Neuroprotective potential of adenosine A1 receptor partial agonists in experimental models of cerebral ischemia

Article abstract of DOI:10.1111/jnc.14660

Cerebral ischemia is the second most common cause of death and a major cause of disability worldwide. Available therapies are based only on anticoagulants or recombinant tissue plasminogen activator. Extracellular adenosine increases during ischemia and a

Full text of DOI:10.1111/jnc.14660

DR. ALBERTO MARTIRE (Orcid ID : 0000-0003-0538-4704)
Article type : Original Article
Neuroprotective potential of adenosine A1 receptor partial agonists in experimental
models of cerebral ischemia
Alberto Martire1*§, Catia Lambertucci2§, Rita Pepponi1, Antonella Ferrante1, Nicholas
Benati1, Michela Buccioni2, Diego Dal Ben2, Gabriella Marucci2, Karl-Norbert Klotz3,
Rosaria Volpini2, Patrizia Popoli1.
1National Center for Drug Research and Evaluation, Istituto Superiore di Sanità, Roma, Italy
2School of Pharmacy, Medicinal Chemistry Unit, University of Camerino, Camerino, Italy
3Institut für Pharmakologie und Toxikologie, Universität Würzburg, Würzburg, Germany
* Corresponding Author
Postal address: Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy
E-mail address: alberto.martire@iss.it
Telephone: +390649902050
§ First co-authors equally contributed to the work.
Running title: A1R partial agonists in cerebral ischemia.
Keywords: Cerebral ischemia; Hippocampus; Adenosine A1 receptor; Partial agonists;
Neuroblastoma cells.
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been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/jnc.14660
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Abbreviations: A1R, adenosine A1 receptor; AC, adenylyl cyclase; ACSF, artificial cerebro-
spinal fluid; ADAC, adenosine amine congener; AMPA, alpha-amino-3-hydroxy-5-
methylisoxazole-4-propionate; ATP adenosine 5’-triphosphate; BBB, blood brain barrier;
cAMP, cyclic AMP; CCPA, 2-chloro-N6-cyclopentyladenosine; CHO, chinese hamster
ovary; CNS, central nervous system; CPA, N6-cyclopentyl-adenosine; DMSO, dimethyl
sulfoxide; DPCPX; 8-cyclopentyl-1,3-dipropylxanthine; fEPSP, field excitatory postsynaptic
potential; HEMADO, 2-(1-Hexynyl)-N6-methyladenosine; i.p., intraperitoneally; min,
minute; NECA, 5'-N-Ethylcarboxamidoadenosine; NMDA, N-methyl-D-aspartic acid; OGD,
oxygen-glucose deprivation; PKA, protein kinase A; PPF, paired-pulse facilitation; PPR,
paired-pulse ratio; PPS, paired-pulse stimulation; R-PIA, (R)-phenyl-isopropyl-adenosine;
RRID, Research Resource Identifier; SEM, standard error of mean.
Abstract
Cerebral ischemia is the second most common cause of death and a major cause of disability
worldwide. Available therapies are based only on anticoagulants or recombinant tissue
plasminogen activator. Extracellular adenosine increases during ischemia and acts as a
neuroprotective endogenous agent mainly by activating adenosine A1 receptors (A1Rs) which
control calcium influx, glutamate release, membrane potential, and metabolism.
Accordingly, in many experimental paradigms it has been already demonstrated that the
stimulation of A1R with full agonists is able to reduce ischemia-related structural and
functional brain damage; unfortunately, cardiovascular side effects and desensitization of
A1R induced by these compounds have strongly limited their exploitation in stroke therapy so
far. Among the newly emerging compounds, A1R partial agonists could be almost free of side
effects and equally effective. Therefore, we decided to evaluate the neuroprotective potential
of two A1R partial agonists, namely 2’-dCCPA and 3’-dCCPA, in in vitro and ex vivo
experimental models of cerebral ischemia. Within the experimental paradigm of oxygen-
glucose deprivation (OGD) in vitro in human neuroblastoma (SH-SY5Y) cells both A1R
partial agonists increased cell viability. Considering the high level of expression of A1Rs in
the hippocampus and the susceptibility of CA1 region to hypoxia, we performed
electrophysiological experiments in this subfield. The application of 7 min of OGD
constantly produces an irreversible synaptic failure in all the C57Bl/6 mice hippocampal
slices evaluated; both tested compounds allowed a significant recovery of synaptic
transmission. These findings demonstrate that A1R and its partial agonists are still of interest
for cerebral ischemia therapy.
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INTRODUCTION
In the last report of World Health Organization on noncommunicable diseases (WHO
GLOBAL STATUS REPORT, 2014) some impressive numbers clearly show that ischemic
brain injury is a widespread pathological condition with deleterious consequences both on
quality of life for individual patients and on related costs of health care and social services.
Cardiovascular disease was the leading cause of noncommunicable diseases deaths in 2012
and was responsible for 17.5 million deaths. Of these deaths, 6.7 million were due to strokes
and ischemic stroke represents approximately 80% of all cases. The majority of cerebral
strokes are ischemic in nature and result from the occlusion of a major cerebral artery by a
thrombus or an embolism, which leads to loss of blood flow, subsequent OGD and, finally,
tissue death in the affected region (Gibson, 2013).
Cerebral ischemia, leading to serious CNS damage (Honig and Rosenberg, 2000), is the
second most common cause of death and a major cause of disability worldwide (Macrez et
al., 2011).
Unfortunately, very few therapeutic options are available to date: 1) acute treatment with
recombinant tissue plasminogen activator in the restricted therapeutic time-window (0-6
hours) after the ischemic attack (Beresford et al., 2003; Chen et al., 2014); 2) aspirin and
anticoagulants as preventive therapy (Young et al., 2007; Ginsberg, 2009; Moskowitz et al.,
2010; Albers et al. 2011; Macrez et al. 2011).
Such a limited panel of therapeutic options demonstrates the difficulties in the translation
from basic research to clinical application in this field: during the last years more than 1000
molecules with brain-protective effects have been identified, targeting and correcting
different pathophysiological manifestations of brain ischemia. Approximately 250 molecules
deserved a dedicated clinical trial, but, unfortunately, none of them has so far successfully
completed phase III. Despite such a discouraging score, looking for, and testing new
therapeutic agents for cerebral ischemia remains an absolute priority for global health.
Hippocampus is a brain area particularly vulnerable to ischemia (Yue et al., 1997).
Hypoxic/ischemic damage, induced by the reduction of the blood flow, causes generally
necrosis, but in most cases there is also a process of slower apoptotic neuronal death in the
region (penumbra) surrounding the core of the damaged area (Honig and Rosenberg, 2000;
Yuan and Yankner, 2000; Schaller et al., 2003; Lo, 2008).
A rapid consequence of OGD in neuronal tissues is a failure in the production of ATP and in
the support of the energetic state of neurons, which allows to maintain membrane ionic
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gradients; as a consequence, there is a loss of membrane potential and an uncontrolled
depolarization of nervous connections. Voltage-dependent calcium channels are activated and
excitatory amino acids are released into the extracellular space. A further cytotoxic overload
of calcium is probably responsible for the beginning of other nuclear and cytoplasmic
deleterious pathways, including apoptotic events. The glutamate released in excess binds to
ionotropic NMDA and AMPA receptors, inducing a further increase in intracellular calcium.
Then, the condition known as “excitotoxicity” (Dong et al., 2009) is the first that takes place
after the ischemic attack in the brain and plays a central role in its pathogenesis (Choi and
Rothman, 1990).
In addition to the increase of glutamate, hypoxic/ischemic conditions trigger also a robust and
sustained enhancement of the extracellular levels of adenosine in the brain parenchyma, but
also in in vitro brain preparations or in cultured neurons (van Wylen et al., 1986; Fredholm et
al., 1994). Adenosine is an endogenous neuromodulator involved in the regulation of many
functions within the CNS (Phillis and Wu, 1981) and whose effects are mediated by at least
four distinct receptors: A1, A2A, A2B, and A3 (Fredholm et al., 2001). Of the four adenosine
receptors, the A1R is the most abundant and widespread in the brain (Fastbom et al., 1987).
The increase in extracellular adenosine early after ischemia is considered an endogenous
neuroprotective response aimed at stimulating A1Rs. Indeed, this receptor is crucial to control
several events that have been associated with ischemic damage: first of all, the glutamate
release, but also the calcium influx, the alteration of the membrane potential and of
metabolism (reviewed in Rudolphi et al., 1992; de Mendonça et al., 2000). In contrast, the
blockade of the A1R generally tends to exacerbate ischemic brain damage (reviewed in de
Mendonça et al., 2000). A1R stimulation prevents excitotoxicity both at the presynaptic
(inhibition of glutamate release) and at the postsynaptic (inhibition of AMPA/NMDA
currents) level. Furthermore, A1R stimulation is also able to decrease neuronal excitability by
activating G-protein-coupled inwardly rectifying potassium channels (Sickmann and
Alzheimer, 2003; Kim and Johnston, 2015), which, in turn, mediate hyperpolarization of the
membrane. Since A1R activation has clear protective effects in brain ischemia, A1R agonists
have been historically considered very promising drugs for this condition.
Selective A1R agonists, like CPA in acute administration, protect neurons against the damage
induced both by focal and by global ischemia (Rudolphi et al., 1992; von Lubitz et al., 1995).
Unfortunately, after chronic administration full A1R agonists lead to desensitization of A1Rs.
Furthermore, the activation of A1Rs generally induces relevant cardiovascular side effects
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including bradycardia and hypotension (Park etꢀal., 2012). Finally, puzzling results about the
ability of some full A1R agonists to cross the BBB have been obtained (Gervitz et al., 2002).
All these undesirable effects strongly mitigated their clinical implementation (White et al.,
1996; Fredholm et al., 2005).
However, it appears reasonable to explore the efficacy of A1R partial agonists, low efficacy
ligands which elicit only a submaximal response of the receptor in contrast to a full agonist:
they should have less pronounced cardiovascular effects and seem to act more selectively
(Mathot et al., 1996; IJzerman et al., 1996); they should induce less receptor
downregulation/desensitization and, finally, they should be equally effective in acute as well
as in chronic administration (Albrecht-Kupper et al., 2012). Given the higher level of
expression of A1Rs in the brain (particularly in the hippocampus and prefrontal cortex)
compared to peripheral organs like the heart (Fredholm et al., 2001; Fredholm et al., 2011),
partial agonists should elicit a maximal response only in tissues in which A1Rs are largely
expressed, where a submaximal receptor occupancy leads to a maximal functional effect
(Srinivas et al., 1997; van Schaick et al., 1998). These drugs have thus the potential to induce
limited cardiovascular side effects and to cause less pronounced receptor desensitization than
full agonists after chronic exposures (Parsons and Stiles, 1987; Longabaugh et al., 1989). The
efficacy of a A1R partial agonist in angina pectoris treatment has been proven in a clinical
trial with capadenoson (Tendera et al., 2007), a non-nucleoside molecule that mediates
cardioprotection in the absence of adverse hemodynamic effects (Albrecht-Kupper et al.,
2012; Sabbah et al., 2013). Therefore, the aim of the present study was to characterize, for the
first time, the possible neuroprotective potential of two A1R partial agonists, namely 2’-
dCCPA and the newly developed 3’-dCCPA, in in vitro and ex vivo experimental models of
ischemia.
MATERIALS AND METHODS
Animals
All animal procedures were carried out according to the principles and procedures outlined in
the European Community Guidelines for Animal Care, DL 26/2014, application of the
European Communities Council Directive, 2010/63/EU, FELASA and ARRIVE guidelines.
All animal procedures were approved by the Italian Ministry of Health and by the local
Institutional Animal Care and Use Committee (IACUC) at Istituto Superiore di Sanità
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(Rome, Italy; protocol number n. 7496) and at University of Camerino (Camerino, Italy;
protocol in agreement with the Article 3, paragraph 1, of the DL 26/2014). All functional
assays experiments were conducted on male BALB/c mice (CAT# JAX:000651,
RRID:IMSR_JAX:000651; 25.5
0.5 g weight, 8 weeks old) bred at the University of
Camerino. Electrophysiology and behavioral experiments were performed on male C57Bl/6
mice (CAT# JAX:000664, RRID:IMSR_JAX:000664; 25.5 2 g weight, 8 to 16 weeks-old)
purchased from Charles River (Calco, Italy). No sample calculation was performed to
estimate the number of mice used in each experimental group. No randomization was
performed to allocate subjects in the study. Anyway, we allocated arbitrarily the animals to
the different experimental groups on which to perform the euthanasia and the withdrawn of
organs (ileum, heart, brain) for ex vivo evaluations, or the i.p. injection of A1 agonists and the
following behavioral test. Additionally, no blinding was performed. The animals were kept in
standard cages (48 x 26 x 20 cm, 4 mice per cage) under standardized temperature (22°C),
humidity (55%) and lighting conditions (12:12 h light:dark cycle, with lights on at 6 am and
euthanasia/withdrawn of organs and in vivo experiments conducted as soon as possible after
the start of the light phase; usually, behavioral experiments were conducted between 7:30 am
and 10:00 am) with free access to water and food. For animals used in the present study,
proper treatment, care, and humane conditions have been provided. All efforts were made to
reduce the number of animals used and to minimize their pain and discomfort. Permanent
veterinary surveillance and animal welfare evaluation have been provided by the host
Institutions. As for behavioral experiments, we did not apply any inclusion or exclusion
criteria, and no animals died during experiments. A time-line diagram of animal experiments
is provided in Figure 1.
Drugs
The A1R partial agonists 2’-dCCPA and 3’-dCCPA are indicated below as compounds 4 and
7. CPA (A1R agonist, Cat# 1702), CCPA (A1R agonist, Cat# 1705), DPCPX (A1R antagonist,
Cat# 0439), forskolin (adenylyl cyclase, AC, activator, Cat# 1099) were purchased from
Tocris Biosciences (Bristol, UK). All pharmacological agents (CPA, DPCPX, compounds 4
and 7) were dissolved in DMSO or in distilled water to obtain a stock solution of 5 mM (CPA
and DPCPX) and 50 mM (4 and 7). Stock solutions were made to obtain concentrations of
DMSO lower than 0.001% in the superfusing ACSF and in cell cultures. This DMSO
concentration did not affect cell viability of SH-SY5Y or basal synaptic transmission in
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hippocampal slices. For behavioral experiments, compound 7, CPA, and CCPA were
dissolved in DMSO and then diluted in saline (0.9% NaCl), to obtain a solution for i.p.
injection with a 5% maximal concentration of DMSO. This DMSO concentration induced a
slight and non-significant reduction of mouse locomotor activity. The synthesis of compound
4 has been previously reported, hence, it will not be described here (Fig. 2, scheme 1, Vittori
et al., 2000). The custom-made compounds used in this study will be shared upon reasonable
request.
Synthesis of compound 7
General chemistry. Melting points were determined with a Büchi apparatus and are
1
uncorrected. H NMR spectra were obtained with Varian Mercury 400 MHz spectrometer; δ
in ppm, J in Hz. All exchangeable protons were confirmed by addition of D2O. Mass spectra
were recorded on an HP 1100-MSD series instrument. All measurements were performed
using electrospray ionization (ESI-MS) on a single quadrupole analyzer. Thin Layer
Chromatography (TLC) was carried out on precoated TLC plates with silica gel 60 F-254
(Fluka). For column chromatography, silica gel 60 (Merck) were used. Elemental analyses
were determined on Fisons Instruments Model EA 1108 CHNS-O model analyzer and are
within 0.4% of theoretical values. The purity of the compounds was ≥ 95% according to
elemental analysis data.
(2S,4R,5R)-4-acetoxy-5-(2,6-dichloro-9H-purine-9-yl)tetrahydrofuran-2-yl)methyl benzoate
(6):
A solution of 1,2-di-O-acetyl-5-O-benzoyl-3-deoxy-ribofuranose (Rizzo et al., 1992) (5; 0.28
g; 0.96 mmol) in 5 mL of anhydrous CH3CN was added of desiccated 2,6-dichloropurine (1;
0.20 g; 1.03 mmol) and cold in an ice-bath. When the mixture is at 0 °C, SnCl4 (1.1 mL) is
added and the reaction is left at r.t. under stirring for 16 h. After this time, cold saturated
solution NaHCO3 is added to neutralize the reaction pH and the crude extracted with CH2Cl2
(3x 100 mL). Organic extracts were collected dried over Na2SO4, filtered and concentrated to
dryness under vacuum. The residue was chromatographed on silica gel gravity column,
1
eluting with CHCl3 (100%), to obtain 6 as pure vitreous solid with 84% yield. H NMR
(DMSO-d6) δ 2.10 (s, 3H, COCH3), 2.32 (dd, 1H, J = 6.3 and 13.5 Hz, H-3’a), 2.70 (m, 1H,
H-3’b), 4.45 (m, 1H, H-CH-5’), 4.61 (m, 1H, H-CH-5’), 4.70 (m, 1H, H-4’), 5.76 (d, 1H, J =
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5.7 Hz, H-2’), 6.25 (d, 1H, J = 1.2 Hz, H-1’), 7.45 (t, 2H, J = 7.6 Hz, H-Ph), 7.63 (t, 1H, J =
7.5 Hz, H-Ph), 7.81 (m, 2H, H-Ph), 8.84 (s, 1H, H-8).
(2R,3R,5S)-2-(2-chloro-6-(cyclopentylamino)-9H-purin-9-yl)-5-(hydroxymethyl)tetra-
hydrofuran-3-ol (7):
To 0.25 grams (0.55 mmol) of 6, cyclopentylamine (5 ml) was added and the mixture let to
react at r.t. for 1 h. Then the residual amine was removed under vacuum and, to the crude
mixture, methanolic ammonia (25 mL) was added. The reaction was left at 75°C for 20 h.
Volatiles were removed under vacuum and the residue was chromatographed on silica gel
flash column, eluting with CHCl3-cC6H12-MeOH (70:27.5:2.5), to obtain 7 as a white solid
after crystallization from CH3OH-Et2O with 93% yield. M.p.: 161-164 °C. 1H NMR (DMSO-
d6): δ 1.56 (m, 4H, H-cyclohexyl), 1.72 (m, 1H, H-cyclohexyl), 1.91 (m, 3H, H-3’ and H-
cyclohexyl), 3.19 (m, 1H, H-3’), 3.54 (m, 1H, H-CH-5’), 3.68 (m, 1H, H-CH-5’), 4.36 (m,
1H, H-4’), 4.40 (m, 1H, CH-NH), 4.52 (m, 1H, H-2’), 5.02 (m, 1H, OH), 5.69 (d, 1H, J = 5.0
13
Hz, OH), 5.82 (bs, 1H, H-1’), 8.30 (1H, m, NH), 8.40 (s, 1H, H-8); C NMR (DMSO-d6): δ
23.9; 34.3; 52.1; 62.8; 75.3; 81.5; 91.1; 118.5; 136.5; 139.5; 153.6; 155.1.
Binding assay of compounds 4 and 7
Dissociation constants of unlabeled compounds (Ki values) were determined in competition
experiments in 96-well microplates as described earlier (Klotz et al., 1998).
Membrane preparation
Membranes for radioligand binding were prepared from CHO cell line stably transfected with
the four human adenosine receptor subtypes. The cell line used was provided by Dr. Klotz
from Wurzburg University (among the authors; details available in the reference Klotz et al.,
1998) and was not further authenticated. Anyway, the original CHO cell line is not listed as a
commonly misidentified cell line by the International Cell Line Authentication Committee
(ICLAC; http://iclac.org/databases/cross-contaminations/). Crude membranes were prepared
by collecting cells in ice-cold hypotonic buffer (5 mM Tris/HCl, 2 mM EDTA, pH 7.4). The
cell suspension was homogenized on ice (Ultra-Turrax, 2 x 20 sec at full speed) and the
homogenate was spun for 10 min (4 °C) at 1,000 x g in order to remove cell fragments and
nuclei. The supernatant was then centrifuged for 50 min at 100,000 x g at 4°C. The
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membrane pellet was resuspended in the specific binding buffer used for the binding
experiments, frozen in liquid nitrogen and stored at -80 °C (ref Klotz et al., 1998). The
preparation of a membrane fraction for the measurement of AC activity followed a simplified
protocol with only one high-speed centrifugation of the homogenate. The resulting crude
membrane pellet was resuspended in 50 mM Tris/HCl, pH 7.4 and immediately used for the
cyclase assay without freezing.
Radioligand binding
For competition binding the following radioligands and binding buffers were used: 1 nM
[3H]CCPA for A1Rs (50 mM TRIS/HCl, pH 7.4), 10 nM [3H]NECA for A2ARs (50 mM Tris,
pH 7.4, 10 mM MgCl2), 1 nM [3H]HEMADO for A3Rs (50 mM TRIS/HCl, pH 8.25, 10 mM
MgCl2, 1 mM EDTA) (Klotz et al., 2007). Binding assay was not performed at A2B receptors
since in our hands the available radioligands to target these receptors did not gave
reproducible results. Nonspecific binding of [3H]CCPA was determined in the presence of 1
mM theophylline, 100 µM R-PIA was used in the case of [3H]NECA and [3H]HEMADO
binding. Dissociation constants (Ki-values) were calculated from competition experiments
utilizing the program SCTFIT (De Lean et al., 1982) and are given as geometric means with
95% confidence intervals (n ≥ 3).
Functional assays (in vitro and ex vivo) of compounds 4 and 7
Adenylyl Cyclase Activity.
The functional activity of new derivatives was determined in AC activity evaluation
experiments. Therefore, receptor-mediated stimulation of AC activity was measured to
determine agonist potency given in EC50-values. The stimulation of AC via hA2BRs was
measured as described earlier (Klotz et al., 1998).
Functional interaction with A1Rs was studied in receptor-mediated inhibition of cyclase
activity. AC was inhibited with the receptor agonists after stimulation with 10 µM forskolin.
Inhibition with selected compounds was compared to CCPA-induced cyclase inhibition as a
full agonist. For details see Klotz et al., 1985, 1998.
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Mouse Ileum Assay
Animals were sacrificed by cervical dislocation; the ileum was isolated, cleaned of the
connective tissue and dipped into Krebs solution of the following mM composition: NaCl
119, KCl 4.5, MgSO4 2.5, NaHCO3 25, KH2PO4 1.2, CaCl2 2.5, glucose 11.1. The 20 mm
ileum segments were suspended in organ baths containing 10 ml of Krebs solution kept at 37
°C and aerated with 5% CO2 : 95% O2. The proximal end was connected with a silk thread to
an isometric force transducer (Cat# 7003/4/5/10, Ugo Basile, Biological Research Apparatus,
VA, Italy). Mechanical activity was digitized by an A/D converter, visualized, recorded, and
analyzed on a personal computer using the PowerLab/400 system.
Longitudinal preparations were subjected to an initial tension of 200 mg and were allowed to
equilibrate for at least 30 min. Rhythmic spontaneous contractions of varying amplitude
developed in all preparations. After the equilibration time, preparations were challenged with
the known agonist CCPA, which triggers a gradual spontaneous contraction reduction in a
concentration-dependent manner. Concentration–response curve for CCPA was constructed
by cumulative addition of the drug. In order to validate the assay, the well-known A1R
antagonist DPCPX in the presence of CCPA was studied and the shift on the right of CCPA
concentration-response curve confirmed the involvement of A1R in mouse ileum rhythmic
spontaneous contractions. Time control experiments showed that a second curve of the
agonist was reproducible. A1R ligands under study were evaluated as for CCPA and the curve
registered. The agonist potency was expressed as ED50 as previously reported (Lambertucci
et al., 2011).
Mouse heart rate
Animals were sacrificed by cervical dislocation, the heart was rapidly removed and mounted
in Krebs solution at 30°C and aerated with 5% CO2 : 95% O2. Chronotropic activity was
recorded isometrically. Tissues were equilibrated for at least 30 min and a cumulative
concentration-response curve to CCPA was constructed. The concentration of agonist in the
organ bath was increased approximately 3-fold at each step, with each addition being made
only after the response to the previous addition had attained a maximal level and remained
steady. Following 30 min of washing, tissues were incubated with the antagonist DPCPX (30
nM) for 60 min, and a new concentration-response curve to the agonist was obtained. When
studying the activation of the receptor, after 30 min washing, a cumulative concentration-
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response curve of the agonist under study was constructed. Responses were expressed as a
percentage of the maximal response obtained in the control curve. Contractions were
recorded by means of a force displacement transducer connected to the MacLab system
PowerLab/800. In addition, parallel experiments in which tissues did not receive any
antagonist were run in order to check any variation in sensitivity. In all cases, parallel
experiments in which tissues received only the reference agonist were run in order to check
any variation in sensitivity. The agonist potency was expressed as ED50 and represents the
dose which triggers 50% of the maximum effect.
Cell cultures
SH-SY5Y human neuroblastoma cells (RRID: CVCL_0019; Sigma-Aldrich from The
European Collection of Authenticated Cell Cultures, ECACC, Public Health England) are not
listed as a commonly misidentified cell line by the International Cell Line Authentication
Committee (ICLAC; http://iclac.org/databases/cross-contaminations/) and were not further
authenticated. Cells were cultured in Dulbecco modified Eagle’s medium (DMEM)
supplemented with 10% fetal bovine serum, penicillin (100 IU/ml), streptomycin (100 μg/ml)
and L-glutamine (2 mM). The culture was maintained at 37°C in 95% air- 5% CO2 in a
humidified incubator. Experiments were performed next day after seeding with 80% of
confluence.
In vitro OGD
The in vitro model of ischemia was achieved maintaining the cells under OGD for 4h
followed by 24 h of recovery. Briefly, SH-SY5Y neuroblastoma cells were washed once with
pre-warmed (37 °C) OGD medium (in mM, 154 NaCl, 5.4 KCl, 0.8 MgSO4, 1 NaHPO4, 1.8
CaCl2, 26.2 NaHCO3) that has been bubbled with an anaerobic gas mixture (95% N2, 5%
CO2) for 30 min to remove residual oxygen. Cells were incubated with this medium at 37 °C
for 4 h in an anoxia chamber [Modular incubator chamber (Cat# MIC-101) Billups-
Rothenberg Inc] to produce lethal oxygen deprivation. Then, the OGD medium was replaced
by an oxygenated minimum essential medium with Earle salts (L-glutamine-free)
supplemented with 11 mM glucose and cells were further incubated in a humidified aerobic
incubator at 37 °C for 24 h of recovery. Control cells were incubated with OGD medium with
glucose (11 mM) for 4 h at normoxia.
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In vitro treatment
A1R full (CPA) and partial (4 and 7) agonists were added to the cultures in a similar manner,
1 h prior, during OGD exposure and also during the subsequent 24 h (recovery period). The
treatment with the selective A1R antagonist DPCPX (100 nM) or the AC activator forskolin
(1 µM) was performed by adding the drug 30 min before full or partial agonists and keeping
it in the culture medium until the cell survival evaluation.
Cell viability measurement
Trypan blue staining was carried out to assess the extent of cell death occurring after 24 h of
recovery from OGD exposition. Cells were trypsinized and incubated with 0.1% trypan blue.
A hemocytometer was used to count stained and unstained cells.
Electrophysiological experiments
Slice preparation and recordings
Hippocampal slices were prepared as follows. After sacrifice of animals by cervical
dislocation, the hippocampus was removed and 450-μm slices cut with a McIlwain tissue
slicer (Cat # TC752, The Mickle Lab). Slices were maintained at room temperature (22–
24°C) in ACSF containing (in mM): 126 NaCl, 3.5 KCl, 1.2 NaH2PO4, 1.2 MgCl2, 2 CaCl2,
25 NaHCO3, 11 glucose (pH 7.3) saturated with 95% O2 and 5% CO2. After incubation in
ACSF for at least 1 h, a single slice was transferred to a submerged recording chamber and
continuously superfused at 32–33°C with ACSF at a rate of 2.6 ml/min. The drugs were
added to this superfusion medium. The perfusion apparatus was made of chemically inert
materials (i.e., silicone tubing). Extracellular fEPSPs were recorded in stratum radiatum of
the hippocampus with a glass microelectrode filled with 2 M NaCl solution (pipette
resistance 2–5 MΩ) upon stimulation of Schaffer collaterals with an insulated bipolar twisted
NiCr electrode (50 μm OD). Each pulse was delivered every 20s (square pulses of 100 μs
duration at a frequency of 0.05 Hz), and three consecutive responses were averaged. The
stimulation intensity used in the fEPSP recordings was always adjusted to obtain a
submaximal fEPSP slope (~60% of maximum) with minimum population spike
contamination. Signals were acquired with a DAM-80 AC differential amplifier (Cat# 3484,
WPI) and analyzed with the LTP program (Anderson and Collingridge, 2001). At least
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10 min of stable baseline recording preceded drug application. Data were expressed as
mean SEM of n experiments (one slice tested per experiment. Slices were obtained from at
least two animals for each set of the experiment). To allow for comparisons between different
experiments, slope values were normalized, taking the average of the baseline values to be
100%. The drug effect was expressed as the mean percentage variation of the slope from
baseline over the last 5 min of drug perfusion. The washout period lasted at least 30 min.
Paired-pulse stimulation
In PPS protocol, two consecutive pulses are applied 50-msec apart. In control condition, this
protocol elicits a PPF, in which the response elicited by the second stimulus (R2) is greater
than that elicited by the first stimulus (R1). The degree of PPF is quantified by the R2/R1
ratio and a modification of this ratio is an indication of a presynaptic action on
neurotransmitter release (Schulz et al. 1994).
OGD in hippocampal slices
In hippocampal slices, OGD was obtained by superfusing the slice with ACSF with sucrose
instead of glucose and gassed with nitrogen (95% N2 – 5% CO2). After 7 min of OGD, the
slice was again superfused with normal, glucose-containing, oxygenated ACSF (Pugliese et
al., 2003). The effects of drugs on OGD were expressed as the mean percentage variation of
the slope from baseline over the last 5 min of washout.
In vivo evaluation of compound 7
In behavioral experiments we decided to evaluate how much compound 7, the most
promising of the two A1 partial agonists tested, affect mouse locomotor activity in vivo, in
comparison to the well-known A1 full agonists CPA and CCPA. To this end we performed
the open field test on C57Bl/6 mice (25.5 2 g weight, 16 weeks-old) injected i.p. with
different doses (0.1, 1, and 10 mg/kg) of compound 7. The effects induced by compound 7
were compared to the decrease of locomotor activity induced by CPA and CCPA (0.1 and 1
mg/kg). We failed to compare the effects at 10 mg/kg because, given their high potency, the
full agonists gave already the maximal response at 1 mg/kg.
After acclimation to the behavioral testing room for at least 30 min, mice were administered
with vehicle, CCPA, CPA or compound 7 and, after 5 minutes from injection of drugs, placed
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into the center of a Plexiglas chamber (43x43x20cm) for 30 minutes. During the first 5
minutes after the administration of compound 7 mice were awake and maintained a normal
posture while CPA and CCPA seemed to affect mice wakefulness and posture soon after their
administration (personal observation). Mouse movement was monitored with an overhead
camera and total distance traveled (measure of locomotion) was measured by using the Any-
maze video tracking system (Cat# 60000, UGO BASILE).
Statistics
Results from in vitro experiments were expressed as a percentage of control, which was
considered as 100%, and as mean SEM values of at least 3 independently performed
experiments (each independent experiment corresponds to an independent cell culture
preparation). In vitro data were first subjected to normality test (D’Agostino and Pearson
Omnibus Normality test); since we found that parameters did not regularly follow a normal
distribution, we applied Mann-Whitney test. Results from electrophysiological experiments
were expressed as mean SEM from n slices. In the Legends the number of animals from
which the slices have been obtained are reported for each data set. Results from behavioral
experiments were presented as mean SEM from n mice. A p-value <0.05 was considered to
indicate a significant difference. Statistical analysis of the data was performed using Mann-
Whitney or Wilcoxon tests for electrophysiology experiments and for behavioral
experiments. Statistical analyses and curve fittings were obtained by using GraphPad Prism
software (version 6.05, GraphPad Software, San Diego, CA, US). Test for outliers (i.e.
Grubbs test) were not applied.
Results
Synthesis of deoxynucleosides 4 and 7
The 2’- and 3’-deoxy derivatives were synthesized using a convergent approach: the suitable
sugars were synthesized and then they were reacted with the appropriate purine base. The
obtained products were further modified to obtain the final desired nucleosides (Fig. 2,
schemes 1 and 2).
The synthesis of 2-chloro-2’-deoxy-N6-cyclopentyladenosine (4) has been previously
reported, hence, it will not be described in detail here (Fig. 2, scheme 1, Vittori et al., 2000).
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The synthesis has been carried out by reacting 2,6-dichloropurine (1) with 1-chloro-2-deoxy-
3,6-ditolylribose (2) in anhydrous conditions in the presence of sodium hydride to get the
protected nucleoside 3 (Fig. 2, scheme 1). This intermediate was then reacted with
cyclopentylamine and then with ammonia saturated in methanol to remove the protecting
groups at the sugar moiety. The suitable nucleoside 4 (2’-dCCPA) has been obtained in good
yield (Fig. 2, scheme 1). The 2-chloro-3’-deoxy-N6-cyclopentyladenosine (7) was prepared
by reaction of the protected 3-deoxy-ribofuranose 5, prepared by the yet reported procedure,
(Rizzo et al., 1992) with the commercially available 2,6-dichloropurine (1, Fig. 2, scheme 2).
The glycosylation reaction was performed using tin tetrachloride in anhydrous acetonitrile.
The protected 2,6-dichloropurine glycoside 6 is obtained in 84% yield after silica gel
chromatography. The latter compound 6 was first reacted with cyclopentylamine and then
with methanolic ammonia to completely remove protecting groups at the sugar moiety of the
obtained nucleoside. The desired 3’-deoxynucleoside 7 (3’-dCCPA) was obtained in 93%
yield after silica gel chromatography and crystallization (Fig. 2, scheme 2).
Binding assay of compounds 4 and 7
The deoxy nucleosides 4 and 7 were evaluated at the human recombinant ARs, stably
transfected into CHO cells, using radioligand binding studies (A1R, A2AR, A3R, Table 1 in
Supporting information) or AC activity assay (A2BR) to assess their affinity for A1R and their
binding selectivity for the other adenosine receptor subtypes. Receptor binding affinity was
determined using [3H]CCPA as a radioligand for A1R, whereas [3H]NECA was used for
A2AR and [3H]HEMADO for A3R subtype (Table 1 in Supporting information; Klotz et al.,
1998; Klotz et al., 2007). In the case of A2BR, potential stimulation or inhibition (EC50 and
IC50 values) was determined by measurement of AC activity or of inhibition of NECA-
stimulated AC activity, respectively. Both deoxy nucleosides showed no measurable
interaction with the A2B subtype (EC50 and IC50 >30,000 nM), hence, these data are not
reported in the table 1. The full A1R agonist CCPA was reported as a reference compound
(Cristalli et al., 1998).
The reference A1R agonist CCPA is endowed with high affinity for A1Rs, with Ki = 0.83 nM,
and high selectivity versus the A2AR subtype of about 2,770 fold, while it presents a moderate
selectivity versus the A3Rs (51 fold). Removal of the 2’ or 3’-hydroxyl group of the sugar
moiety reduced the affinity for the A1Rs. In particular, the 2’-hydroxyl group seemed to be
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essential for the receptor binding, in fact, its lack caused a reduction of affinity by more than
600 fold of affinity (2’-dCCPA, 4, Ki A1 = 550 nM). On the contrary, the lack of the 3’-
hydroxyl group of the sugar moiety seems to be better tolerated leading only to a 12 fold
decrease in A1 affinity (3’-dCCPA, 7, Ki A1 = 10 nM). The lack of the hydroxyl groups
caused also a consistent reduction in affinity at the two other adenosine receptor subtypes,
which causes, in general, a reduction of the A1R selectivity. An exception is represented by
the 3’-deoxy derivative 7, which showed an increased selectivity versus the A3R sub type
compared to the reference compound CCPA (select. A3/A1 = 530 vs 51, respectively). Hence,
the 3’-dCCPA showed the best balance of A3 affinity and selectivity (7, Ki = 10 nM, sel
A2A/A1 = 1,040, select. A3/A1 = 530).
Functional assays (in vitro and ex vivo) of compounds 4 and 7
Functional activity of compounds 4 and 7 was assessed both in vitro, on CHO cells stably
transfected with human A1 adenosine receptors, and in ex vivo assays on mouse ileum and on
mouse heart (Table 2 in Supporting information; Fig. 3).
Functional assays at human A1R stably transfected in CHO cells
Firstly, functional activity of compounds 4 and 7 at A1Rs was studied at human adenosine
receptors stably transfected in CHO cells by evaluating the inhibition of AC activity. AC was
first stimulated by forskolin at the concentration of 10 µM and the inhibition of the enzyme
activity caused by agonists was evaluated (Klotz et al., 1985, 1998). Inhibition of AC by
compounds 4 and 7 was compared to that induced by the full agonist CCPA whose intrinsic
activity (α) was set equal to 1 (Table 2 in Supporting information; Fig. 3, panel a).
In this assay, as expected, CCPA was able to induce the maximal response with an EC50 of 15
nM. Compounds 4 and 7 showed a different behavior. In fact, while 4 was not able to induce
the maximal effect (α = 0.70, EC50 = 832), compound 7 behaved as a full agonist with an
EC50 = 269 nM. It is worthwhile to note that in this in vitro assay at hA1Rs the 3’-deoxy
derivative resulted to be a full agonist while the 2’-deoxy analog presented as a partial
agonist.
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Mouse Ileum Assay
This assay was performed on a segment of the intestine where the A1R subtype is highly
expressed, and it allows the monitoring of the spontaneous variation of intestinal contractions
after agonist stimulation of the A1R (Lambertucci et al., 2011). After the equilibration time,
preparations were challenged with the full agonist CCPA, which triggers a gradual
spontaneous reduction of contraction in a concentration-dependent manner (Fig. 3, panel b).
Furthermore, the A1R antagonist DPCPX was studied in the presence of CCPA and the right-
shift of the CCPA concentration-response curve confirmed the involvement of A1R in mouse
ileum rhythmic spontaneous contractions (data not shown).
The two deoxy derivatives 4 and 7, tested at increasing concentrations, were not able to reach
the reduction of intestinal contraction induced by CCPA, hence, they acted as A1R partial
agonists. It is worthwhile to note that both their intrinsic activity and potency were very
similar (4, α = 0.75, EC50 = 1,380 nM vs 7, α = 0.78, EC50 = 1,318 nM).
Heart rate
To assess the effect induced by the administration of the compounds on a cardiovascular
parameter (heart rate), compounds 4 and 7 were evaluated in an ex vivo model represented by
the mouse isolated heart. The effect of the two deoxy nucleosides has been investigated in
comparison with the A1R full agonist CCPA which caused a decrease of heart rate as a
consequence of its potent inhibitory activity. CCPA, at 10 µM, reduced the normal mouse
heart rate by 80%, while compounds 4 and 7 reduced the heart rate by 32 and 42%,
respectively (Fig. 3, panel c). Both the deoxy nucleosides 4 and 7 behaved as partial A1R
agonists at mouse receptors, showing potency in the micromolar range on heart rate (Table 2;
4: EC50 = 1,698 nM; 7: EC50 = 1,548 nM).
Nucleosides 4 and 7 protect SH-SY5Y from OGD injury
The SH-SY5Y cell line is extensively used as an in vitro model to study neuronal injury or
death because of its similarity to neurons in morphological, neurochemical and
electrophysiological properties (Dal-Cim et al., 2012; Jantas et al., 2015; Zhang et al., 2011,
Hermann et al., 2013). Moreover, these cells constitutively express moderate levels of A1Rs
(Salim et al., 2000; Canals et al., 2005).
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SH-SY5Y cells were exposed to OGD for 4 h, followed by 24 h of reoxygenation; under this
experimental condition, their viability was reduced to ~60% (third column in every panel,
63.2 3.7% in Fig. 4a, 59.1 1.4% in 4b, 60.4 2.1% in 4c) in comparison to control-normoxia
(99.7 1.1%, first column of each panel in Fig. 4a, 4b, 4c). To evaluate a possible
neuroprotective effect, the A1R partial agonists were applied to SH-SY5Y culture 1h before,
during OGD treatment and during the recovery period (24 hours), until the assessment of cell
viability. To compare the effects of our drugs to a full A1R agonist, CPA was used at
concentrations already known as selective (Vissiennon et al., 2006). Compound 4 exerted a
protective effect toward OGD exposure at concentration of 0.5, 1, 10 μM (78.3 4.5%,
85.8 3%, 82.6 4% respectively, as compared to the control; Fig. 4a); compound 7 showed a
protective effect only at 0.5 and 1 µM (76.7 6.4%, 73.6 3.5 of the control; Fig 4b). Both
effects were blocked by the A1R antagonist DPCPX (0.1 µM, Fig. 4a, 4b). CPA showed a
significant protective effect toward OGD damage at concentrations of 0.5 and 1 μM
(94.2 8.2% and 86.7 5.9% of the control, respectively; Fig. 4c), whereas at a concentration
of 0.1 µM it was ineffective. DPCPX completely blocked CPA neuroprotection (Fig. 4c).
DPCPX alone did not affect cell viability. Compound 4 (1 µM), compound 7 (0.5 µM), and
CPA (0.5 µM), did not show any effect on normoxia cell viability. Further data about the
effect of forskolin versus A1R partial agonists are reported in Supporting information (Fig.
S1).
Effects of nucleosides 4 and 7 on synaptic transmission in hippocampal slices and on
presynaptic neurotransmitter release
A1Rs are largely distributed in the brain, especially in the hippocampal area (Fastbom et al.,
1987; Wakabayashi et al., 2000; Fukumitsu et al., 2003); furthermore, given the well-known
inhibitory action of A1R full agonists on synaptic transmission (Dunwiddie and Fredholm,
1989; Wu and Saggau, 1994; Johansson et al., 2001), we started to explore the effects of 4
and 7 on fEPSPs recorded in extracellular electrophysiology performed in hippocampal CA1
area. Slices were perfused with 4 or 7 at a concentration of 2 μM over 20 min, since lower (1
μM) or higher (10 μM) concentrations of the two compounds failed to clearly affect fEPSP
(data not shown). Treatment with 4 induced a brief but significant reduction of the fEPSP
slope (62.01 10.51% of the basal at the end of the 20 min application, n = 6, *p < 0.05 vs
basal, according to Wilcoxon test, Fig. 5a, 5b). Perfusion with 7 provoked a more pronounced
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reduction of fEPSP slope (50.65 6.12% of the basal at the end of the 20 min application, n =
5, *p < 0.05 vs basal, according to Wilcoxon test, Fig. 5d, 5e) lasting throughout the washout
period (30 min). The effects of both A1R partial agonists were fully prevented by DPCPX
(500 nM, 30 min; 100.60 1.83%, n = 4, °p < 0.05 vs 4 and 107.9 5.28%, n = 4, °p < 0.05 vs
7 respectively, according to Mann-Whitney test, Fig. 5b, 5e). In our experimental conditions,
DPCPX, when given alone, induced a slight and non-significant increase of the fEPSP slope
(data not shown).
Since the A1R-mediated inhibitory effect on neurotransmission predominantly results from a
presynaptic reduction of neurotransmitter release (Manabe et al., 1993; Calabresi et al., 1997;
Lupica et al., 1992; Prince and Stevens, 1992) accompanied by changes of the PPR (Schulz et
al., 1994), we analyzed the fEPSP slope ratios of paired pulses delivered with a 50-ms
interval. Perfusion with 4 (2 µM, 20 min) induced a slight and non-significant increase of
mean PPR (Fig. 5c), suggesting a low effect on presynaptic release, at least in our
experimental conditions. Conversely, the application of compound 7 (2 µM, 20 min) clearly
increased the mean PPR (Fig. 5f), suggesting a strong effect of this drug on presynaptic
release.
CPA was used as a positive control. The drug (30 nM, given over 20 min) induced a
significant reduction of neurotransmission (52.92 13.70% of the basal at the end of the 20
min application, n = 5, *p < 0.05 vs basal, according to Wilcoxon test, Fig. 5g, 5h). The
effect of CPA was fully prevented by DPCPX (500 nM, 30 min; 93.81 3.14%, n = 4, °p <
0.05 vs CPA, according to Mann-Whitney test, Fig. 5h). As expected, the application of CPA
clearly increased the mean PPR (Fig. 5i), confirming the strong effect of this drug on
presynaptic release.
Effects of compounds 4 and 7 on OGD in hippocampal slices
Hippocampus, and particularly the CA1 area, is more sensitive to ischemic insult than other
areas of the brain (Schmidt-Kastner, 2015). We applied an OGD protocol of 7 min to
hippocampal slices to produce an irreversible depression of synaptic transmission (Pugliese
et al., 2003), eliminating transient reappearance of synaptic potentials linked to
hyperexcitability phenomena (Zhu and Krnjevic, 1999; Fowler, 1990; Luhmann and
Heinemann, 1992; Doolette and Kerr, 1995) in order to point out the possible protective
effect of A1R partial agonists. CPA was used as a control to confirm, in our experimental
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conditions, the well-known protective effect of the A1R full agonists on OGD treatment. The
drug (30 nM, given over 10 min before OGD) allowed a significant recovery of
neurotransmission (62.95 10.61%, n = 5, *p < 0.05 vs OGD, according to Mann–Whitney
test, Fig. 6a, 6b). In the presence of CPA, recovery was observed in 5 out of 7 slices tested.
Compound 4 (2 µM, given over 10 min before OGD) induced a partial but significant
recovery of neurotransmission (38.82 13.53%, n = 6, *p < 0.05 vs OGD according to Mann–
Whitney test, Fig. 6c, 6d).
Analogously, we tested compound 7 in similar experiments in which the drug seemed to be
very promising in terms of protection against OGD. Indeed, it allowed, in 4 out of 8 slices
tested, a strong and clear recovery of synaptic activity after OGD, in a range between 27 and
160% of basal slope (86.62 29.05%, n = 4, *p < 0.01 vs OGD according to Mann–Whitney
test, Fig. 6e, 6f). Despite the huge variability observed in the effect of compound 7, which
prompted us to show the time course of single experiments, the neuroprotective potential of
the drug in ischemia seems to be clear and to deserve further experimental evaluation.
Effects of nucleoside 7 on mouse locomotor activity
In behavioral experiments we decided to evaluate the potency of compound 7, the most
promising of the two A1 partial agonists tested, in terms of effect on mouse locomotor
activity in vivo, in comparison to the well-known inhibitory action of A1 full agonists CPA
and CCPA on the same parameter. To this end we performed the open field test in C57Bl/6
mice injected i.p. with different doses (0.1, 1, and 10 mg/kg) of compound 7. The effects
induced by compound 7 were compared to the decreased locomotor activity induced by CPA
and CCPA (0.1 and 1 mg/kg). We failed to compare the effects of the different compounds at
10 mg/kg because, given their high potency, the full agonists gave already the maximal
response at 1 mg/kg.
In more detail, as can be seen in Figure 7, panel a, the compound 7 at the highest doses tested
(i.e 1 and 10 mg/kg) induced a slight and non-significant reduction in locomotor activity, in
terms of total distance traveled by mice, with respect to vehicle (Compound 7 at 10 mg/kg:
34.55 4.04m, n=4; Vehicle 42.09 4.96m, n=3; p>0.05 according to Mann–Whitney test); on
the contrary, at the dose of 1 mg/kg either CCPA and CPA significantly reduced mice activity
(CCPA: 2.96 0.51m, n=3; CPA: 6.49 2.74m, n=3; p<0.05 vs. Vehicle in both cases
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according to Mann–Whitney test). When activity was analyzed over time, it was even more
evident that none of the tested drugs at the dose of 0.1mg/kg was able to significantly affect
locomotor activity of mice, although a trend to a reduction was induced by the full agonist
CPA (Fig. 7 panel b). Oppositely, both CCPA and CPA administered at 1 mg/kg completely
inhibited mice locomotor activity soon after ten minutes from the start of the test (Fig. 7
panel c, p<0.05 vs. Vehicle according to Mann–Whitney test); at the same dose, compound 7
induced a slight reduction of mice locomotor activity, quite evident after 20 minutes spent in
the field. The weaker effect exerted by the compound 7 on locomotor activity, in comparison
with CPA or CCPA given at the same dose, is in agreement with its effect on synaptic
transmission in hippocampal slices; in these experiments a comparable depression of the
fEPSP slope was obtained with the compound 7 at 2 μM (Fig. 5d) and with CPA at 30 nM
(Fig. 5g).
DISCUSSION
The neuroprotective effects of A1R agonists have been widely demonstrated in several
cerebral ischemia models, both in vitro (Goldberg et al., 1988) and in vivo (Rudolphi et al.,
1992; von Lubitz et al., 1995). These effects are mostly attributable to the ability of A1Rs to
reduce, once activated, the presynaptic intake of calcium and, as a consequence, to inhibit the
release of glutamate, the neurotransmitter responsible for excitotoxicity (Choi and Rothman,
1990). Unfortunately, the clinical exploitation of such protective effects has been strongly
limited by several disadvantages typically associated with A1R full agonists, first of all, their
serious cardiovascular side effects (Belardinelli et al., 1995; Olsson et al., 1990).
Furthermore, other therapeutic tools currently available so far did not meet the expectations.
Firstly, ADAC, very promising at the beginning, gave controversial results in terms of ability
to cross the BBB (Gervitz et al., 2002). Secondly, the inhibition of adenosine kinase, and the
consequent increase of adenosine, despite its protective action shown in a model of in vivo
focal ischemia (Jiang et al., 1997), would expose to a risky non-selective stimulation of other
P1 purinergic receptors. Finally, the allosteric modulators of A1Rs were already successfully
tested in vitro (Romagnoli et al., 2014), but are still under evaluation and their application in
therapy is yet to come.
Based on their pharmacological properties, A1R partial agonists should induce fewer side
effects than full agonists and show comparable efficacy both in acute and chronic treatments.
Even though such an issue remains to be fully elucidated, in the present study we evaluated,
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for the first time, the neuroprotective potential of two A1R partial agonists, namely
compounds 4 and 7, in experimental models of cerebral ischemia, in vitro and ex vivo.
In a first screening, these compounds showed interesting features in terms of A1R selectivity
(Table 1) and intrinsic activity, which resulted to be low in mouse cardiac tissue and higher in
mouse ileum (Table 2). Such an observation could encourage a possible use of such
compounds in ischemia therapy, eventually in chronic treatments and, hopefully, with
reduced cardiovascular side effects. First experimental results on partial agonists of the
present study have been achieved in an in vitro experimental model of ischemia obtained in
human neuroblastoma cells (SH-SY5Y) and support such a notion.
Once we got convincing protective effects in in vitro ischemia model, the efficacy of partial
agonists 4 and 7 was verified in extracellular electrophysiological experiments performed in
mouse hippocampal slices subjected to OGD; this ex vivo model is highly informative
because it is characterized by a level of integration between pre- and postsynaptic neuronal,
and also neuronal/glial, components, that is not reproducible in cell cultures. Furthermore,
given the high level of A1R expression in the hippocampus, and the well-known susceptibility
of CA1 region to hypoxia, the OGD ex vivo model is particularly suitable for a preclinical
evaluation of protective properties of new candidate compounds for therapy in cerebral
ischemia. Moreover, such a model has been extensively developed and validated in previous
extracellular electrophysiological studies performed by Pugliese and collaborators (Pugliese
et al., 2003, 2009, Traini et al., 2011), in which the exposure of the hippocampal slice to
seven-minutes OGD induces a huge, reproducible, and irreversible depression of synaptic
transmission. The choice of such a drastic protocol has the advantage to confidently reveal
any possible protective effect of tested drugs against OGD. Conversely, these effects may not
be observed in the totality of slices challenged, and then it is a good rule to specify, as we
also did in the results section, the number of slices that positively responded to the treatment.
First of all, we started by confirming the protective effect of the A1R full agonist CPA on
OGD in hippocampal slices under our experimental conditions. As expected, this drug
significantly increased the recovery of fEPSP after OGD, in addition to having clearly
reduced the basal synaptic transmission in the very first minutes of the application at a very
low concentration (30 nM, Fig. 6a). Such a high responsivity of the hippocampus to A1R
stimulation confirms both the high expression of the receptor in this specific brain area and
its inhibitory properties on synaptic activity. After the refinement of the protocol obtained by
using CPA, we moved to the evaluation of the effects of A1R partial agonists 4 and 7 in
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similar extracellular electrophysiology experiments. The two compounds were able to induce,
as expected after A1R stimulation, the reduction of both basal synaptic transmission and
presynaptic neurotransmitter release; these effects were particularly evident and long lasting
in the case of compound 7 (Fig. 5d, 5e, 5f), in agreement with its higher affinity and
selectivity for A1R compared to 4 (Tables 1 and 2 in Supporting information). The ability to
reduce the presynaptic neurotransmitter release is the main mechanism by which the A1R
exerts its control on synaptic transmission (O’Kane and Stone, 1998), and it is a fundamental
requirement for a potential therapy of cerebral ischemia, in which the excessive glutamate
release induces most of the tissue damage (Choi and Rothman, 1990). Finally, the more
relevant results of the present study regard the protective effects shown by 4 and 7 in the
OGD experimental paradigm. Both compounds induced a significant recovery of
hippocampal synaptic transmission after the irreversible disappearance of fEPSP due to
OGD. In particular compound 7, in addition to the clear effect of reduction of presynaptic
neurotransmitter release (Fig. 5f), allowed a very good recovery of hippocampal synaptic
transmission, partial or even full depending on the single experiments (Fig. 6e, 6f); such a
result is very promising given the severity of the OGD protocol applied to hippocampal
slices.
It should be noted that the performance of the compound 7 in terms of protection against
OGD in slices (Fig. 6e, 6f) seems to be proportional, analogously to the full A1 agonist CPA
(Fig. 6a, 6b), to the magnitude and duration of the reduction of the basal synaptic
transmission and presynaptic release (Fig. 5d, 5e, 5f), which are both maintained all along the
washout period.
Anyway, it is likely that the neuromodulation exerted by A1R on presynaptic release is not
sufficient to explain the neuroprotective effects of adenosine or partial agonists. Indeed,
immediately after few minutes of hypoxia, a large and long-lasting neuronal depolarization
occurs (Zhang and Krnjevic, 1993; Kaminogo et al., 1998); this seems to depend not only on
the adenosine-induced inhibition of presynaptic release but also on the failing energy
potential in cells, which represents an extreme attempt to protect neurons from a harmful
condition (Fowler and Li, 1998; Balestrino et al., 1999). Furthermore, A1R stimulation is also
able to activate G-protein-coupled inwardly rectifying potassium channels (Sickmann and
Alzheimer, 2003), which, in turn, mediate hyperpolarization of the membrane and decrease
neuronal excitability; such an effect is particularly evident in CA1 dorsal hippocampal
pyramidal cells (Kim and Johnston, 2015). Differently from adenosine which exerts its
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protective function after the ischemic episode, in a time window of hours, in our experimental
condition the efficacy of partial agonists depends mainly on the fact that they are applied
before the OGD. It will be necessary, in future studies, to evaluate the therapeutic potential of
these compounds when administered after the ischemic attack. In any case, even if their
efficacy is confirmed only in pretreatment, these compounds may still be useful alternatively
to anticoagulant drugs currently used to prevent relapse in patients already affected by mild
stroke (Young et al., 2007; Ginsberg, 2009; Moskowitz et al., 2010; Albers et al., 2011;
Macrez et al., 2011).
In the present work we started to explore, to a limited extent, the behavioral profile in mice of
the compound 7, the most promising of the two A1 partial agonists tested, by using the open
field test. In such a model, it has been previously demonstrated that A1 agonists (i.e. CPA) are
able to reduce spontaneous motor activity by acting at central, and not peripheral, level
(Marston et al., 1998). Similarly, the reduction in locomotor activity induced by the
compound 7 (Fig. 7) should be due to a central effect, and suggests an ability of the drug to
cross the BBB.
By the way, a further evaluation of the effects of compounds 4 and 7 in in vivo models of
cerebral ischemia is mandatory in order to confirm their ability to cross the BBB and to
induce central effects, like the full agonist CCPA (from which they are derived) does; it will
be necessary to have confirmation, both ex vivo and in vivo, of a reduced receptor
desensitization induced by compounds 4 and 7, a key requirement for chronically
administering these substances; finally, and above all, it will be necessary to get confirmation
of a reduced cardiovascular effect caused by these partial agonists, only suggested, at the
moment, by the lowest intrinsic activity they showed in mouse heart tissue compared to other
isolated organs.
Although further studies are needed to fully elucidate their neuroprotective potential, our
results indicate that A1R partial agonists 4 and 7 may represent a potential alternative to the
treatment of cerebral ischemia, given alone or in combination with other substances such as
antagonists of the A2AR, which in hippocampus inhibits the modulation of synaptic
transmission mediated by A1Rs (Cunha et al., 1994).
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Pre-registration of the study
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Acknowledgments
This work was supported by the University of Camerino (Fondo di Ricerca di Ateneo) and by
a grant from the Italian Ministry of Research (PRIN N° 2015E8EMCM_008, 2015). We
acknowledge Sonja Kachler for her technical assistance in setting up experiments. We
acknowledge Antonella Pezzola and Dr. Zaira Boussadia for their help in behavioral
experiments.
Conflict of interest
The authors declare no issues regarding conflicts of interest.
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Legends
Figure 1
Time-line diagram of animal experiments and analysis.
Figure 2
Synthetic schemes for the synthesis of nucleosides 4 and 7.
The new nucleosides were obtained through a convergent approach by reaction of 1 (2,6-
dichloropurine) with the suitable sugar 2 (1-chloro-2-deoxy-3,6-ditolylribose) or 5 (3-deoxy-
ribofuranose). The two intermediates 3 or 6
were, then, treated, in turn, with
cyclopentylamine and methanolic ammonia to furnish final compounds 4 (2-chloro-2’-deoxy-
N6-cyclopentyladenosine) and 7 (2-chloro-3’-deoxy-N6-cyclopentyladenosine).
Scheme 1, reagents and conditions: (a) NaH, CH3CN anhydrous; (b) i. cC5H9NH2, Et3N, Δ, ii.
NH3/CH3OH.
Scheme 2, reagents and conditions: (a) SnCl4, CH3CN anhydrous; (b) i. cC5H9NH2, Et3N, Δ,
ii. NH3/CH3OH.
Figure 3
Functional activity of compounds 4 and 7 versus the activity of the reference agonist
CCPA. Panel a: concentration-response curve of compounds 4 and 7 in comparison with that
of the reference agonist CCPA at CHO cells stably expressing the hA1AR; changes in cAMP
levels are expressed as % decrease of luminescence intensity.
Panel b: concentration-response curves of compounds 4, 7, and CCPA obtained by inhibition
of mouse ileum rhythmic spontaneous contractions.
Panel c: concentration-response curves of compounds 4, 7, and CCPA obtained by inhibition
of mouse heart rate. Each point represents the mean of four or five experiments with a
maximum SE lower than 10.
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Figure 4
Effect of A1R partial (4 and 7) and full (CPA) agonists on SH-SY5Y cell death induced
by OGD.
Compound 4 (a), compound 7 (b), and CPA (c) were applied to SH-SY5Y culture 1h prior,
during OGD treatment, and during the recovery period. DPCPX was added 30 min before the
drugs and kept throughout the OGD time and recovery time. Cell viability was evaluated by
using trypan blue dye exclusion. In the dot-plot graphs, single values are indicated; the
mean SEM of at least 3 independent experiments (each one performed with an independent
cell culture preparation) is expressed as a percentage of control which was considered to be
§
100%. p < 0.01 vs CTR-normoxia group; *p < 0.05 vs OGD group; °p < 0.05 vs
corresponding drug/OGD group (in every case according to Mann-Whitney test). Compound
4 (1 µM), compound 7 (0.5 µM), and CPA (0.5 µM), did not show any effect on normoxia
cell viability.
Figure 5
Effects of compounds 4 and 7 on synaptic transmission and on presynaptic
neurotransmitter release in hippocampal slices
The time course of the fEPSP slope during the perfusion with compounds 4 (n = 4 animals, 6
slices), 7 (n = 5 animals, 5 slices), and CPA (n= 3 animals, 5 slices) is shown respectively in
panels a, d, and g. The related dot-plot graphs (panels b, e, and h) represent the mean values
of the fEPSP slope (taken at the end of the 20 min application) in slices perfused with
compounds 4, 7, and CPA, in the presence (n = 4 animals, 4 slices for compound 4; n = 3
animals, 4 slices for compound 7; n = 4 animals, 4 slices for CPA) or absence of DPCPX. *p
< 0.05 versus basal; °p < 0.05 versus 4, 7, and CPA.
The time course of the PPR (R2/R1) during the application of compounds 4 (n = 6), 7 (n = 5),
and CPA (n=5) is shown respectively in panels c, f, and i. PPR has been evaluated with the
same animals/slices used for the time course of the fEPSP slope.
Figure 6
Effects of compounds 4 and 7 on hippocampal slices subjected to OGD damage
The time course of the fEPSP slope before, during and after reperfusion in oxygenated ACSF
(panels a, c, and e, black dots, n = 4 animals, 7 slices) shows the irreversible synaptic failure
induced by OGD protocol.
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CPA (panel a, white dots, and panel b) was used as a control to confirm the neuroprotection
induced by A1R full agonists toward the OGD effect (n = 5 animals, 5 slices, *p < 0.05 vs
OGD). Insets in panel a show representative fEPSP recordings in correspondence of baseline
(a), OGD (b), and washout (c). Calibration bars: 1 mV, 10 ms.
Compounds 4 and 7 were added 10 min before OGD. The time course of the fEPSP slope
during the perfusion with compounds 4 (n = 3 animals, 6 slices, white dots) and 7 (n = 4
animals, 4 slices) is shown respectively in panels c and e.
Insets in panels c and e show representative fEPSP recordings in correspondence of baseline
(a), OGD (b), and washout (c). Calibration bars: 0.5 mV, 10 ms in panel c and 1 mV, 10 ms
in panel e (in which the representative fEPSPs are referred to the time course of experiment
4).
The dot-plot graphs show the mean values of the fEPSP slope obtained in slices subjected to
OGD in presence of compounds 4 (panel d, *p < 0.05 vs OGD) and 7 (panel f, *p < 0.01 vs
OGD).
Figure 7
Effects of compounds 7, CPA, and CCPA on locomotor activity in C57Bl/6 mice
The compound 7 at the highest doses tested (i.e 1 and 10 mg/kg) induced a slight and non-
significant reduction in locomotor activity, in terms of total distance traveled by mice (n = 3
animals at 1 mg/kg, n = 4 animals at 10 mg/kg; panel a), with respect to vehicle (n = 3
animals, panel a); on the contrary, at the dose of 1 mg/kg either CCPA and CPA significantly
reduced mice activity (CCPA: n=3, p<0.05 vs. vehicle; CPA: n=3, p<0.05 vs. vehicle). In
panel b activity was analyzed over time; none of the tested drugs at the dose of 0.1 mg/kg (n
= 6 animals for compound 7; n = 3 animals for CCPA; n = 3 animals for CPA) was able to
significantly affect locomotor activity of mice. CCPA and CPA administered at 1 mg/kg
completely inhibited mice locomotor activity soon after ten minutes from the start of the test
(panel c, p<0.05 vs. Vehicle); at the same dose, compound 7 induced a slight reduction of
mice locomotor activity, quite evident after 20 minutes spent in the field (panel c).
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