRS-measured outcomes

Study design

In order to achieve the maximum power while limiting the amount of participants
necessary, this will be a prospective cross-over study with repeated
measurements where healthy volunteers their dominant forearm will be randomized
to a random order of three arms consisting of NPWT on the forearm with either:

* No challenge agent
* Topical Diclofenac challenge
* Oral Diclofenac challenge

Participants will receive ± 4 gram of gel in this study to their forearm during
the topical challenge, which equals around 40 mg Diclofenac, around the
approximate location where the probe holder will be placed. During the oral
challenge, participants will receive 100 mg Diclofenac.

This study design will facilitate investigation whether ASIC3-mediated PIV is
indeed involved during the blood flow changes seen during NPWT. This will also
facilitate investigation of the influence of Diclofenac, as well as any
potential differences between topically and orally administered Diclofenac.

This study is investigator-initiated and will be performed in the Academic
Medical Center only.

Three sessions with Negative Pressure Wound Therapy, once with oral Diclofenac,
and once with Diclofenac gel.

5. TREATMENT OF SUBJECTS

5.1 Treatment

Healthy volunteers their dominant forearm will be randomized to a random order
of three arms:

* No challenge agent
* Topical Diclofenac challenge
* Oral Diclofenac challenge

Participants will receive ± 4 gram of gel in this study to their forearm during
the topical Diclofenac challenge, which equals around 40 mg Diclofenac, around
the approximate location where the probe holder will be placed. During the oral
Diclofenac challenge, participants will receive 100 mg Diclofenac.

According to the summary of product characteristics, the maximum plasma
concentration is reached 1-4 hours after oral administration. Topical
administration of 2.5 gram of Diclofenac 5% gel 1 hour before measurement was
able to completely abolish PIV in humans in the experiment of Fromy et al.38
The gel and medication will therefore be supplied to the participant sixty
minutes prior to the measurements, during which the patient will be sitting
comfortably in a chair in order to have an acclimatisation period of 50 minutes
in order to adjust to the conditions in the room where the measurements will be
performed. During the last ten minutes, the gel will be removed gently and NPWT
and measurement probes will be applied. The summary of product characteristics
of the gel that we will use (Diclofenac HTP 1%, gel (Healthypharm B.V.,
Etten-Leur, The Netherlands) mentions that the amount of gel needed to treat an
area of 4 to 8 dm2 is 2-4 gram. Therefore we will apply ± 4 gram of gel in this
study to the patients their forearm, around the approximate location where the
probe holder will be placed. Subsequently, the dominant forearm of the
participant will be subjected to 5 minutes of cutaneous NPWT with -25 mm Hg of
subatmospheric pressure, during which perfusion will be measured to attain a
baseline measurement. The -25 mm Hg of subatmospheric pressure is to ensure the
equipment is held in place correctly onto the skin.7 After 5 minutes, the
amount of subatmospheric pressure will be increased to -200 mm Hg
subatmospheric pressure. After 20 minutes of -200 mm Hg NPWT, the NPWT device
will be turned off and the equipment removed. During the measurement sessions
and acclimatisation periods, participants will be instructed to relax, breathe
as normal, and will be assured to be free from distractions. Participants will
be instructed to not move their upper extremities in order to stabilize the
probes during the measurements. If a measurement session is hampered by leaks
from the NPWT, the measurement will be attempted again at a later point in time
during another week, at approximately the same time of day. During each
measurement session, all participants will receive 20 minutes of cutaneous NPWT
with -200 mm Hg with a foam pad of 7 x 12 x 2 cm.
5.2 Use of co-intervention

Participants will be instructed to abstain from eating or drinking in the 2
hours before and during the experiment. Participants will be instructed to
abstain from smoking, caffeine, alcohol, and use of other pharmaceuticals
during participation in the experiment. Participants will only receive one dose
of 100 mg of oral Diclofenac during the complete duration of the study. The
study population will consist of healthy volunteers between 18 and 35 of age
free from a history of gastro-intestinal complaints. Co-medication such as
proton pump inhibitors aimed at preventing gastro-intestinal adverse events is
therefore not considered to be indicated in this experiment.

5.3 Escape medication

Not applicable.



13. STRUCTURED RISK ANALYSIS

13.1 Potential issues of concern

a. Level of knowledge about mechanism of action

NPWT:
NPWT is considered to result in increased wound healing as a result of the
mechanical stimulation of the tissue and removal of wound fluid.80 The
mechanical stimulation is thought to result in augmentation of perfusion and
granulation tissue proliferation. Moreover, the foil of the NPWT provides a
wound with a sealed-off environment where the wound can remain moist.

Diclofenac:
Diclofenac is a phenyl acetic acid that inhibits cyclooxygenase activity, which
can result in a reduction of the synthesis of prostaglandins and other
inflammatory mediators.

b. Previous exposure of human beings with the test product(s) and/or products
with a similar biological mechanism

NPWT:
There are no known risks of cutaneous NPWT, other than a possible allergic skin
reaction to polyurethane foam or the foil that will be used in this experiment.
Several studies have demonstrated cutaneous NPWT application to volunteers in
an experimental setting to be safe and did not report any adverse events
related to their study7,27,29-31,46, one of which performed in the Leiden
University Medical Center.7 This study will not convey any benefits to its
participants other than providing an interesting experience and the knowledge
they will contribute to increased scientific knowledge and ultimately maybe
even more effective and safer health care.

Diclofenac:

A Cochrane review (350 studies, around 35000 participants) from 2015,
investigating the peri-operative incidence of adverse events with single-dose
analgesics when compared with placebo, indicated that single-dose NSAIDs do not
seem to convey additional risks, suggesting that the risk of taking a
single-dose of an NSAID (such as Diclofenac) is comparable to normal daily
life.70 Moreover, a Cochrane review investigating the incidence of adverse
events specifically with single-dose Diclofenac, concluded that adverse event
rates were low in these single-dose studies, with no difference between
diclofenac and placebo (moderate quality evidence).1

Although these reviews concern populations that underwent surgery, the
observation that Diclofenac did not appear to convey risks to these patients
suggests a single-dose of oral Diclofenac is safe for volunteers as well.
Studies that investigated Diclofenac in a population of healthy volunteers have
not reported any noticeable adverse events during their experiments.75-79

c. Can the primary or secondary mechanism be induced in animals and/or in
ex-vivo human cell material?

In animals, yes.

d. Selectivity of the mechanism to target tissue in animals and/or human beings

NPWT:
NPWT is able to increase perfusion all over the body.

Diclofenac:
Diclofenac is thought to act all over the body. Please be referred to the
summary of product characteristics for details.

e. Analysis of potential effect

Participants will receive a single-dose of oral Diclofenac, and a single-dose
of topical Diclofenac. Because our study design incorporates a *wash-out*
period of >3 days, plasma concentrations will be lower than during regular
clinical treatment with Diclofenac.
It is considered highly unlikely that the administration of Diclofenac will
result in harmful effects in this study, as substantiated by the review of
Moore1, and the Summary of Product Characteristics document.

f. Pharmacokinetic considerations

Diclofenac:
Resorption: Fast and complete.
F: 50%
Tmax: 1-4 hours.
Metabolism: Mainly through hydroxylation, followed by binding with glucuronic
acid
Elimination: As metabolites, 60% in urine, rest in feces.
T* 1-2 hour

g. Study population

Healthy volunteers.

h. Interaction with other products

The interaction of Diclofenac with NPWT is the subject of this study. This
interaction is not considered harmful in healthy volunteers.

i. Predictability of effect

Not applicable.

j. Can effects be managed?

If any adverse events will be detected during the conduct of the study, NPWT
will be removed immediately.

In case of any unexpected emergencies, participants will be brought to the
medical support when deemed necessary, for example at the emergency department.

13.2 Synthesis

In order to reduce the risk of this study as much as possible, we have chosen
to select a study population of healthy volunteers. Moreover, candidates with
an elevated risk will be excluded according to our exclusion criteria.
Additionally, we do not dose the Diclofenac above the maximum dose according
the Summary of Product Characteristics document.

Diclofenac is available over-the-counter and is one of the most frequently
prescribed pharmaceuticals. This suggests that the information regarding safety
is relatively reliable.

If this study shows Diclofenac impairs blood flow increases during NPWT, this
is relevant information to healthcare providers caring for thousands of
patients receiving both NPWT and Diclofenac concomitantly. This could have
substantial clinical impact. Thus, this study is evidently warranted, supported
by a design aimed to limit any risk as much as possible.

The risks of this study are deemed very limited and thus acceptable for
subjects. Yet, this will be their decision because every participant will
receive appropriate information regarding the study during recruitment. Only
after informed consent has been obtained will any candidate be included into
this study.