Carbonic anhydrase inhibition for the management of cerebral ischemia: in vivo evaluation of sulfonamide and coumarin inhibitors

Article abstract of DOI:10.3109/14756366.2015.1113407

Ischemia of brain areas is a global health problem, causing death or long-term disability. Current pharmacological options have limited impact on ischemic damages. Recently, a relationship between hypoxia and carbonic anhydrase (CA) over-expression has be

Full text of DOI:10.3109/14756366.2015.1113407

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Carbonic anhydrase inhibition for the
management of cerebral ischemia: in vivo
evaluation of sulfonamide and coumarin inhibitors
Lorenzo Di Cesare Mannelli, Laura Micheli, Fabrizio Carta, Andrea Cozzi,
Carla Ghelardini & Claudiu T. Supuran
To cite this article: Lorenzo Di Cesare Mannelli, Laura Micheli, Fabrizio Carta, Andrea
Cozzi, Carla Ghelardini & Claudiu T. Supuran (2015): Carbonic anhydrase inhibition for the
management of cerebral ischemia: in vivo evaluation of sulfonamide and coumarin inhibitors,
Journal of Enzyme Inhibition and Medicinal Chemistry, DOI: 10.3109/14756366.2015.1113407
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2015 Taylor & Francis. DOI: 10.3109/14756366.2015.1113407
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RESEARCH ARTICLE
Carbonic anhydrase inhibition for the management of cerebral
ischemia: in vivo evaluation of sulfonamide and coumarin inhibitors
Lorenzo Di Cesare Mannelli1, Laura Micheli1, Fabrizio Carta2, Andrea Cozzi3, Carla Ghelardini1, and Claudiu T.
Supuran2,4
1Dipartimento di Neuroscienze, Psicologia, Area del Farmaco e Salute del Bambino - NEUROFARBA - Sezione di Farmacologia e Tossicologia,
2
Universita degli Studi di Firenze, Florence, Italy, Dipartimento Di Chimica, Laboratorio Di Chimica Bioinorganica, Universita Degli Studi Di Firenze,
`
`
3
Sesto Fiorentino, Florence, Italy, Dipartimento di Medicina Clinica e Sperimentale - DMSC - Universita degli Studi di Firenze, Florence, Italy, and
`
4
`
Dipartimento Neurofarba, Sezione Di Scienze Farmaceutiche, Polo Scientifico, Universita Degli Studi Di Firenze, Sesto Fiorentino, Florence, Italy
Abstract
Keywords
Ischemia of brain areas is a global health problem, causing death or long-term disability.
Current pharmacological options have limited impact on ischemic damages. Recently, a
relationship between hypoxia and carbonic anhydrase (CA) over-expression has been
highlighted suggesting CA inhibition as a possible target. This study aimed to evaluate the
pharmacological profile of sulfonamide and coumarin CA inhibitors in rats underwent
permanent middle cerebral artery occlusion (pMCAO). The neurological score of pMCAO rats
was dramatically reduced 24 h after occlusion. Repeated subcutaneous injections of the CA
inhibitors 4 and 7 (1 mg kgꢀ1) were able to increase the neurological score by 40%. Compound
7 showed the tendency to reduce the volume of hemisphere infarction. The standard CA
inhibitor acetazolamide was ineffective. The properties of novel CA inhibitors to improve
neurological functionalities after cerebral ischemic insult are shown. The CA involvement in
cerebral hypoxic phenomena deserves deeper investigations.
Acetazolamide, carbonic anhydrase,
coumarin, neurological score, pMCAO,
sulfonamide
History
Received 21 October 2015
Revised 24 October 2015
Accepted 24 October 2015
Published online 25 November 2015
Introduction
The evidence that hypoxic microenvironments elicit the
expression of specific isoforms of carbonic anhydrase (CA)
(in particular CAIX and CAXII) through the hypoxia inducible
factor5,6 allows to hypothesize a possible CA relevance in ischemia.
The aim of this study was the evaluation of the pharmaco-
logical profile of a new class of CA inhibitors in a rat model of
cerebral ischemia.
Cerebral ischemia occurs when blood flow to the brain is
insufficient to meet the metabolic demand. Occlusion of cerebral
arteries interrupts blood flow, limits oxygen and glucose supply to
the central nervous system (CNS) and causes infarction/ischemic
stroke1. Preclinical results individuate candidate targets for CNS
protection: glutamate release, glutamate receptor, excitotoxicity,
mitochondrial dysfunction, nitric oxide, inflammation, necrosis,
apoptosis and redox unbalance. In spite of advances in the
pathophysiology of cerebral ischemia, therapeutic options remain
very limited2.
Materials and methods
Chemistry
Anhydrous solvents and all reagents were purchased from Sigma-
Aldrich (Milan, Italy), Alfa Aesar (Milan, Italy) and TCI (Milan,
Italy). All reactions involving air- or moisture-sensitive compounds
were performed under a nitrogen atmosphere using dried glassware
and syringes techniques to transfer solutions. Nuclear magnetic
resonance (1H NMR, 13C NMR) spectra were recorded using a
Bruker Advance III 400 MHz spectrometer in DMSO-d6 or CDCl3.
Chemical shifts are reported in parts per million (ppm) and the
coupling constants (J) are expressed in Hertz (Hz). Splitting
patterns are designated as follows: s, singlet; d, doublet; sept,
septet; t, triplet; q, quadruplet; m, multiplet; brs, broad singlet; dd,
double of doublets. The assignment of exchangeable protons (OH
and NH) was confirmed by the addition of D2O. Analytical thin-
layer chromatography (TLC) was carried out on Merck silica gel F-
254 plates. Flash chromatography purifications were performed on
Merck Silica gel 60 (230–400 mesh ASTM) as the stationary phase
and ethyl acetate/n-hexane were used as eluents. Melting points
(mp) were measured in open capillary tubes with a Gallenkamp
Up to now, only one pharmacological treatment for acute
ischemic stroke was approved by FDA, the intravenous administra-
tion of recombinant tissue plasminogen activator (rt-PA)3. rt-PA
delivered within 3 h after the onset of symptoms, reduces neuro-
logical deficits and improves functional outcome in stroke patients.
Unfortunately, the large majority of patients with ischemic stroke are
not able to reach hospital within 3 h, as a result rt-PA can be
exploitable by a very limited number of patients and most of them do
not receive treatment4. Consequently, successful treatments of
ischemic stroke remain a major challenge in clinical practice.
Address for correspondence: Lorenzo Di Cesare Mannelli, Department of
–
Neuroscience, Psychology, Drug Research and Child Health
NEUROFARBA – Pharmacology and Toxicology Section, University of
Florence, 50139 Florence, Italy. Tel: +39-055-2758395. E-mail:
lorenzo.mannelli@unifi.it

2
L. Di Cesare Mannelli et al.
J Enzyme Inhib Med Chem, Early Online: 1–6
MPD350.BM3.5 apparatus and are uncorrected. All compounds
reported here were 495% HPLC pure.
Freshly prepared 1-azido-2-bromobenzene 3 (0.44 g, 1.1 eq)
and 7-(prop-2-ynyloxy)-2H-chromen-2-one 2 (0.4 g, 1.0 eq) were
dissolved in tert-ButOH/H2O 1/1 (2.0 ml) and then TMACl (0.4 g,
1.0 eq) and copper(0) nanosize (5% mol) were added. The
suspension was stirred at 60 ꢁC until starting materials were
consumed (TLC monitoring), then quenched with H2O (10 ml)
and the formed precipitate was filtered-off. The obtained residue
was purified by silica gel column chromatography eluting with
33% ethyl acetate in n-hexane to afford 4 as a white solid.
Synthesis of 7-(prop-2-ynyloxy)-2H-chromen-2-one 27
3'
OH
4
5
8
3
2
6
7
Ph P, DIAD
3
2'
1'
THF dry
Sonication
0°C--r.t.
HO
O
O
O
O
O
7-[(10-(2-Bromophenyl)-1H-10,20,30-triazol-40-yl]methoxy)-
2H-chromen-2-one 4
1
2
Hydroxy coumarin 1 (1.0 g, 1.0 eq), propargyl alcohol (1.0 eq)
and triphenylphoshine (1.0 eq) were dissolved in dry THF (90 ml).
Then the temperature was lowered to 0 ꢁC and diisopropylazadi-
carboxylate (DIAD; 1.1 eq) was added drop-wise under sonic-
ation. The orange solution was sonicated at r.t. under a nitrogen
atmosphere until starting material was consumed (TLC monitor-
ing). Solvents were removed under vacuo to give a white solid that
was recrystallized from MeOH to afford the titled compound as
white solid.
Fifty-percent yield; m.p. 133–134 ꢁC; silica gel TLC Rf 0.16
(Ethyl acetate/n-hexane 33% v/v); ꢀmax (KBr) cmꢀ1, 1770
(C ¼ O), 1560 (aromatic); dH (400 MHz, DMSO-d6) 5.41 (2H, s,
100-H2), 6.35 (1H, d, J 9.6, 3-H), 7.11 (1H, dd, J 8.8, 2.4, 6-H),
7.26 (1H, d, J 2.4, 8-H), 7.67 (4H, m, Ar-H), 7.96 (1H, dd, J 8.8,
2.4, 5-H), 8.04 (1H, d, J 9.6, 4-H), 8.78 (1H, s, 50-H); dc
(100 MHz, DMSO-d6) 162.0, 161.2, 156.2, 145.2, 142.9, 137.0,
134.5, 133.0, 130.5, 129.9, 129.7, 128.1, 119.8, 113.9, 113.7,
113.6, 102.6, 62.4.
7-(Prop-2-ynyloxy)-2H-chromen-2-one 2
65% yield; m.p. 118 ꢁC (Lit.6 120 ꢁC); silica gel TLC Rf 0.53
(Ethyl acetate/n-hexane 50% v/v); ꢀmax (KBr) cmꢀ1, 3310
(C ꢂ C–H), C2160 (C ꢂ CH), 1765 (C ¼ O), 1604 (Aromatic);
dH (400 MHz, DMSO-d6) 3.69 (1H, t, J 2.4, 30-H), 4.97 (2H, d, J
2.4, 10-H2), 6.36 (1H, d, J 9.6, 3-H), 7.03 (1H, dd, J 8.5, 2.3, 6-H),
7.09 (1H, d, J 2.3, 8-H), 7.69 (1H, d, J 8.5, 5-H), 8.03 (1H, d, J
9.6, 4-H); dC (100 MHz, DMSO-d6) 161.1 (C-2), 161.0 (C-7),
156.0 (C-8a), 145.1 (C-4), 130.4 (C-5), 113.9 (C-3), 113.8 (C-4a),
113.7 (C-6), 102.7 (C-8), 79.8 (C-20), 79.4 (C-30) and 57.0.
Experimental in agreement with reported data8.
CA inhibition
An Applied Photophysics stopped-flow instrument (Leatherhead,
UK) has been used for assaying the CA catalyzed CO2 hydration
activity11. Phenol red (at a concentration of 0.2 mM) has been
used as indicator, working at the absorbance maximum of 557 nm,
with 20 mM Hepes (pH 7.5) as buffer, and 20 mM Na2SO4 (for
maintaining constant the ionic strength), following the initial rates
of the CA-catalyzed CO2 hydration reaction for a period of 10–
100 s. The CO2 concentrations ranged from 1.7 to 17 mM for the
determination of the kinetic parameters and inhibition constants.
For each inhibitor at least six traces of the initial 5–10% of the
reaction have been used for determining the initial velocity. The
uncatalyzed rates were determined in the same manner and
subtracted from the total observed rates. Stock solutions of
inhibitor (0.1 mM) were prepared in distilled-deionized water and
dilutions up to 0.01 nM were done thereafter with the assay buffer.
Inhibitor and enzyme solutions were preincubated together for
15 min – 6 h at room temperature (15 min) or 4 ꢁC (6 h) prior to
assay, in order to allow for the formation of the E-I complex. Data
from Table 1 were obtained after 6 h incubation of enzyme and
inhibitor. The inhibition constants were obtained by non-linear
least-squares methods using PRISM 3 and the Cheng-Prusoff
equation, as reported earlier8,11, and represent the mean from at
least three different determinations. All CA isoforms were
Synthesis of 2-bromophenylazide 39
NH
N
2
3
Br
NaNO , NaN
Br
2
3
HClaq 2 M
3
The commercially available 2-bromo aniline (0.5 g, 1.0 eq) was
dissolved in a 2 -M aqueous hydrochloric acid solution (15 ml) at
0 ꢁC and then was treated with NaNO2 (1.2 eq) and NaN3 (1.5 eq)
dissolved in H2O (15 ml each). The reaction mixture was stirred at
r.t. for 15 min and then extracted with ethyl acetate (2ꢃ 15ml) and
the combined organic layers were dried over Na2SO4, filtered-off
and the solvent evaporated in vacuo to afford the titled compound
which was used without further purification.
recombinant ones obtained in-house as reported earlier12–16
.
Animals
2-Bromophenylazide 3
Male Sprague–Dawley rats (Harlan, Varese, Italy), weighing 200–
250 g at the beginning of the experimental procedure, were used for
all the experiments. Animals were housed in the Centro
Stabulazione Animali da Laboratorio, University of Florence and
used no earlier than 1 week after their arrival. Four rats were housed
per cage (size 2 6 ꢃ 41 cm); animals were fed with standard
laboratory diet and tap water ad libitum, and kept at 2 3 1 ꢁC with
89% yield; dH (400 MHz, CDCl3) 7.55 (app d, J ¼ 8.0 Hz, 1H),
7.35 (app t, J ¼ 7.7 Hz, 1H), 7.17 (app d, J ¼ 8.0 Hz, 1H), 7.01
(app t, J ¼ 7.7 Hz, 1H); dC NMR (100 MHz, CDCl3) 138.6, 133.8,
128.5, 125.9, 119.4, 113.8. Experimental in agreement with
reported data9.
Synthesis of 7-[(10-(2-bromophenyl)-1H-10,20,30-triazol-
40-yl]methoxy)-2H-chromen-2-one 410
N3
Br
4
5
8
3
2
6
1"
Cu(0) nanosized, TMACl
5'
7
4'
H2O/tBuOH 1/1
O
O
O
O
O
O
N
N
N
2'
3'
Br
2
3
4

DOI: 10.3109/14756366.2015.1113407
CA inhibition for the management of cerebral ischemia
3
Table 1. hCA I, II, VII, IX and XII inhibition data of the newly synthesized compounds 4–7 by a stopped flow CO2 hydrase assay11
.
SO2NH2
SO2NH2
O
N
S
NH2
O
O
O
S
O
BnO
O
N
4
7
N
N
N
ClO4
Br
5
6
KI (nM)*
hCA VII
No.
hCA I
hCA II
hCA IX
hCA XII
4**
5
6***
7***
AAZ
40600
4000
762
48.1
250
4100000
21
15.3
15
8.3
7.7
2.5
6800
14
17.9
0.89
25
27000
7
20.3
7.6
12
10.5
2.5
5.7
Acetazolamide (AAZ) inhibition data are included for comparison reasons.
*Mean from three different assays, errors in the range of 10% of the reported values.
**From Ref25
***From Ref26
.
.
a 12-h light/dark cycle, light at 7a.m. All animal manipulations were TWEEN 80 and per os or subcutaneously (s.c.) administered,
carried out according to the European Community guidelines for respectively. The AAZ injections were performed 30 min before,
animal care (DL 116/92), application of the European Communities 15 and 120 min after pMCAO. Compounds (1 mg kgꢀ1) were
Council Directive of 24 November 1986 (86/609/EEC). The ethical dissolved in 1% DMSO/1% PEG/0.2% TWEEN 80 and s.c.
policy of the University of Florence complies with the Guide for the administered at 5 and 20 min after the induction of cerebral
Care and Use of Laboratory Animals of the US National Institutes ischemia.
of Health (NIH Publication No. 85-23, revised 1996; University of
Florence assurance number: A5278-01). Formal approval to conduct Neurological test
the experiments described was obtained from the Animal Subjects
Neurological evaluation of motor sensory functions was carried
Review Board of the University of Florence. Experiments involving
out 24 h after pMCAO. The examiners were blind as to the
animals have been reported according to ARRIVE guidelines17. All
procedure that the rat had undergone. Evaluations were always
efforts were made to minimize animal suffering and to reduce the
performed between 10 and 11 a.m. to exclude behavioral changes
number of animals used.
based on circadian rhythm. The neurological examination
consisted of six tests19,20
(1) Spontaneous activity
:
Permanent middle cerebral artery occlusion (pMCAO) in rats
pMCAO was induced by using a previously described, relatively (2) Symmetry in the movement of four limbs
non-invasive technique18 in Sprague–Dawley rats. In these (3) Forepaw outstretching
experiments, the animals were randomly picked up from the (4) Climbing
cages and allocated to the vehicle or treated groups. The (5) Body proprioception
researchers were aware of the treatment allocation. Anesthesia (6) Response to vibrisse touch.
was induced with 2% isoflurane in air and maintained with the
The score assigned to each rat at completion of the evaluation
lowest active concentration of anesthetic. Body temperature was equals the sum of all six test scores. The final minimum score was
measured with a rectal probe and kept at 37 ꢁC with a heating pad. 3, and the maximum was 18.
The external and internal right carotid arteries were dissected
under an operating microscope, and a silk suture was tied loosely Evaluation of infarct volume
around the external carotid stump. A silicone-coated nylon
Twenty-four hour after injury, rats were killed and their brains
filament (diameter: 0.28 mm) was then inserted through the
were carefully removed. The brains were cut into five coronal
external into internal carotid artery up to the Willis circle to
slices of 2-mm thickness. Slices were incubated in 2% solution of
occlude the right middle cerebral artery. The silk suture was
2,3,5-triphenyltetrazolium chloride (TTC) at 37 ꢁC for 30 min and
finally tightened around the intraluminal filament to prevent
immersion fixed in 10% buffered formalin solution. TTC stains
bleeding. To maintain the animals at standard temperature
the viable brain tissue red, while infracted tissue remains
conditions, a heating pad was used for the entire duration of the
unstained21,22. For quantification of infracted area and volumes,
surgery. In this pMCAO severe model, the nylon filament was not
the brain slices were photographed using a digital camera and the
remove and the animals were killed 24 h after surgery.
Image analysis was performed on a personal computer with an
image analysis software program (using ImageJ for Windows)23.
Compound administration protocol
To compensate the effect of brain edema which distorts and
Acetazolamide (AAZ; 30–60 mg kgꢀ1) was suspended in 1% enlarges the infarcted tissue and surrounding white matter, infarct
carboxymethilcellulose or dissolved in 1% DMSO/1% PEG/0.2% areas were calculated subtracting the area of intact tissue in the

4
L. Di Cesare Mannelli et al.
J Enzyme Inhib Med Chem, Early Online: 1–6
ipsilateral hemisphere from the area of the contralateral hemi-
sphere. Infarct volumes were calculated multiplying the infarct
area by the distance among sections (2 mm) as described
previously24.
Statistical analyses
Statistically significant differences in the neurological scores
among vehicle-treated, CA inhibitors-treated and sham-operated
rats were evaluated by one-way ANOVA followed by the post hoc
Newman–Keuls multiple comparison test. Statistically significant
differences in brain damage were evaluated by unpaired Student’s
t-test. At least five rats were examined per group. A p value of
50.05 was considered significant.
Results
Three sulfonamide and one coumarin CA inhibitors (CAIs) were
included in the study (Table 1)25,26. Except the coumarin 4, which
is reported here for the first time (see ‘‘Materials and methods’’
section for details), the other compounds were reported earlier by
our groups26.
Figure 1. Evaluation of neurological score. Measurements were carried
out 24 h after pMCAO. The protocol consisted of six tests in order to
evaluate spontaneous activity, symmetry in the movement of four limbs,
forepaw outstretching, climbing, body proprioception and response to
vibrisse touch. The score assigned to each rat at completion of the
evaluation equals the sum of all six test scores. The final minimum score
was 3, and the maximum was 18. Each value represents the mean SEM
of five rats per group. **p50.01 versus sham + vehicle-treated rats;
^p50.05 versus pMCAO + vehicle-treated rats.
The main differences between coumarins and sulfonamide
CAIs is that the first class tends to show isoform selective
inhibition profile27–30, whereas the sulfonamides generally inhibit
indiscriminately most CA isoforms31,32
.
This is also observed for the data of Table 1: indeed the
coumarine 4 is a hCA VII-selective inhibitor (with an inhibition
constant of 15.3 nM against this brain-specific isoform), being an
ineffective inhibitor of isoforms hCA I, II (cytosolic) as well as IX
and XII (transmembrane CAs).
treated with 7 induced a tendency to infarct volume decrease
(Figure 2).
The positively charged sulfonamide 5 is a membrane-
impermeant, pyridinium derivative which does not cross plasma
Discussion and conclusions
membranes33. This compound is a poor hCA I inhibitor but To the best of our knowledge, this study is the first preclinical
effectively inhibits CA II, VII, IX and XII (Table 1), with KIs evidence of CAI efficacy in cerebral ischemia treatment. Four
ranging between 7 and 21 nM. The remaining two sulfonamides, 6 synthetic CAIs 4–7 were compared with AAZ about the property
and 7, are lipophilic, membrane-permeant derivatives. They are to ameliorate the neurological score and the brain infarct volume
effective inhibitors of all five CA isoforms investigated here after pMCAO. While AAZ administration was not active neither
(except compound 6 against hCA I), with inhibition constants to increase neurological score nor to reduce brain damage, 4 and 7
between 0.89 and 48.1 nM. As all these five isoforms are (both 1 mg kgꢀ1) were able to significantly improve motor and
presumably present in the brain, and two of them (CA IX and sensory functions 24 h after injury. Moreover, a tendency to
XII) may be over-expressed in hypoxic conditions, it may be decrease the infarct volume was highlighted after compound 7
rather difficult to assess the role of precise CA isoforms in treatment.
ischemic processes, and this was also the reason why compounds
CA reversibly converts carbon dioxide to H+ and bicarbonate
with such a variable inhibition profile were included in this study. and contributes to several biological processes, including pH
AAZ, the classical, standard sulfonamide CAI was also included regulation, anion transport and water balance. It plays a pivotal
in the study as a control compound and its inhibition profile are role both in physiological and pathological conditions also
also shown in Table 1.
concerning the CNS34. In astroglia (CA high expressing cells35),
The neurological score was evaluated in sham operated and in both intracellularly and membrane-bound (acting interstitially)
pMCAO rat groups 24 h after occlusion (Figure 1). The neuro- CA isoforms cooperatively couple intercellular CO2 shuttling to
logical score of pMCAO vehicle-treated rats was significantly the acid/base transporter activities36,37. Glial CA converts neuron-
reduced (9.5 1.3) in comparison to sham-operated rats derived CO2 to bicarbonate and protons which are driven out
(17.5 0.2). AAZ, administered per os (50 mg kgꢀ1) or subcuta- of the glial cell by a Na+/HCO3ꢀ cotransporter and monocarbox-
neously (30 mg kgꢀ1) 30 min before, 15 and 120 min after ylate transporters. In the extracellular space, CA catalyzes
occlusion did not improve the neurological deficit induced by the reaction of bicarbonate with a proton to recycle CO2,
cerebral stroke. Repeated subcutaneous injections, 5 and 20 min which is instrumental for buffering extracellular pH36. pH
after surgery, of the CAIs 4 and 7 (both 1 mg kgꢀ1) were able to dysregulation has been related to a spectrum of pathological
significantly increase the neurological score up to 40%. The other conditions38, in particular in vitro studies showed that hypoxia/
two compounds tested (5 and 6) did not reduce the neurological anoxia decreases pH in neurons and glial cells38 possibly by
damages induced by cerebral ischemia.
alterations in the Na+/H+ exchange activity39,40. Moreover, the
The cerebral infarction was examined on slices obtained lack of oxygen caused by hypoxia leads to a switch from aerobic
from cerebrum 24 h after pMCAO (Figure 2). pMCAO- to anaerobic glucose metabolism, characterized by increased
vehicle-treated rats showed a large infarct volume in comparison production of lactic acid and a lowered intracellular pH41. CAIs
to sham-operated rats (data not shown). The treatments with the intervene to reduce H+ concentration contributing to the pH
analyzed compounds were not able to significantly reduce homeostasis42,43
the volume of total hemisphere infarction 24 h after occlusion
in comparison to pMCAO + vehicle group. Nevertheless, animals fluids42 offers a further opportunity to modulate ischemia-induced
.
Moreover, the CA ability to regulate the volume of body

DOI: 10.3109/14756366.2015.1113407
CA inhibition for the management of cerebral ischemia
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treatment with sulfonamide/coumarin CAIs, our data cannot
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This research was funded by the Italian Ministry of Instruction, University
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Declaration of interest
The authors declare no conflict of interest.

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