FTY720 is an oral sphingosine-1-phosphate (S1P) receptor modulator (Baumruker et al., 2007) under development for the treatment of multiple sclerosis (MS), representing the first of a new class of immunomodulatory agents. Promising results in phase II trials with relapsing MS patients (Kappos et al., 2006) mirror the striking efficacy of FTY720 in MS models of experimental autoimmune encephalomyelitis (EAE), shown by preventive and therapeutic treatment (Brinkmann et al., 2002; Fujino et al., 2003; Webb et al., 2004; Kataoka et al., 2005; Balatoni et al., 2007). FTY720 is converted in vivo to its biologically active phosphate ester metabolite (FTY720-P), which acts as a high-affinity agonist for four of the five known G-protein-coupled S1P receptors, namely S1P₁ and S1P_3–5 (Brinkmann et al., 2002; Mandala et al., 2002). Sphingosine kinase (SPHK) 2 is the primary enzyme required for FTY720-P formation, as we and others subsequently confirmed in SPHK2 knockout mice (Kharel et al., 2005; Zemann et al., 2006). The fact that SPHK1 null mice become lymphopenic after FTY720 administration further supports the view that SPHK2 is sufficient for the functional activation of FTY720 (Allende et al., 2004).

Emerging evidence suggests that the effectiveness of FTY720 in the central nervous system (CNS) extends beyond immunomodulation to encompass other aspects of MS pathophysiology, including an influence on the blood-brain barrier and glial repair mechanisms that could ultimately contribute to restoration of nerve function (Baumruker et al., 2007; Jung et al., 2007; Osinde et al., 2007). A key consideration behind this concept is the finding that FTY720 distributes to the brain (Meno-Tetang et al., 2006), which contains endogenous SPHK2 for the phosphorylation of FTY720 (Billich et al., 2003). Moreover, neurons and glial cells (astrocytes, microglia, oligodendrocytes) in the brain differentially express S1P receptors (Fig. 1), thus raising the possibility for receptor activation in situ by FTY720-P. So far, there is no information on the presence of FTY720-P in the brain or potential concentrations therein. Our primary aim was to investigate the distribution of FTY720 and its phosphorylated form in the CNS after clinically relevant doses in two different EAE models. We provide preclinical evidence that the bioactive metabolite FTY720-P distributes to the CNS white matter, suggesting the potential for functional interaction with glial cells bearing S1P receptors in the brain and spinal cord.

Materials and Methods

Animals. For EAE, female RT1¹ Lewis rats from Charles River (Sulzfeld, Germany) and Dark Agouti (DA) rats from Harlan Winkelmann (Borchen, Germany) were kept under standardized light- and climate-controlled conditions with free access to food and water. Age-matched rats were acclimatized for at least 1 week before distribution into the experimental groups. For autoradiography, male pigmented LE/CR WIGA rats (Charles River; 198–238 g) were housed individually in metabolism cages. All experiments conformed to Novartis animal care regulations and were approved by the Austrian and Swiss health authorities in compliance with international animal welfare standards according to the European Communities Council Directive and the guidelines set forth in the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, 1996).

EAE Induction and Clinical Scoring. Animals were lightly anesthetized by isoflurane inhalation (0.5% Forane; Abbott Laboratories, Vienna, Austria) and given a single intradermal 200-μl inoculation in the dorsal base of the tail root. The immunization mixture for DA rats consisted of syngeneic CNS antigen (4 parts brain to 6 parts of spinal cord) in phosphate-buffered saline emulsified 1:1 in incomplete Freund's adjuvant supplemented with 200 μg of heatinactivated Mycobacterium tuberculosis (strain H37 RA; DIFCO, BD Diagnostics, Oxford, UK); the adjuvant is henceforth referred to as complete Freund's adjuvant (CFA). For Lewis rats, the inoculum contained guinea pig spinal cord in phosphate-buffered saline emulsified 1:1 in CFA. As an adjuvant control for all EAE studies, animals were injected with CFA alone and vehicle-treated. The rats were weighed every other day and scored daily for neurological signs as follows: 0, no clinical deficit; 1, complete loss of tail tonus; 2, limb weakness or ataxia; 3, full paralysis of hind or forelimbs; or 4, tetraparalysis or moribund. Animals with a score of 4 were sacrificed if weight loss indicated little chance of recovery, in accordance with animal welfare standards. Mortality due to sacrifice or spontaneous EAE-related death was indicated (•) and recorded as a 4 on the given day; this death score continued to be included in the clinical assessment, but body weight measurements were not carried forward.

Test Compounds for in Vivo Evaluation. Unlabeled and ¹⁴C-labeled FTY720, as well as cyclosporine A (CsA), were supplied by Novartis Pharma AG (Basel, Switzerland). The radiochemical purity of [¹⁴C]FTY720, which was labeled in position 2, was shown by high-pressure liquid chromatography to be 98% with a specific activity of 35 μCi/mg. FTY720 was dissolved in water, and CsA was dosed in the Neoral vehicle. Both drugs were freshly prepared and given p.o. once daily by gavage at a dosing volume of 5 ml/kg body weight. For prophylactic and therapeutic treatment, oral dosing started on day 0 at immunization and at the peak of disease in fully established EAE, respectively.

Peripheral Leukocyte Counts. Rats were lightly anesthetized by isoflurane inhalation and 100 μl of blood from the retro-orbital venous plexus was collected in EDTA-coated tubes (Sarstedt AG, Nümbrecht, Germany). Automated differential leukocyte analysis was performed on the HESKA Vet ABC-Diff Hematology Analyzer (Heska Corp., Fort Collins, CO).

Autoradiography of ¹⁴C-Labeled FTY720. Quantitative whole-body autoradiography (QWBA) and light microscopic autoradiography were performed to assess the uptake and tissue distribution of [¹⁴C]FTY720 radioactivity in male pigmented rats (n = 6) following seven oral doses at 7.5 mg/kg/d. At 8, 24, and 168 h after the last dose, the animals were deeply anesthetized with isoflurane and submerged in a dry ice-hexane bath at –70°C for at least 20 min. The frozen carcasses were rapidly shaven and stored below –20°C until embedment in an ice-cold aqueous solution of 2% carboxymethylcellulose. They were then frozen for approximately 30 min in a dry ice-hexane mixture at –70°C, followed by an overnight stabilization at –20°C. Lengthwise sections (40 μm thick) were obtained in a cryomicrotome (Leica Microsystems, Nussloch, Germany) at –20°C. Whole-body autoradiograms were obtained by autoradioluminography. Briefly, sections with a paper backing were placed on Fuji BASIII imaging plates (Fuji Photo Film, Tokyo, Japan) for 1 day at room temperature in a lead shielding box. After exposure (detection of approximately 1.5 dpm/mg), the imaging plates were first kept in the dark for 3 to 5 min and then transferred to a Fuji BAS 2000 TR phosphorimaging device (Fuji Photo Film) for scanning at a 100-μm step with a 1024 gradation. Images were prepared by re-exposing the sections onto Super Resolution storage phosphor screens (PerkinElmer, Shelton, CT) for 1 day at room temperature and scanning at a 42-μm step (Cyclone PhosphorImager; Packard Instrument, Meriden, CT). The image files were processed using Photoshop Elements 2.0 software (Adobe Systems, San Jose, CA). Levels of radioactivity in the tissues were determined by comparative densitometry, as described previously (Schweitzer et al., 1987).

For light microscopy, brain and spinal cord samples from all animals were fixed in 3% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, for 2 days at 4°C. Postfixation was performed with 1% osmium tetroxide in 0.1 M cacodylate buffer, pH 7.4, for 2 h at 4°C. The tissues were dehydrated in graded acetone solutions and embedded in Epon. Semithin sections were dipped in Ilford L4 emulsion (Ilford, Mobberley, Cheshire, UK) in the dark at 4°C. Dipped sections were developed in Kodak D19 (Eastman Kodak, Rochester, NY) after 31 weeks exposure, stopped in distilled water, fixed in Ilford Hypam rapid fixer (Ilford), and counterstained with toluidine blue. Light microscopic examination of sections was performed independently by three pathologists. Labeling was identified by more silver grains over the cells than the background without tissue.

Quantification of FTY720 and FTY720-P in the Blood, Brain, and Cerebrospinal Fluid. Plasma, whole blood, brain, and cerebrospinal fluid (CSF) were collected from EAE rats at 24 h after the last FTY720 dose to obtain trough levels. Concentrations of FTY720 and FTY720-P were determined by high-pressure liquid chromatography (Agilent 1100; Agilent, Waldbronn, Germany) with mass spectrometric detection as described previously for serum and other tissues (Zemann et al., 2006). For measurements in plasma, heparinized blood, or CSF, 20- to 100-μl aliquots were spiked with internal standards (final concentration, 0.5 μg/ml) and extracted with chloroform/methanol at acidic pH; extracts were dried and reconstituted in methanol/0.2% formic acid. Samples were chromatographed on a Luna C8 column (3 μ, 2 × 50 mm; Phenomenex, Torrence, CA) equipped with a C4 wide-bore precolumn. The analytes were eluted with a gradient (eluent A, 10 mM ammonium acetate containing 0.08% HCOOH in water; eluent B, 10 mM ammonium acetate containing 0.08% HCOOH in MeOH; 50–98% B in 14 min) at a flow of 0.4 ml/min at 40°C. Analytes were detected by electrospray-ionization liquid chromatography with tandem mass spectroscopy using an API 4000 QTrap instrument (MDS Sciex, Concord, ON, Canada). The optimal collision energies for FTY720 and FTY720-P were 23 and 25 V, respectively. The multiple reaction monitoring transitions monitored for FTY720 and FTY720-P were m/z 308/255 and 388/255, respectively.

For CNS determination, half of a rat brain (approximately 750 mg) was emulsified 1:1 in 2 ml of MeOH/H₂O (1:1) with a glass homogenizer (Potter S; Braun, Melsungen, Germany). The homogenate was divided for separate determination of FTY720 and FTY720-P, then aliquots were spiked with their respective internal standards (35 μl) to give a final concentration of 0.5 μg/ml. Methanol (400 μl) was added to 0.5 ml of homogenate, followed by 20 min of mixing on a rotary shaker and 5 min of sonication at room temperature. Supernatants were harvested after centrifugation at 12,000g; the pellet was extracted once with 200 μl of methanol, then the supernatant was harvested after centrifugation. Combined supernatants were subjected to solid-phase extraction using STRATA-X-C 33 μm Cation Mixed-Mode Polymer Phase columns (Phenomenex). The columns were eluted with 3 ml of MeOH/1% NH₄OH. The eluate was dried, its residue was dissolved in a 35:65 mixture of eluents A and B (see above), and then samples were chromatographed as described above.

An alternative method for blood or homogenized brain comprised two extractions with acetonitrile/methanol/ethyl acetate (5:3:2), dissolution of the residue of the evaporated organic phase in methanol/water/ammonia (50:48.75:1.25), and chromatography on a Zorbax SB-C8 0.5- × 75-mm column (3.5 μm; Agilent Technologies) at 50°C with a linear gradient from 0 to 95% B within 10 min at 20 μl/min (solvent A, 0.2% HCOOH in water; B, 0.2% HCOOH in acetonitrile). In this case, a Micromass Micro triple quadrupole mass spectrometer with electrospray source (Waters AG, Rupperswil, Switzerland) was used, and multiple reaction monitoring transitions were monitored for FTY720 and FTY720-P at m/z 308/105 and 388/255, respectively. Both analytical methods yielded identical results.

Statistical Analysis. A one-way analysis of variance (ANOVA) was used to compare all data sets using SigmaStat for Windows, version 3.11 (Systat Software Inc., Richmond, CA). Differences between groups were analyzed using the post hoc Tukey test for pairwise multiple comparison. For EAE, area under the curve (AUC) values for body weight loss and clinical grade scores were evaluated during the entire prophylactic treatment period or after the initiation of therapeutic dosing. Probabilities (p) ≤ 0.05 were considered to be statistically significant.

Results

FTY720 Provides Sustained Protection in EAE. Two-week therapeutic treatment with 0.03 to 0.9 mg/kg FTY720 dose dependently inhibited progression of established disease in the DA rat model of chronic EAE compared with vehicle controls, which exhibited sustained neurological deficits throughout the 2-month observation (Table 1; Fig. 2). Evidence for rapid and full disease suppression was consistently observed after administration of 0.3 mg/kg FTY720, whereas the minimum effective dose providing almost complete protection even 1 month after discontinuation was 0.1 mg/kg (p = 0.00002). A plateau in efficacy appeared to be reached by 0.3 mg/kg since there was no difference in the cumulative disease score between this dose and the 3-fold higher one of 0.9 mg/kg (Table 1). The very low dose of 0.03 mg/kg also tended to diminish the overall disease burden and prevent a marked rebound. In contrast, severe paralysis reoccurs following cessation of classic immunosuppressive agents, such as CsA at 25 mg/kg (Fig. 2) or FK506 at 4 mg/kg (data not shown), that were used as reference compounds. All doses of FTY720 completely prevented moribund incidence compared with 18 and 22% with vehicle and CsA, respectively (Fig. 2).

Prolongation of Low-Dose FTY720 Steadily Reduces EAE Signs after Therapeutic Treatment. To further explore the long-term efficacy of low-dose FTY720 in a more clinically relevant setting, 0.03 to 0.3 mg/kg was administered for 3 weeks in DA rats with established EAE (Fig. 3). By the last week of therapeutic treatment, three different studies consistently demonstrated a significant decrease in disease signs with 0.03 mg/kg versus the vehicle. Higher doses of 0.1 mg/kg (data not shown) and 0.3 mg/kg were even more efficacious. Moreover, the mortality rate was markedly reduced with FTY720 at 0.03 mg/kg (8.9% death), with complete protection at 0.1 and 0.3 mg/kg compared with 29.2% deaths in the positive control.

In contrast, 2-week prophylactic treatment with 0.03 mg/kg FTY720 in the Lewis rat model of acute EAE merely delayed the onset of paralysis by approximately 1 day but had no protective effect on disease development (Fig. 4). Increasing the 2-week dose to 0.3 mg/kg markedly prevented neurological deficits during the 1-month study (p = 0.0003); however, restricting the treatment to days 0 to 6 failed to stop EAE induction, with clinical signs appearing 1 week after drug cessation.

Influence of FTY720 on Circulating Lymphocyte Counts during EAE. Given that the above disparities in EAE-efficacy appeared to be less related to the FTY720 dose but more to its duration, we sought to investigate the temporal relationship between peripheral lymphocyte counts and EAE treatment regimens. Earlier studies showed that 0.03 and 0.3 mg/kg FTY720 can decrease the peripheral lymphocyte count by approximately 20 and 70%, respectively, within 6 h after a single oral dose in naive Lewis rats (Brinkmann et al., 2002), resulting in an ED₅₀ of 0.09 ± 0.01 mg/kg by 48 h. Likewise during EAE, 0.3 mg/kg FTY720 already reached maximum reduction (approximately 90%) of lymphocytes by 6 h in the Lewis rat (Fig. 5A). Furthermore, it was already recognized that daily doses of 0.03 mg/kg FTY720 for 1 week can reduce lymphocyte counts by up to 80% compared with placebo-treated Lewis rats in a heart allograft model (Nikolova et al., 2000). We have extended these findings to EAE and demonstrate that 0.03 mg/kg therapeutic dosing for 3 weeks in DA rats leads to a highly significant reduction in circulating lymphocytes by days 14 and 33 versus vehicle (Fig. 5B), i.e., 52 and 67%, respectively. At the early time point, 0.3 mg/kg FTY720 led to over twice the reduction in lymphocyte numbers compared with 0.03 mg/kg (p < 0.001), but by day 33, there was no difference between these two doses (Fig. 5B).

Taking into account that 7-day preventive dosing with 0.3 mg/kg FTY720 failed to suppress EAE (Fig. 4), yet lymphocytes were reduced by 75% versus vehicle (Fig. 5A), we agree with previous suggestions (Webb et al., 2004) that FTY720 or its phosphate are apt to exert additional effects beyond the induction of peripheral lymphopenia. For example, although 0.03 mg/kg FTY720 decreased the circulating lymphocytes by at least 50% shortly after drug initiation (Fig. 5B), this low dose failed to significantly prevent EAE signs when treatment is limited to 2 weeks in a prophylactic setting (Fig. 4). To explore whether additional effects of FTY720 and FTY720-P in the brain are possible at all, based on available drug concentrations in that tissue, we examined the distribution of FTY720 in the rat and determined levels of FTY720/FTY720-P in the brain.

Quantitative Whole-Body Autoradiography and Myelin Sheath Distribution of [¹⁴C]FTY720. First, QWBA was used to investigate the distribution of [¹⁴C]FTY720-related radioactivity in vivo and, in particular, its uptake into the CNS. Pigmented rats received [¹⁴C]FTY720 for 1 week at a high oral dose of 7.5 mg/kg/d. By 24 h after the seventh and last dose (Fig. 6A), elevated amounts of extravascular radioactivity were detected in the adrenal cortex, kidney (cortex-medullary junction), nasal turbinates, pituitary gland, preputial gland, and stomach (glandular mucosa); maximal levels of radioactivity occurred in the brain, epididymis, eye (ocular membranes, vitreous body), and testis. At 168 h, residual radioactivity was still observed in most of these tissues, equivalent to approximately 1.4% of the administered dose, but the highest concentrations were found in the brain (reticular nucleus, corpus callosum, cerebellar white matter), preputial gland, and spinal cord (Fig. 6B). The distribution pattern after multiple doses was similar to that after a single dose (data not shown), especially the preferential localization to brain and spinal cord at 168 h. The accumulation factor (i.e., ratio between the tissue concentration after multiple versus single dosing) was 3.2 for brain and 3.7 for spinal cord at 24 h after the seventh dose compared with the single dose.

Light microscopic evaluation showed that the ¹⁴C labeling in brain and spinal cord was confined to the myelin sheets (Fig. 7). Neurons were free of grains, except for some background labeling.

Levels of FTY720 and FTY720-P in Blood and Brain. Given that exposure of the CNS after oral dosage of FTY720 to normal rats was shown in the above QWBA study and in a previous pharmacokinetic analysis (Meno-Tetang et al., 2006), we next sought to determine concentrations of FTY720 and its phosphorylated form in blood and brain of rats diseased with EAE. In fact, levels of FTY720-P in brain have so far not been reported at all. Samples from both acute and chronic models of EAE (in Lewis and DA rats, respectively) were obtained 24 h after the last dose to determine trough levels. FTY720 and FTY720-P were found both in the blood and brain, with ratios between approximately 0.8 and 3 (Table 2). Brain levels of both forms were considerably higher (by factors of 10 to 27 after 23 doses) than those in blood, as seen before for FTY720 in normal rats (Meno-Tetang et al., 2006). Brain concentrations of the two forms were of comparable magnitude. The observed amounts of both FTY720 and its phosphorylated form in the DA rat brain increased with the oral dose of FTY720, as did the blood levels; thus, the brain/blood ratio was relatively constant in DA rats (21–27 for FTY720; 14–17 for FTY720-P). We also followed the time course of FTY720 and FTY720-P concentrations in blood and brain (Table 2). Although blood levels were constant over 21 days, those in the brain increased over time and, hence, the brain/blood ratio.

FTY720 and FTY720-P concentrations in plasma were lower than in whole blood due to binding of the compounds to blood cells (Table 3). Levels of FTY720 and FTY720-P in the CSF were 30 to 80-fold lower in CSF than in plasma, indicating their almost exclusive association with the CNS tissue rather than the extracellular space.

Discussion

Our findings clearly demonstrate for the first time that FTY720 localizes to the CNS white matter, with autoradiography depicting preferential distribution along the myelin sheath. Pharmacokinetic analysis during EAE further showed a dose- and time-dependent uptake into the CNS. In the DA rat model, a progressive rise in brain trough levels of both FTY720 and FTY720-P was associated with an increase in the oral dose. CNS concentrations of the parent drug and its metabolite reached comparable levels, suggesting that an equilibrium was established due to SPHK2-mediated phosphorylation in situ. Since the brain contains endogenous SPHK2 (Billich et al., 2003), and since FTY720-P, as a charged phosphate, is unlikely to cross the blood-brain barrier by itself, we assume that FTY720-P is formed from FTY720 within the CNS. Interestingly, the brain/blood ratio remained relatively constant (21–27 and 14–17 for FTY720 and FTY720-P, respectively) after 3 weeks of treatment despite the dose range (0.03–0.3 mg/kg; Table 2).

Regardless of how the FTY720-P levels are attained in the brain, we speculate that this phosphorylated metabolite may promote endogenous repair mechanisms in the CNS via S1P receptors on glial and/or neuronal cells. It is noteworthy that the nervous system is a major locus for constitutive S1P receptor expression in glial cells and neurons (Ishii et al., 2004). Four of the five known S1P receptor subtypes display a distinct distribution pattern within specific brain regions and cell lineages, as illustrated in Fig. 1. CNS expression of S1P₅, for example, is restricted to oligodendrocytes (Terai et al., 2003) and expressed throughout development to the mature myelin-forming cell. Subsequent to the discovery that S1P acts as an important regulator of cell growth, it has become increasingly clear that this sphingolipid mediator may induce the survival of such cells in the CNS (Ishii et al., 2004). Indeed, recent studies have demonstrated that FTY720-P promotes the survival of oligodendroglial lineage cells in vitro (Jung et al., 2007). Moreover, FTY720-P ligation of S1P receptors on astrocytes (Osinde et al., 2007) could contribute to its known enhancement of endothelial barrier function (Abbott et al., 2006; Baumruker et al., 2007) and possibly to myelination (Talbott et al., 2005; Ishibashi et al., 2006). Further studies are needed to directly elucidate the in vivo consequences of FTY720-P signaling on CNS cells, which is underscored by the recent in vitro observation that S1P activation of S1P₁ and S1P₃ receptors can inhibit gap junctions in astrocytes (Rouach et al., 2006).

The signature feature of FTY720 is its ability to rapidly reduce blood lymphocytes as a consequence of S1P₁-mediated retention in the peripheral lymph nodes (Lo et al., 2005). It is notable that FTY720 not only spares CD4⁺CD25⁺ T-regulatory cells (T_reg) but also induces their functional activity (Daniel et al., 2007). Other mechanisms that act independently of S1P receptors may also be part of the activity of FTY720, such as suppression of eicosanoid production due to inhibition of cytosolic phospholipase A2 (Payne et al., 2007). Importantly, FTY720 neither inhibits the activation of lymphocytes at therapeutically relevant concentrations nor overtly alters their effector function, including antibody responsiveness and T cell cytokine secretion (Brinkmann et al., 2001; Diaz-Romero et al., 2001; Habicht et al., 2006; Kabashima et al., 2006). Reduction of circulating lymphocyte numbers by FTY720 is thought to be the driving force behind its efficacy in allograft rejection (Nikolova et al., 2000) and various autoimmune disease models. Even though the IC₅₀ for FTY720-mediated lymphodepletion is somewhat lower in the rat compared with monkey, which is assumed to be similar to human (Meno-Tetang and Lowe, 2005), we also believe that lowering the lymphocyte count plays a role in EAE as well as MS. Further studies are justified to exactly determine the maximal extent of lymphocyte reduction that may be required for full efficacy with FTY720, assuming that retention of naive and central memory T cells in the peripheral lymph nodes is a necessary event. In any case, our findings complement those of other investigators (Webb et al., 2004) in pointing at a disconnect between a significant decrease in circulating lymphocytes by FTY720 (Fig. 5) but yet a lack of EAE efficacy, i.e., restricting the 0.03 mg/kg dose to 2 weeks of prophylactic treatment and limiting the 0.3 mg/kg dose to days 0 to 6 (Fig. 4); in the latter case, it may be relevant that despite a 75% reduction in the peripheral lymphocytes by day 6 (Fig. 5), the brain/blood ratio was almost 6 and 2 times lower for FTY720 and FTY720-P, respectively, compared with a 21-day treatment (Table 2). Our preclinical data also indicate that a plateau in long-term EAE efficacy was achieved by FTY720 doses in the range of 0.1 to 0.3 mg/kg, compared with no additional improvement with 0.9 mg/kg. These findings may be insightful in determining the maintenance dose for FTY720 in MS patients.

Various exploratory compounds and registered drugs for MS are known to differentially affect the number of circulating lymphocytes. For example, immunosuppressants like mitoxantrone and azathioprine reduce the total white blood cell count via cytotoxic mechanisms (Fernández et al., 2002; Jeffery et al., 2005). Nonmitogenic anti-CD3 mAb blockade induces a profound lymphopenia via alterations in lymphocyte trafficking (Kohm et al., 2005). On the other hand, mAb blockade of α4β1 integrin (Tysabri) induces lymphocytosis (Polman et al., 2006). FTY720 is mechanistically unique to such agents in that its ability to lower lymphocyte counts is not due to cytotoxic effects; in addition, it has the potential for a central influence on S1P receptors in the CNS. During established and ongoing EAE, lymphocytes would already have infiltrated the CNS parenchyma. FTY720 is known to markedly reverse the number of inflammatory cells in the brain and spinal cord upon therapeutic treatment in the DA rat model of EAE (Balatoni et al., 2007). This is further evidence that, over the long term, another aspect of the pathogenesis of demyelinating diseases should be considered as a therapeutic target.

We favor the hypothesis that the ongoing clinical efficacy of FTY720 in CNS diseases like MS (Kappos et al., 2006) is due to a culmination of immunomodulatory mechanisms involving reduction of autoreactive memory T cells, sparing of the protective T_reg population, neuroprotective influences of FTY720-P in the CNS (Fig. 1), and inhibition of inflammatory factors such as eicosanoids. The latter, along with numerous other immune response mediators, was recently confirmed by gene expression in the CNS using the DA rat model of EAE (C.A. Foster and D. Mechtcheriakova, manuscript in preparation). In addition, preliminary studies have already indicated that i.c.v. application of FTY720 during EAE reduces disease severity without significantly affecting the peripheral lymphocyte count (Schubart et al., 2007). Additional experiments, such as using organ culture with living nerve tissue slices exposed to activated lymphocytes, would be necessary to further test the hypothesis that FTY720-mediated suppression of CNS inflammation occurs via a novel mechanism involving direct effects on glial and/or neuronal cells.