Selective Sphingosine 1-Phosphate Receptor 1 Agonist Is Protective Against Ischemia/Reperfusion in Mice
Background and Purpose—Growing evidence supports that the immunomodulatory drug fingolimod is protective in stroke. Fingolimod binds to 4 of 5 sphingosine-1-phosphate (S1P) receptors and, among other actions, it induces lymphopenia. In this study, we investigated whether a selective S1P1 agonist is protective in experimental stroke.
Methods—Drug selectivity was studied in vitro in cells overexpressing the human S1P receptors. Mice (n=54) received different doses of LASW1238 (3 or 10 mg/kg), fingolimod (1 mg/kg), or the vehicle intraperitoneal, and lymphopenia was studied at different time points. After intraluminal middle cerebral artery occlusion for 45 minutes and immediately after reperfusion, mice (n=56) received the drug treatment. At 24 hours, a neurological test was performed and infarct volume was measured. Treatment and all the analyses were performed in a blind fashion.
Results—In vitro functional assays showed that LASW1238 is a selective agonist of the S1P1 receptor. At 10 mg/kg, this compound induced sustained lymphopenia in mice comparable with fingolimod, whereas at 3 mg/kg it induced short-lasting lymphopenia. After ischemia, both LASW1238 (10 mg/kg) and fingolimod reduced infarct volume, but only LASW1238 (10 mg/kg) showed statistically significant differences versus the vehicle. The neurological function and plasma cytokine levels were not different between groups.
Conclusions—The selective S1P1 agonist LASW1238 reduces infarct volume after ischemia/reperfusion in mice, but only when lymphopenia is sustained for at least 24 hours. S1P1 and lymphocytes are potential targets for drug treatment in stroke. Defining the best drug dosing regimens to control the extent and duration of lymphopenia is critical to achieve the desired effects.
Fingolimod is an oral drug approved for the treatment of relapsing-remitting multiple sclerosis.1 Several studies reported beneficial effects of fingolimod in experimental models of brain ischemia2 and in ischemic stroke patients.3,4 Fingolimod is the prodrug of fingolimod phosphate (fingolimod-P), an agonist of 4 of the 5 sphingosine-1-phosphate (S1P) receptors (all except S1P2). S1P receptors are G-protein–coupled receptors regulating diverse cellular functions, including survival, proliferation, and migration.5 S1P regulates lymphocyte migration from lymphoid tissue to blood.6 Synthetic S1P1 agonists behave as functional antagonists by inducing lymphocyte receptor internalization, leading to receptor unavailability at the cellular membrane.7 Therefore, cells are retained in lymphoid organs causing lymphopenia.8
Fingolimod penetrates and accumulates in the brain,9 where S1P receptors are expressed.10 The contribution of S1P receptors other than S1P1 on the beneficial effects of fingolimod in stroke is not completely understood. We addressed this question and the mechanism behind the efficacy by studying the effects of a selective S1P1 agonist in mouse ischemic stroke.
See the online-only Data Supplement for further details.
Animal work was conducted in adult male C57BL/6J mice (Charles River) after the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments) and with approval from the Animal Experimentation Ethical Committee of Almirall (Barcelona, Spain) or the University of Barcelona (CEEA).
Chemicals and Reagents
LASW1238 and fingolimod were synthesized by the Department of Medicinal Chemistry (Almirall R&D Center, Barcelona, Spain). Drugs were suspended in vehicle (0.5% carboxymethyl cellulose, 0.1% Tween-80 in purified water) and were administered intraperitoneally.
Target Selectivity Profile
Assays were performed on membranes of Chinese hamster ovary (CHO) cells transfected with human S1P receptors. Agonist potencies were measured by 35S-GTPγS binding after drug stimulation of the receptors (n=5). Potential off-target effects of LASW1238 (10 µmol/L) were studied on a panel of G-protein–coupled receptors and enzymes.
Mice were randomized to receive the vehicle, LASW1238 (3 or 10 mg/kg) or fingolimod (1 mg/kg; n=54), and blood cell counts were obtained in a hemocytometer.
Focal Brain Ischemia/Reperfusion
The mouse right middle cerebral artery was intraluminally occluded for 45 minutes followed by reperfusion (n=56). Cerebral blood flow (CBF) was measured with laser Doppler flowmetry (Perimed AB, Järfälla, Sweden). CBF data were used as criteria to exclude animals (n=16) from the study if they did not show: (1) a drop in CBF after middle cerebral artery occlusion >75% from basal values; (2) recovery in CBF at reperfusion >70% of basal. The mice were randomized, and treatments were given after reperfusion in a blinded fashion. At 24 hours, a neurological score was performed, and brain infarction was studied with the 2,3,5-triphenyltetrazolium chloride technique.
One-way ANOVA or the Kruskal–Wallis test followed by post hoc analyses were performed using GraphPad Prism software.
In Vitro Studies
Evaluation of drug affinity for human S1P receptors showed that LASW1238 is a highly selective S1P1 agonist compared with S1P3 and S1P5 and had no effect on S1P2 (Table).
LASW1238 did not show cytotoxicity in Chinese hamster ovary cells at doses of up to 40 µmol/L, whereas fingolimod showed cytotoxicity at 8.5 µmol/L. Moreover, LASW1238 (10 μmol/L) did not show activity on any of the >100 different targets tested, except for a 55% inhibition of binding to the M2 muscarinic receptor (not shown).
In Vivo Pharmacology: Lymphopenia
All the treatments reduced the numbers of circulating lymphocytes by ≈80% at 4 hours (Figure 1). Lymphopenia was sustained for at least 24 hours with LASW1238 (10 mg/kg) and fingolimod (1 mg/kg). Dose differences between these drugs needed for this effect agree with the 10-fold difference in their affinities for S1P1. The low-dose LASW1238 (3 mg/kg) induced transient lymphopenia at 4 hours that was not sustained at 24 hours.
LASW1238 Reduces Infarct Volume
Mice of all groups showed similar CBF changes after middle cerebral artery occlusion (Figure 2A). High-dose (10 mg/kg) LASW1238 produced a smaller infarct volume at 24 hours (P<0.05; Figure 2B and 2C) than both the vehicle and the low-dose (3 mg/kg) LASW1238. Fingolimod (1 mg/kg) tended to produce a smaller infarct volume, but this did not reach statistical significance (Figure 2C). Edema volume (Figure I in the online-only Data Supplement) and the neurological score (Figure 2D) did not differ between groups. There was no mortality in any of the groups.
This study shows that the selective S1P1 agonist LASW1238 reduced infarct volume after brain ischemia/reperfusion in mice, suggesting that S1P1 is a relevant target in ischemic stroke rather than other S1P receptors that are also targets of fingolimod. This finding is in agreement with a previous study, showing that another selective S1P1 agonist was protective when given after ischemia/reperfusion in rats and suggested that the drug acted on neuronal S1P1.11 Although actions of S1P1 agonists on brain or vascular cells cannot be ruled out, our study suggests that lymphopenia is important for the beneficial effect of the S1P1 agonist because LASW1238 reduced infarct volume only at the dose able to sustain lymphopenia for 24 hours, but not at a lower dose causing short-lasting lymphopenia. In line with our results, fingolimod was not protective after ischemia/reperfusion in lymphocyte-deficient mice.12 The current results show that drug-induced lymphopenia needs to be sustained for at least 24 hours to translate into benefits after brain ischemia.
Bradycardia is observed in multiple sclerosis patients treated with fingolimod, and it can cause atrioventricular blockade because of S1P1 direct agonistic action on cardiomyocytes.13 This side effect requires patient monitoring for 6 hours after the first drug administration to avoid complications, whereas full lymphopenia is only achieved hours later. Although a quick onset of lymphopenia is not required in a chronic disease, such as multiple sclerosis, it is currently unknown how fast after stroke onset and for how long lymphopenia should be maintained. Also, any potential adverse effects of lymphopenia should be investigated, for instance regarding the risk of infection associated with the naturally occurring stroke-induced immunodepression.14 Effective drug doses and time window would need careful assessment in stroke patients to minimize cardiac issues or infections.
We postulate that S1P1 is a candidate target for drug treatment in acute stroke. Prevention of lymphocyte migration to the central nervous system potentially underlies the beneficial effect of S1P1 agonists, although additional effects on S1P1 in other compartments cannot be excluded. Translation of these results into human stroke therapy would require establishing the adequate timing and extent of drug-induced lymphopenia needed for efficacy, as well as determining for how long lymphopenia should be maintained after stroke onset to ensure benefits minimizing any potential adverse effects.
We acknowledge the contribution of Teresa Domènech for compound characterization and the significant technical support of Luis Boix and Jose Luis Gómez (Almirall) and Alba Hernández (Institut d’Investigacions Biomèdiques August Pi i Sunyer).
Sources of Funding
Supported by the Spanish Ministerio de Economia y Competitividad (SAF2014-56279-R).
Drs Tarrasón, Gavaldà, and Godessart are full time employees of Almirall. The other authors report no conflicts.
Guest Editor for this article was Christoph Kleinschnitz, MD.
The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.116.015371/-/DC1.
- Received June 21, 2016.
- Revision received September 7, 2016.
- Accepted October 6, 2016.
- © 2016 American Heart Association, Inc.
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