SPHINGOSINE-1-PHOSPHATE, ANALOGS AND ANTAGONISTS FOR USE AS MEDICAMENTS

Field of the invention

The present invention relates to compounds for treating and/or preventing cardiac and/or vascular symptoms in individuals suffering from Fabry disease. More generally, the present invention relates to compounds treating and/or preventing vascular and/or cardiac hypertrophy and/or hyperplasia, in particular in smooth muscle tissue / cells, such as vascular smooth muscle tissue. The present invention further relates to SlP and/or its agonists in methods for the treatment of atherosclerosis.

Background of the Invention and Problems to be Solved

Fabry disease was first described more than 100 years ago. Patients have characteristic skin lesions, known as angiokeratoma corporis diffusum universal. It is an X-linked inborn error metabolism, related to glycosphingolipid catabolism abnormality due to a wide variety of mutations in the gene that encodes the lysosomal enzyme α-galactosidase A. α- galactosidase A is one of the hydrolytic enzyme located in the lysosomes. Lysosomal enzymes are very specific regarding their substrate, and a deficient activity of one of them prevents a breakdown of the intermediates, which results in their accumulation. The glycosphingolipid substrate of α-galactosidase A is globotriaosylceramide (Gb3). Gb3 accumulates within tissues and organs of affected patients. Sphingolipids are located in all human tissues, but their concentrations vary from one tissue to another. This is reflected by a variety of manifestation in different organs. The most affected cells include: endothelial cells, pericytes, vascular smooth muscle cells (VSMC), renal epithelial cells, myocardial cells, and dorsal ganglia neuronal cells. The pathogenesis of Fabry disease is not well understood. It is in general assumed that the clinical manifestations are the direct result of Gb3 accumulation within a range of cell types and tissues, leading to cell/organ dysfunction.

Until recently, heterozygous females were considered as asymptomatic « carriers ». Recent analysis of the Fabry Out-come Survey database, has demonstrated that heterozygous females are affected and may even exhibit the full range of disease manifestations MacDermot KD, et al. J Med Gene (2001) 38(11):769-75.

Several reports have focused on the cardiac and vascular complications of Fabry disease. A high prevalence of left ventricular hypertrophy (LVH), which is mainly progressive, homogeneous and concentric, with well-preserved ejection fraction, and mild to moderate degrees of diastolic filling impairement, has been observed in male and female patients with Fabry disease (Linhart A et al. Am Heart J. (2000) 139: 1101-1108).

Macrovascular involvement has been reported in hemizygote patients, this includes a marked and accelerated increase in intima-media thickness (IMT) of the radial artery, a

medium-size muscular artery, and of the common carotid artery (CCA). Boutouyrie P et al. Acta Paediatr Suppl 2002; 91 : 62-66. Barbey F. et al. Arterioscler Thromb Vase Biol. (2006) 26:839-844.

The mechanism leading to progressive LV hypertrophy and increased radial and CCA IMT remains unclear. Only 2% of the increased myocardial mass has been attributed to a storage material. Elleder M. et al. Virchows Arch A Pathol Anat Histopathol (1990) 417:449- 455; von Scheidt W. et al. N Engl J Med. (1991) 324:395-399.

The deposits are also disproportionately small compared to the increase of arterial wall mass. Elleder M. Acta Paediatr Suppl (2003) 92(443):46-53; discussion 45. Barbey F. et al. Arterioscler Thromb Vase Biol. (2006) 26:839-844 have shown that the increase of CCA IMT was present in both males and females. There was observed a strong correlation between CCA IMT and LV mass index.

In view of the current standard of knowledge it is an objective of the present invention to identify the cause and underlying molecular mechanism of the cardiac and vascular implications mentioned above in Fabry patients. Accordingly, it is an objective of the present invention to identify receptors and/or other cellular and extracellular substances implied in the cardiac and vascular implications in Fabry patients and other diseases having similar implications. In particular, it is an objective to control extent of cardiac and vascular symptoms such as hypertrophy and/or hyperplasia by acting upon said receptors, cellular and/or extracellular substances playing a key role in the development of these symptoms.

It is also an objective of the present invention to target specific cellular or extra cellular binding sites in order to reduce and/or prevent cardiac and/or vascular hypertrophy and/or hyperplasia and associated conditions.

It is an objective of the present invention to provide prophylactic and/or therapeutic treatments for hypertrophy of cardiac and vascular tissue, in particular patients suffering from Fabry disease.

It is another objective of the present invention to provide prophylactic and/or therapeutic treatments for cardiac arrhythmia, smooth muscle hypertrophy and/or smooth muscle hyperplasia. It is a still further objective of the present invention to provide prophylactic and/or therapeutic treatments against vascular and/or cardiac symptoms associated with one or more of the following conditions: vascular hypertrophy, vascular hyperplasia, cardiac hypertrophy, cardiac hyperplasia, cardiac arrhythmia, smooth muscle hypertrophy, smooth muscle hyperplasia, Fabry disease, ftbromuscular dysplasis, Buerger's disease, Ehler-Danlos vascular syndrome, Takayasu disease, and vascular stenosis following angioplasty

It is also an objective of the present invention to provide prophylactic and/or therapeutic treatment against atherosclerosis.

Summary of the Invention

Remarkably, the present inventors have purified and identified sphingosine- 1 - phosphate (SlP) as circulating factor capable of inducing hypertrophy and proliferation of vascular smooth muscle cells. Furthermore, the inventors were able to reproduce the effects of sphingosine- 1 -phosphate with agonists, and, importantly, prevent or reduce its effects with antagonists binding to the same receptors. The receptor(s) involved in the vascular effects of SlP have been identified. Furthermore and surprisingly, SlP and its agonists were observed to exhibit anti-atherosclerotic properties.

In general, the present invention provides SlP antagonists for use as medicaments, for the applications reported herein, in particular for treating and/or preventing one or more selected from smooth muscle hypertrophy and/or hyperplasia, vascular and/or cardiac hypertrophy, vascular and/or cardiac hyperplasia, hypertension, and cardiac arrhythmia.

In an aspect, the present invention provides the compounds / SlP antagonists for treating and/or preventing hypertrophy and/or hyperplasia. In another aspect, the present invention provides compounds / SlP antagonists for treating and/or preventing smooth muscle hypertrophy and/or hyperplasia.

In yet another aspect, the present invention provides compounds / SlP antagonists for treating and/or preventing vascular hypertrophy and/or hyperplasia.

In yet another aspect, the present invention provides compounds / SlP antagonists for treating and/or preventing vascular smooth muscle hypertrophy and/or hyperplasia.

In yet another aspect, the present invention provides compounds / SlP antagonists for treating and/or preventing hypertension.

In yet another aspect, the present invention provides compounds / SlP antagonists for treating and/or preventing cardiac arrhythmia. More specifically, the present invention provides a method of treating and/or preventing symptoms and/or conditions, in particular vascular symptoms and/or conditions, occurring in individuals suffering from Fabry disease, said method comprising the step of administering, to said individual, a composition comprising an efficient amount of an sphingosine- 1 -phosphate antagonist. The present invention provides methods (a) of treating and/or preventing cardiovascular conditions in an individual suffering from Fabry disease, and/or (b) of treating and/or preventing symptoms of Fabry disease in an individual suffering from Fabry disease, said methods comprising the step of administering, to said individual, a composition comprising an effective amount of an sphingosine- 1 -phosphate antagonist and/or a compound capable of blocking one or more of the receptors selected from SlPl, S1P2, S1P3, S1P4 and/or S1P5.

The present invention further provides one or more methods selected from the method (a) of treating and/or preventing vascular and/or cardiac hypertrophy and/or hyperplasia, (b) of treating and/or preventing cardiac arrhythmia, (c) treating and/or preventing smooth muscle

hypertrophy and/or hyperplasia, (d) of treating and/or preventing vascular and/or cardiac symptoms of one or more of the diseases selected from Fabry disease, fϊbromuscular dysplasis, Buerger's disease, the Ehler-Danlos vascular syndrome, the Takayasu disease, vascular stenosis following angioplasty, and (e) of treating and/or preventing hypertension; wherein said methods comprise the step of administering, to an individual in need thereof, a composition comprising an effective amount of a compound capable of blocking one or more of the receptors selected from SlPl, S1P2, S1P3, S1P4 and/or S1P5, and/or an sphingosine- 1 - phosphate antagonist.

More generally, the present invention also provides compounds, in particular SlP antagonists, for the treatment and/or prevention of one or more of the diseases selected from Fabry disease, fϊbromuscular dysplasis, Buerger's disease, the Ehler-Danlos vascular syndrome, the Takayasu disease, vascular stenosis following angioplasty. In particular, the present invention provides the treatment and/or prevention of vascular and/or cardiac symptoms of these diseases. More particularly, the present invention provides compounds / SlP antagonists for treating and/or preventing one or more of the conditions selected from: increased intima- media thickness of arteries, ventricular hypertrophy, and diastolic filling impairment.

The present invention also provides SlP and its agonists for use as medicaments, in particular for the treatment and/or prevention of atherosclerosis. The present invention also provides a method of treating and/or preventing atherosclerosis, the method comprising the step of administering, to an individual in need thereof, an effective amount of a compound selected from: sphingosine- 1 -phosphate and/or a sphingosine- 1 -phosphate agonist.

The present invention further provides compounds and methods as defined in the appended claims.

The present invention further provides a method for improving effectiveness and/or efficacy of enzyme administration in a patient undergoing an enzymotherapy, the method comprising the step of administrating to the patient undergoing enzymotherapy an effective amount of a sphingosine- 1 -phosphate (SlP) antagonist. The present invention further provides Sphingosine- 1 -phosphate antagonist for use in enzymo therapies .

The present invention also relates to inhibitors of kinases, in particular MAP kinases, for the same purposes (medicaments, therapeutic and/ prophylactic treatments) as the SlP antagonists as disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

In the figures,

Figure 1 shows the effect of varying concentration of plasma of Fabry patients on proliferation of Vascular Smooth Muscle Cells (VSMC). Figure 2 shows proliferation of Vascular Smooth Muscle Cells (VSMC) in response to exposure to a medium containing the upper lipid extract of plasma of healthy subjects and Fabry patients, respectively.

Figure 3 shows the effect on proliferation of VSMC of various concentrations of plasma extracts of the upper lipid fraction of healthy subjects and Fabry patients, respectively, in the cultivating medium.

Figure 4 shows VSMCs metabolic activity in the presence of extracted plasma after fractionation on Sep-Pak C-18. Plasma of Fabry patients (F) and healthy controls (H) were extracted and fractionated at various concentrations of acetonitrile in water. The graph shows that the main metabolic effect is found in the range of 0 to 20 % acetonitrile. Figure 5 A shows a representative chromatogram of HPLC fractionation on a Cl 8 column of the active fractions (0 and 20% acetonitrile) shown in Figure 4, further fractionated on tendem column.

Figure 5 B shows the effect of various different fractions obtained by HPLC (Figure 5 A) on VSMC proliferation. Figure 6 shows a MS product ion spectrum of [M+H]+ of the highest active fraction

(fractions 8-10 in Figure 5 B) of plasma of Fabry patients. Fractions were analysed with ESI ⁺ - ITMS detector. A mixture of molecular ions with a predominant peak at 380.6 of sphingosine- 1 -phosphate can be seen.

Figure 7 shows sphingosine-1 -phosphate (SlP) levels in plasma of Fabry patients, indicating statistically significantly higher SlP levels in males than in females.

Figure 8 shows sphingosine-1 -phosphate (SlP) levels in plasma of healthy males compared to the male subjects suffering from Fabry disease. The Figure shows significantly higher SlP levels in plasma of Fabry males compared to healthy males.

Figure 9 shows DNA of specific SlP receptors following RT-PCR on RNA of VSMC. Molecular weight markers (M) are shown to the left and housekeeping gene GAPDH to the right. It can be seen that these cells express receptors SlPl and S1P2.

Figure 10 shows the effect of SlP, angiotensin II and endothelin-1 peptide hormones, used alone or in the indicated combinations on VSMC metabolic activity. It can be seen that SlP combined with and either angiotensin II or endothelin-1 synergistically increases metabolic activity.

Figure 11 shows representative images showing the effects of SlP on the enlargement of VSMCs. Cells treated with different concentrations of SlP for 48 h were stained with phalloidin.

Figure 12 shows planar cell surface area of VSMC, determined by image analysis (Figure 11), following exposure to various concentrations of SlP. It can be seen that the cell surface, which can be used as an indicator of hypertrophy, is dependent on the applied SlP concentration. Figures 13 A shows increase of live cells with increased SlP concentration. The maximum differences were observed at 1 μM and 10 μM.

Figure 13 B shows percent of live cells increased with increased concentration of SlP. Forward scatter signal (FSC) mean fluorescence intensity (MFI) decreased with SlP concentration increase. At 10 μM, the differences are statistically significant (p< 0.001) for both percent of live cells and MFI.

Figure 14 shows the mean +SE of aortic intima-media thickness (IMT) of mice obtained by image measurement analysis. It can be seen that Mice treated with SlP and the mouse-model B6 for the Fabry disease have increased IMT if compared to healthy wild type mice. Figures 15 A-F show representative histological sections of the aortas of control, SlP treated and Fabry mice (A, B, and C, respectively). Figures D-F are magnifications of the box shown in Figures 15 A-C, respectively.

Figure 16 A shows cardiac weight index (cardiac weight/body weight) of mice as in Figure 14. SlP treatment induces statistically significant cardiac hypertrophy, while Fabry mice showed a clear tendency for cadiac hyprtrophy without reaching statistically significance.

Figure 16 B shows mean arterial blood pressure +SE in mice as in Figure 14. No statistical significance for differencs was observed between the three groups. A tendency for lower blood pressure is oserved in both groups SlP treated and Fabry. Figures 17 A-C show representative histological cardiac sections of the aortas of control, SlP treated and Fabry mice (A, B, and C, respectively), stained with hematoxylin and eosin. Cardiac hypertrophy can be underlined in both sections SlP treated and Fabry, however for Fabry it is less pronounced.

Detailed Description of the Preferred Embodiments

The present invention relates to compounds that are useful as medicaments in the treatment and/or prevention of various vascular and/or cardiac conditions mainly related to hypertrophy, hyperplasia and/or of cardiac arrhythmia.

For the purpose of the present specification, the use of the plural form when referring to "compounds" refers to a specific compound as well as to chemically different compounds, including combinations of different compounds. Furthermore, for the purpose of the present specification, the terms "comprising" and "comprise" mean "Including, amongst others".

These terms are not intended to mean "consist only of.

The compounds of the present invention are preferably defined by their capacity of blocking one or more of the receptors selected from SlPl, S1P2, S1P3, S1P4 and/or the S1P5 in an individual. The expressions "capable of blocking", "blocking" or "block", for the purpose of the present invention, are not intended to include only the case where any of the indicated receptors is totally and/or permanently blocked by the compound. The expression "capable of blocking" refers to a antagonistic or partially agonistic compound, in the presence of which the hypertrophic and/or hyperplastic effect, in particular S IP-mediated effects, are reduced. Accordingly, the compounds of the present invention are antagonists of SlP, reducing and/or blocking at least to some extent and at least temporarily the activation of the receptor by SlP. The compounds of the present invention also include partial agonists, which have only a partial efficacy at the receptor relative to a full agonist or SlP, the partial agonist acting as a competitive antagonist in presence of SlP and/or a full agonist and thus reducing the effect of SlP and/or a full agonist. The capacity of a compound of blocking any of the indicated receptors is preferably assessed in vitro, in presence of cells, preferably smooth muscle cells, expressing such a receptor, according to the procedure detailed in Examples 1, 6 and 11 below. However, the capacity of blocking the receptor is preferably maintained when administered to an individual and the compound blocks the receptor in the individual.

The receptors SlPl, S1P2, S1P3, S1P4 and S1P5 are known from the literature. SlPl - 5 stands for sphingolipid G-protein coupled receptor 1-5, respectively. Preferably, human variants of these receptors are referred to herein, however, variants isolated from animals or obtained from recombination are also included. SlPl, S1P2, S1P3, S1P4 and S1P5 have been described under accession numbers NMJ)01400, NM_004230, NM_005226, NM_3775, and NMJB0760, respectively.

Preferably, the compound of the present invention is capable of blocking the SlPl and/or the S1P2 receptor in said individual. More preferably, the compound is capable of specifically blocking SlPl and/or S1P2. Most preferably, the compound is capable of specifically blocking S1P2. The expression "specifically blocking", for the purpose of the present invention, refers to the property of a compound to more specifically block one or a couple (1-4) of the five SPl receptors. Alternatively, said SlPl and/or S1P2 receptor is blocked non-specifically, that is, to the same extent as other SlP-receptors by the compound.

According to a preferred embodiment of the present invention, said compound capable of blocking one or more of the receptors selected from SlPl, S1P2, S1P3, S1P4 and/or the S1P5 is a sphingosine-1 -phosphate antagonist.

Specific examples of compounds of the present invention are provided for the purpose of illustrating the present invention. Accordingly, VPC23019 ((i?)-Phosphoric acid mono-[2- amino-2-(3-octyl-phenylcarbamoyl)-ethyl] ester) is a competitive antagonist specifically blocking SlPl (25nM) and S1P3 (30OnM) (Kim et al; J Pharm Pharmacol, 2007 JuI; 59(7):1035-41).

VPC25239 is also a competitive antagonist specifically blocking SlPl and S1P3, but is about 10 times more specific at the S1P3 receptor. (J. Biol Chem. 2005 Mar 18; 280(11):9833-41).

Sanna et al. (Nat. Chem Biol 2006 Aug 2(8):434-41 disclose (i?)-3-amino-4-(3- hexylphenylamino)-4-oxobutylphosphoric acid as an antagonist specifically acting on SlPl.

Preferably, the compound of the present invention is JTE013 (l-[l,3-dimethyl-4-(2- methylethyl)- 1 H-pyrazolo [3 ,4-b]pyridin-6-yl] -4-(3 ,5 -dichloro-4-pyridinyl)-semicarbazide), which is a SlP antagonist specifically blocking the S1P2 receptor (FASEB J 2007 Sept ; 21(11) :2818-28, Cardiovasc res 2003 April ; 58(1) : 170-7). The compound VPCO 1091 is an S1P3 antagonist, but an SlPl agonist, and can thus also be used as an agonist against the SlPl for the prupose of the present invention. VPC01091 relates to the compounds ((li?,3 _l S)-l-Amino-3-(4-octylphenyl)cyclopentyl)methyl, (( IR,3R)- 1 -Amino-3-(4-octylphenyl)cyclopentyl)methanol Hydrochloride, ((Ii?, 3S)- 1 -Amino- 3-(4-octylphenyl)cyclopentyl)methyl Dihydrogen Phosphate, ((li?,3i?)-l-Amino-3-(4- octylphenyl)cyclopentyl) methanol Dihydrogen Phosphate, as well as derivatives prepared on the basis af the aforementioned compounds as well as pharmaceutically acceptable salts of the aforementioned.

The compound VPC44116 (3-Amino-3-(3-octylphenylcarbamoyl)propyl]-phosphonic acid) is an SlPl antagonist. The R stereoisomer of SlP also functions as an SlPl antagonist.

The S IP-antagonist for treating or preventing cardiac and vascular implications, such as hyperplasmia and hypertrophy in Fabry disease, for example, may be administered in quantities in the range of 0.1-3, preferably 1 to 2 mg/kg/day. Optimal quantities need, however, to be defined by the skilled person. The compounds of the present invention are useful in the treatment in the vascular and/or cardiac implications reported herein, in particular those occurring in Fabry disease. Accordingly, the compound are useful in treating, preventing and/or in any way alleviating, for example, ventricular hypertrophy, in particular left ventricular hypertrophy, and diastolic filling impairment. Macro vascular complications and/or conditions observed in Fabry patients, which can be treated or prevented with the compounds of the present invention, include increased intima-media thickness of arteries, such as muscular and elastic arteries, for example of the radial artery and the common carotid artery, for example.

The method of treating and/or preventing symptoms of Fabry disease and/or cardio vascular conditions in individuals suffering from Fabry disease preferably further comprises the step of administering to said individual, an effective amount of an enzyme catalysing the hydrolysing the terminal alpha-galactosyl moieties from glycolipids and/or glycoproteins. For example the method comprises the step of administering to said individual, an effective

amount of agalsidase, alpha-galactosidase and/or functional derivatives of any of the aforementioned. Such enzymes, for example agalsidase alpha or agalsidase beta, are commercially and are known, for example, under tradenames Replagal® and Fabrazyme®. These enzymes are preferably administered parenterally and have the purpose of fulfilling the function of alpha-Galactosidase A, encoded by the GLA-gene. The enzyme is deficient and/or at least partially unfuctional in individuals suffering from Fabry disease. By providing enzyme treatment along with administration of an SlP analog as defined herein, it is believed that symptoms of the disease can be treated and/or alleviated more holistically.

The present invention is also based on the surprising finding that the SlP activity is mediated by MAP kinases. Accordingly, the present invention provides the use of inhibitors of such kinases to the same end as the S IP-antagonists (the latter blocking any one selected from SlPl, S1P2, S1P3, S1P4, and S1P5). "To the same end" refers to all therapeutic, prophylactic and/or medical purposes as provided herein. Examples of MAP kinases are the MTOR, P38 and/or ERK kinase. Examples of MTOR inhibitors are Rapamycin and Everolimus and derivatives of these; an example of a P38 inhibitor is SB203580 and its derivatives; an example of an ERK inhibitor is PD098059 and its derivatives.

The present invention is also based on the surprising finding that SlP and peptide hormones angiotensin II and/or endothelin, such as endothelin 1, 2 and 3, but preferably endothelin-1, synergistically increase cell metabolic activity, hypertrophy and/or cell proliferation. Accordingly, the present invention also provides a method wherein, besides the SlP antagonists, an angiotensin II antagonist, or an endothelin antagonist, preferably an endothelin-1 antagonist, or a combination of both of them, is administered to an individual, preferably simultaneously.

A large quantity of different angiotensin II antagonists has been described in the literature. For the purpose of providing a few examples of compounds encompassed by the present invention, angiotensin II antagonists blocking Angiotensin II receptor I, Candesartan® and Losartan®, are mentioned. Examples of another type of angiotensin II blockers, namely those inhibiting ACE and rennin, thus indirectly inhibiting angiotensin generation, include Captopril® and Peindotril®. Similarly, a large quantity of different endothelin-A receptor antagonists has been described in the literature. Only a few examples are provided for illustrating the general applicability of the present invention. Examples of endothelin-A receptor antagonist include Bosantan®; BQ123; Atrasentan®; ABT-627; YM 598; AZD4054; S-0139 (27-0-3-[2-(3- carboxy-acryloylamino)-5-hydroxyphenyl]-acryloyloxy myricerone sodium salt) described by Iwasaki T. et al. (Eur J Pharmacol. 2000 JuI 21;400(2-3):255-62); YM598 ((E)-N-[O- methoxy-5-(2-methoxyphenoxy)[2,2'-bipyrimidin]-4-yl]-2-pheny lethenesulfonamide monopotassium salt) described by Yuyama H. et al. (Vascul Pharmacol. 2005 Jun;43(l):40- 6).

Examples of endotheline-B receptor antagonist include Bosantan®; BQ788; A- 182086; BQ788; BQ123 (cycloP-Trp-D-Asp-Pro-D-Val-Leu]); BQ-153 (Fukuroda T. et al, Life Sci. 1992;50(15):PL107-12); ABT 627 ([2R-(2α,3β,4α)]-4-(l,3-benzodioxol-5-yl)-l-[2- (dibutylamino)-2-oxoethyl] -2-(4-methoxyphenyl)-3 -pyrrolidinecarboxylic acid, monohydrochloride, described by Li L. et al. (Hypertension. 2003 Mar;41(3 Pt 2):663-8); A- 147627 ([2i?-(2α,3β,4α)]-4-(l,3-benzodioxo-5-yl)-l-[2dibutyl-ami no-2-oxoethyl]-2-(4- methoxyphenyl)-3-pyrrol-idinecarboxylic acid described by Sasser JM. et al. (J Am Soc Nephrol. 2007 Jan;18(l):143-54), for example.

The review article of Dhaun et al lists selective and mixed endothelin receptor antagonism in cardiovascular disease (Dhaun N. et al., Trends Pharmacol Sci. 2007 Nov;28(l l):573-9)

The angiotensin II antagonist may be administered in quantities in quantities in the range of 10-500 mg/day, depending on various factors, for example on which sartan is used.

The endothelin A or B antagonist may be administered in quantities in the range of 0.5-2 mg/kg/day, for example, depending on which antagonist is used.

The present invention also relates to compounds for treating and/or preventing atherosclerosis. Accordingly, the present inventors found that SlP and its agonists, including partial agonists, have the property of protecting a subject from developing atherosclerotic plaques. Preferably, the SlP agonist used in the treatment and/or prevention of atherosclerosis is capable of activating one or more of the receptors selected from SlPl, S1P2, S1P3, S1P4 and/or the S1P5. More preferably, the S IP-agonist activates these receptors to the same or similar extent as SlP. However, according to a preferable embodiment, the SlP agonist specifically activates one or more of the receptors selected from SlPl and S1P3. The capacity of an agonist of "activating" a receptor, for the purpose of the present invention refers to the contrary of blocking, that is, a given compound that triggers the same or a similar response in the cell as the endogenous ligand, SlP. The agonist thus mimics the action of SlP that binds to the same receptor.

According to a preferred embodiment, the SlP agonist is capable of activating one or more of the receptors selected from SlPl and S1P3. Preferably, it selectively activates the SlPl and/or S1P3 receptor, which means that the agonist is more active on the SlPl and/or S1P3 receptor than on any other of the receptors selected from S1P2, S1P4 and/or S1P5. More preferably, the S IP-agonist has preference on the SlPl and/ or S1P3 receptor if compared to its activity on the S1P2 or S1P5 receptor. An SlP antagonist, which is specific for SlPl and/or S1P3 but does not activate S1P2 and/or S1P5, the cardiovascular implications of SlP reported herein may be selectively avoided and the beneficial anti-atherosclerotic properties of SlP may be obtained.

According to an embodiment, the SlP agonist used for the treatment and/or prevention of atherosclerosis is FTY720 (2-amino-2 [2 - (4-octylphenyl) ethy-1] -1,3 propanediol hydrochloride), which is known to act as agonist on SlP receptors SlPl, S1P3, S1P4, and S1P5, thus on 4 of the 5 known receptors. The phosphorylated form of FTY720, shortly referred to as FTY720-P is an agonist that is equipotent to S1P3, S1P4 and S1P5 receptors, with one log order more potent at SlPl. According to another embodiment, the SlP agonist is AUY954, which is a nonselective SlP agonist that can be orally administered (Chem Biol, 2006; 13(11): 1227-34).

SW-2871 (5-(4-Phenyl-5-(trifluoromethyl)thiophen-2-yl)-3-(3-(trifluo romethyl) phenyl)- 1,2,4-oxadiazole), disclosed by Sanna et al. (Nat Chem Biol 2006 Aug ;2(8) : 434-41) is another SlPl agonist that can be used for the purpose of the present invention.

An SlP agonist being more active on the SlPl and S1P3 receptors is VPC24191 ((S)- phosphoric acid mono-[2-amino-3-(4-octyl-phenylamino)-propyl] ester). An SlP agonist more specifically activating S1P2 is DS-SG-44 ((2S,3R)-2-amino-3-hydroxy-4-(4- octylphenyl)butyl phosphoric acid). Both, VPC24191 and DS-SG-44 are disclosed by Kim K. et al. (Pharm Pharmacol (2007) 59(7): 1035-41).

Another SlP agonist specific for SlPl and which can be used for the purpose of the present invention is SEW2871 (5-[4-phenyl-5-(trifluoromethyl)-2-thienyl]-3-[3- (trifluoromethyl )phenyl]- 1,2,4-oxadiazole), described by Y-HH. Lien, K-C. Yong, C. Cho, S. Igarashi and L-W. Lai, Kidney International (2006) 69, 1601-1608.

As mentioned further above, VPCO 1091 may also act as an agonist on SlPl and can thus be used for the purpose of an agonist as indicated above.

SlP or the SlP agonist for treating and/or preventing atherosclerosis may be administered in quantities that need optimisation by the skilled person, but which may be situated in the range of 0.1-3, preferably 1 to 2 mg/kg/day.

The present invention also relates to a method of screening for a compound having anti-atheroschlerotic properties. In principle, the method comprises the steps of checking the properties of a compound to be screened to function as an agonist of SlP, preferably an agonist that specifically activates SlPl and/or S1P2. To demonstrate that the compound is a molecule that acts as an SlP agonist, it should interact with the receptor (SlPl and/or S1P2) with the same biological effect as SlP. A suitable procedure for demonstrating such an interaction is disclosed by Tani M, Kawakami A, Nagai M, Shimokado K, Kondo K, Yoshida M. Sphingosine 1 -phosphate (SlP) inhibits monocyte-endothelial cell interaction by regulating of RhoA activity. FEBS Lett. 2007;581(24):4621-6. The present specification refers to compounds that are agonists and/or antagonists of

SlP. The present invention also encompasses pharmaceutically acceptable salts as well as derivatives of such compounds, as long as they retain their agonistic and/or antagonistic properties, respectively. For example, the present invention encompasses a compound in

which atoms or functional groups have been substituted (for example hydrogen by halogen or vice versa) and/or compounds that have been alkanoylated, including in particular acetylates of such compounds. In general, a derivative as referred to herein is a compound retaining the basic structure of a compound mentioned herein but which may contain further substituents, but also compounds in which substituents have been replaced by chemically similar substituents or in which substituents that do not have a substantial impact on the activity of the compound have been omitted. Derivative, as incorporated herein, may also be designed to exhibit higher activity, higher selectivity towards a specific receptor, or improved bioavailability at the target cells, for example. The present invention provides a method for improving effectiveness and/or efficacy of enzyme administration in a patient undergoing an enzymotherapy. According to the inventors of the present invention, administration of an effective amount of an SlP antagonist as defined herein improves effectiveness and/or efficacy of an enzymotherapy by improving access of parenterally administered enzymes to the target tissue. The SlP antagonist is administered in sufficient amounts so as to reduce the effect of SlP, the latter tending to diminish permeability of vessels. By administering the SlP antagonist, permeability of vessels is improved, probably by reorganisation of endothelial cell junctions, and the enzymes arrive at target tissues and cells in higher quantities. In this way, effectiveness and/or efficacy of enzyme therapies are increase. This may result in administration of less enzymes while obtaining the same effect as when there was no SlP antagonist administered. The invention may also be used to diminish the frequency of enzyme administration due to the increased effectiveness / efficacy. The present invention may also be used so as to obtain better results with conventional quantities of administered enzymes. The SlP antagonist may be used in enzymotherapies in order to improve permeability of vascular endothelial cells to the enzymes. For the purpose of the present invention, an enzyme administered in enzymotherapy is preferably a recombinent enzyme. Preferably, the enzyme is administered parenterally. The SlP antagonist may be administered parenterally and/or enterally. The SlP antagonist and the enzyme may be administered simultaneously or sequentially. For example, the SlP antagonist may be administered before enzyme administration. In sequential administration, the time delay between administration of SlP antagonist and the enzymes is to be determined by the skilled person in view of obtaining the best results. For example, the SlP may be administered 24 hours to 0.2 hours, 10 hours-0.5 hours, 5 hours to 1 hour before administration of the enzyme. The enzymotherapy may be any kind of therapy by administrating active enzymes. It is not restricted to, but encompasses, the treatment of Fabry disease by enzyme treatment (Gaucher, Cystinose, MPS I (Hurler, Hurler-Scheie, Scheie), MPS VI (Maroteaux-Lamy), Glycogenose type II (Pompe disease), MPS II (Hunter).

The present invention relates to one or more compounds for use in a method of treatment of the human or animal body. A method of treatment may comprise administering

to such an individual a therapeutically-effective amount of the compounds of the present invention, preferably in the form of a pharmaceutical composition. The term treatment as used herein in the context of treating a condition and/or cardiovascular implication, pertains generally to treatment and therapy, whether of a human or animal (e.g. in veterinary applications), in which some therapeutic effect is achieved, for example, the inhibition of the progress of the condition, and includes a reduction in the rate of the progress, a halt in the rate of the progress, amelioration of the condition, cure of the condition, alleviation of symptoms and reduction of symptoms. The condition and/or implication may be associated with suffering, from psychological and/or physical pain, with the individual being in need of a treatment. However, the condition and/or implication need not necessarily be consciously perceived by an individual, but generally has at least some undesired or negative incidence on the overall health of the individual. The condition may increase mortality or increase the susceptibility of suffering from other, in principle unrelated diseases and/or conditions. Treatment as a prophylactic measure (i.e. prophylaxis) is also included. The compound of the present invention may be administered parenterally and/or enterally, or in any other suitable way of administration. While parenteral administration is presently the preferred way of administration under consideration of obtaining a quick, systemic effect, the skilled person may establish other, more convenient routes of administration, depending, amongst other, on the specific chemical nature of the compound to be administered.

Preferably, the compound(s) is (are) administered in the form of a pharmaceutical composition (e.g. formulation) adapted to the way of administration, said composition comprising at least one active compound together with at least one or more pharmaceutically carriers, buffers, as mentioned in WO 2006/018649, p. 16-20, "formulations", which document is expressly incorporated herein by reference. The present invention thus also provides pharmaceutical compositions comprising the compounds disclosed herein.The invention is further elaborated by the following examples. The examples are provided for purposes of illustration to a person skilled in the art and are not intended to be limiting the scope of the invention as described in the claims. Thus, the invention should not be construed as being limited to the examples provided, but should be construed to encompass any and all variations that become evident as a result of the teaching provided herein.

Examples

In the present examples, results are represented for the purpose of statistics as mean ± SEM. GraphPad Prism Software (San Diego, USA)) was used for statistical analysis. Oneway analysis of variance (ANOVA) was performed using unpaired student's t test. Differences were considered significant at a value of p<0.05.

Example 1 : Growth Promoting properties of Fabry Patient Plasma

In order to bring evidence for the presence of growth promoting agent in plasma originating from Fabry patients vascular smooth muscle cells (VSMC) proliferative assays were performed to quantify cells metabolic activities. Supplier indications indicated that metabolic activity is related to cells proliferation. Cells were incubated with DMEM containing different percent of Fabry patient plasma.

VSMC Metabolic Activity Assay:

Vascular smooth muscle cells from Wistar Kyoto rat aorta were isolated by collagenase digestion. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum and antibiotics. VSMCs prepared from these rats were not contaminated with fibroblast or endothelial cells as evidenced by positive immunostaining of smooth muscle α-actin with fluorescein isothiocyanate-conjugated α-actin antibody. VSMCs with early passage (<15) were seeded on 96 well plate (2000 cells/well) and incubated for 24 hours. Subsequently, cells were washed three times with PBS and incubated with 100 μl DMEM containing increased percent of plasma of patient for 16-18 hours, and assayed for cell proliferation.

Cell Proliferation Assay: VSMC proliferation was quantified using the Cell Titer 96 AQueous non-radioactive cell proliferation assay kit (Promega). This assay is based on measuring the cellular conversion of the colorimetric reagent 3, 4-(5-dimethylthiazol-2yl)-5-

(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt into formazan by dehydrogenase enzymes found only in metabolically active, proliferating cells. The absorbance was measured at 490 ran using a 96-well enzyme-linked immunosorbent assay plate reader

The result is shown in Figure 1. It can be seen that culture medium containing 12.5 % plasma (plasma volume / Total volume: DMEM + plasma) had the highest positive effect on in vitro vascular smooth muscle cell proliferation.

Example 2: Preparation and Comparative Testing of Upper Fractions of Plasma

Plasma of Fabry patients and healthy subjects was subjected to classic lipid extraction before testing of metabolic effect as in Example 1.

Lipid fraction extraction: Fresh heparinized human blood was centrifuged at 3000g for 5 min at 4 ⁰ C and the cell free plasma was removed and recentrifuged atl4 000 for 5 min at

4°C to remove any debris. The obtained plasma was aliquoted and stored at -2O ⁰ C until use.

Two tubes of Fifty microliter of each plasma sample was extracted with 20 volume of

CHC13/Methanol 2:1 for 15 min at 37°C with rocking and then centrifuged at 14 000 g for

5min at 4°C. The supernatants were transferred to fresh tubes and partitioned against 1/5 volume of distilled water at 4 ⁰ C for over night for the two phases separation. Following centrifugation, the upper phase was removed and dried under nitrogen. The dry residue was resuspended in 400 microliter of serum free medium and 100 microliter applied on cultured VSMC seeded on 96 well plate. Cells were cultured for 18 hours then assayed with proliferation assay kit (Promega) as indicated in Example 1.

The results can be seen in Figure 2. In can be seen that cells incubated with the upper fraction of human plasma of Fabry patients displayed a significantly increased metabolic activity (p=0.008) when compared to control subjects.

When the upper fraction as obtained above was diluted with culture medium (DMEM containing 10% fetal bovine serum and antibiotics) at various concentrations, the maximum differences of cell proliferate effect could be observed at 25% (v/v) of plasma, as can be seen in Figure 3.

In order to prove that the observed proliferation is specific and related to a possible presence of additional factor in Fabry plasma, the three distinct MAPK pathways were investigated with extracted patient plasma using PD098059, SB203580 and LJNKIl MAPK specific inhibitors. Accordingly, cells were seeded in 96 well plates for 24 hours. Next they were pre-treated with 50 μl of DMEM twice concentrated MAPK inhibitors PD098059, SB203580 and LJNKIl for one hour before the addition of 50 μl twice concentrated dried extracted plasma. The final concentrations of MAPK inhibitors were 50 μM, 10 μM , and 5 μM for PD098059, SB203580 and L-JNKIl, respectively.

Table 1 shows that only SB203580 p38 inhibitor has a specific inhibitor effect of 1% in healthy subject versus 12% in Fabry. This indicates that the identified growth promoting effect is specific and mainly related to p38 MAPK pathway.

Table 1: Effect of Various MAPK Pathway Inhibitors on Growth Promoting Properties of Plasma of Fabr Patients

Example 3: Testing of Various Solid-Phase Extracts

In order to further purify a putative growth promoting agent, plasma from Fabry patients, the upper fraction obtained in Example 2 was further fractionated by solid-phase extraction. Accordingly, 5 ml of heparinized plasma pooled form 5 patients was distributed in 2 ml ependorf tubes in 50 μl aliquots and then extracted by classic lipid extraction as described in Example 2. Each of 20 tubes (1 ml of plasma) of dried residues were resuspended in 1 ml water containing trifluoroacetic acid at l%o (v/v,lμl/lml) and applied on a Cl 8 Sep-Pak® cartridge, which was pre-equilibrated with water trifluoroacetic acid l%o (v/v,lμl/lml). The bound material was eluted with different concentration of acetonirile 0%, 20%, 40% and 80% (v/v). An aliquot of 25 μl from each fraction was dried and re-suspended in DMEM and tested on VSMC as described in Example 1 above. This was carried out in triplicate.

The tested comparative proliferation assay shows, in Figure 4, that the fractions of 0 and 20% acetonitrile possesses the highest prolifrerative effect on VSMC.

Example 4: HPLC Fractionation and Identification of Sphingosine- 1 -Phosphate

The highly active fractions from Fabry extracted plasma 0% and 20% (Example 3) were mixed, dried and resuspended in 40% acetoniril/60% (v/v) water with trifluoroacetic acid in water (v/v,lμl/lml). The obtained material was fractionated by gel filtration high performance liquid chromatography using two columns (two protein-pak 60 in tandem, Macrosphere GPC 60 A 7u Alltech) at flow rate 0.5 ml/min so as to obtain a total of 30 different fractions. An aliquot of 25 μl from each fraction was dried and resuspended in DMEM then tested on VSMC as described in Example 1. This was carried out in triplicate and corresponding control fractions of healthy subjects were conducted in parallel (also in triplicate). The tendem column was calibrated with somatostatin-28 (3000 Da) and somatostatin- 14 (1500 Da).

Those fractions that showed high VSCM-proliferation activity, if compared to the corresponding fractions of healthy subjects (fractions 25-30) were mixed, dried and resuspended in CHC13/Methanol (70/30%). The obtained material was fractionated by HPLC on a Vydac Cn column. Again, corresponding fractions of healthy subjects were used as controls. Elution was performed in isocratic CHC13/methanol (70/30%) for 15 min followed with a linear gradient of methanol at a flow rate of 1 ml/min from 30% to 60% in 5 min, so as to obtain 20 different fractions. A representative chromatogram of theis HPLC is shown in Figure 5 A. Proliferative activity on VSMC of all fractions were again tested, in triplicate, for comparison. As can be seen in Figure 5 B, the comparative VSMC metabolic activity shows a comparatively higher activity in fractions 8-10 and lower activity in fractions 11 and 12.

The highest active fractions 8-11 of the HPLC fractionation on the C 18 column were analysed by ESI-MS (electron spray ion mass spectrum). These fractions 8-10 were mixed,

dried, resuspended in acetoniril/acetic acid and analysed by direct infusion elecrospray ionisation-mass spectometry (ESI-MS).

Direct infusion ESI-MS analysis was performed with a triple-stage quadrupole mass spectrometer, operating in positive ion mode with data collected from 50 to 1000 amu. Analyses were performed by direct infusion of acetoniril/acetic acid resuspended dried collected fraction from HPLC using a seringue pump at 20 μl/min. As is shown in Figure 6, the active fraction showed a mixture of molecular ions with a higher abundance with 380.6. Data base analysis results in the identification of sphingosine- 1 phosphate as possible molecule involved in VSMC proliferation. Example 5: Circulating SlP in Fabry Patients vs Healthy Subjects

In order to prove that sphingosine- 1 phosphate is the agent involved in Fabry patient's cardiac and vascular hypertrophy, a quantification of this molecule in plasma of Fabry and healthy subjects was performed as described in the literature: Butter JJ. et al.: A rapid and validated HPLC method to quantify sphingosine 1 -phosphate in human plasma using solid- phase extraction followed by derivatization with fluorescence detection. (J Chromatogr B Analyt Technol Biomed Life Sci. 2005: 824(l-2):65-70.).

Clinical characteristics of the subject populations have been described earlier (Barbey et al 2006). A group of Fabry patients (n=24) with 12 females and 12 males and a control group 30 healthy subjects (n=30) with 15 females and 15 males was studied. The quantification of SlP in the plasma of the healthy population showed there is no statistically significant difference in between females and males (161.5±8.40 ng/ml versus 175.3±8.32 ng/ml). A small tendency for higher values can be noted for males.

When this analysis is performed in Fabry patients only, higher values were observed in both populations females and males (177.20±11.91 versus 264.80±37) and these are significantly different between gender (p= 0.03), as can be seen from Figure 7.

When healthy female group was compared to Fabry female group the difference was not statistically significant, but a higher values tendency is clearly present (161.5±8.40 versus 177.20±11.9 ng/ml, p=0.23).

Figure 8 shows that in Fabry males sphingosine- 1 phosphate levels are 1.6 time higher that those observed in healthy males (175.3±8.32 versus 280.2±32.71 ng/ml). The statistical analysis showed a highly significant difference (p=0.002). Table 2 summarises the outcome of SlP plasma levels determined in different healthy and Fabry male and Female populations.

Table 2: Plasma sphingosine- 1 phosphate (SlP) levels in controls (healthy subjects) versus Fabry patients, expressed as Mean SlP ± SEM.

Controls (ng/ml) Fabry (ng/ml) p *

Male+Female 173.7±5.49 (n=22) 223.7±21.38 (n=18) 0.017

(MC+FC)/(MF+FF)

Male 175.3±8.32 (n=12) 264.8±37 (n=9) 0.002 MC/MF

Female 161.5±8 .41 (n=12) 177.2±11 .91 (n=10 ) 0.283 FC/FF

0.255 0.031 MC/FC MF/FF

*p<0.05 was considered statisticallly significant

MC: male controls, FC: female controls, MF: male Fabry, FF: female Fabry

From Table 2 it can be seen that statistic significance is also present when considering the entire Fabry-group, including both males and females compared to healthy subjects (males + females) (p=0.017).

These results show that sphingosine-1 phosphate is highly expressed in the Fabry population and this is related to disease manifestation, which is expressed mainly in males.

Example 6: Effect of S lP on Vascular Smooth Muscle Cells Proliferation

The observed circulating sphingosine-1 phosphate concentration (Example 5) can be estimated to be 0.45 μM in healthy subjects and 0.6 μM in Fabry patients. Because the concentration in direct contact with VSMC could not be assessed, we have added sphingosine-1 phosphate to VSMC at different concentrations [0, 1 nM, 10 nM, 100 nM, 1 μM and 10 μM]. Metabolic activity was assessed with MTS (Example 1).

Table 3, first column on the left shows that sphingosine-1 phosphate metabolic activity enhancement on VSMC is concentration dependent. A maximum, statistically significant enhancement of proliferation was observed at 10 μM SlP (p=0.037).

Table 2

SlP mm SlPwiih S IP with LNJKI l SB203580

O nM 1.40S± 0.015 1.338 ± 0.041 1.347 ± 0.053 1.345 ± 0.041 p=0 438 p=0 607 p=0 509

10O nM 1.296 ± 0.060 1.397 ±0.08S 1.244 ± 0.078 1.062 ± 0.065 p=0 602 p=0 666 p=0.923 p=0.129 lμJM 1 495 ± 0 035 1.500 ±0 026 1 477 ± 0 026 1 107 ±0 027 p=0 750 p=0 999 p=0 991 p=0.003

10 μM 1 732 ±0 093 1.851 ±0 144 1 699 ±0 035 1 444 ±0 077 p=ø 037 p=0 713 p=0 988 p=0.144

Example 7: Assessment of SlP Receptors in VSMC

In order to determine, which one of the S IP-receptors is expressed in VSMC used herein as a model for proliferation and hypertrophy, total RNA was extracted from VSMC with the qiagen RNeasy kit (Qiagen).

Genomic DNA was removed by treatment with Dnase I. RNA was then purified by phenol/chloroform extraction. Quantitation of RNA was performed by determining the absorbance at 280 nm and 260 nm. RT-PCR was carried out from 0.5 μg of total RNA with one step Qiagen® RT-PCR kit. RT-PCR was performed by incubating total RNA with forward and reverse primers pairs for SlPl, S1P2, S1P3, S1P4 and S1P5 receptors. Primers and programs for these receptors identification were as follows:

SlPl for: 5 '-GCCTAAGGACTAACTATGCTGCTGTAA-S ' (SEQ ID NO: 1); SlPl rev: 5' -GAGTGTGACCAAGGACAGTCATA-S '(200bp, human cDNA) (SEQ ID NO: 2). S1P2 for: 5 '-CGGAGGCACTGACTAATCAGATT-S ' (SEQ ID NO: 3); S1P2 rev:5'- TCCCAGCACTCAGGACACAGTTA-3' (200bp, human cDNA) (SEQ ID NO: 4). S1P3 for: 5 '-AACCACAACTCCGAGATCCAT-S ' (SEQ ID NO: 5); S1P3 rev:5'- TTGAAGAGGATGGAGC ACTCCTT-3' (200bp, human cDNA) (SEQ ID NO: 6). S1P4 for: 5'-TRCTSAASACSGTGCTGATGAT-3'(SEQ ID NO: 7); S1P4 rev:5'- CKGCTGCGGAAGGAGTAGATGA-3' (200bp, human cDNA) (SEQ ID NO: 8). S1P5 for: 5 '-CGTGTCCTGTGCTTCTGCAA-S ' (SEQ ID NO: 9); S1P5 rev:5'- CTGCAAACTGTTGGAGGAGTCTT-3' (200bp, human cDNA) (SEQ ID NO: 10);

All PCR reactions were performed in Biometra thermocycler. The program performed for RT-PCR amplification include an initial period of 30 min at 50 ⁰ C for RT reaction, followed by 15 min at 95 ⁰ C, followed by 30 cycles of 30 s denaturation at 95 ⁰ C, 30s annealing at 60 ⁰ C, and finally 1 min at 72°C. The program was terminated with a period of 7 min at 72°C.

Figure 9 shows that VSMC (Example 1) express two receptors, S1P2 and S1P2. Accordingly, S IP-mediated cardiovascular implications may be alleviated with SlP antagonists specifically blocking these two receptors.

Example 7: Effect of SlP in vitro in Presence of Inhibitors

The effect of PD098059, SB203580 and LJNKIl MAPK specific inhibitors on the action of sphingosine- 1 -phosphate on VSMC was evaluated. These inhibitors were added at concentrations of 100 μM, 20 μM and 10 μM, respectively. Statistical analysis of the obtained data indicate that the significance is obtained when SB203580 p38 inhibitor is used and only at high concentration of SlP [100 nM, 1 μM and 10 μM] Table 3. This confirmed our previous obtained data with the extracted Fabry plasma (Example 1) where a major effect with the SB203580 p38 inhibitor was observed.

This example shows that p38 phosphorylation is mainly affected by the action of SlP. This does not exclude the action of other kinases we are presently investigating specifically the effect of SlP on ERK and MTOR phosphorylation. The specificity of the action of SlP and its relation to cardiac and vascular hypertrophy in Fabry disease and in cadiomyopathy in

general will be caracterized and the application of their inhibitors will be considered as therapeutical drugs.

Example 8: Synergetic Effect on Proliferation when Combining SlP and Angiotensin II or Endothelin I

Angiotensin II (Ang II) and endothelin- 1 (ET-I) are peptides that induce a prolonged vasoconstriction and enhance proliferation of VSMC. In order to check if the action of sphingosine- 1 phosphate is some how connected to the effect induced by these two hormone peptides we have monitored the action of sphingosine- 1 phosphate in presence and absence of Ang II and ET-I, used effective concentrations 1 μM and 100 nM, respectively, in the same assay of metabolic activity described in Example 1.

Figure 10 shows the effect on treatments based on SlP, Ang II and ET-I, used alone or in combination. A synergistic effect with any one of Ang II or ET-I, if combined with SlP at both low (10 nM) and hight concentration (10 μM), is revealed. These data indicate that the effect of sphingosine- 1 phosphate is not interdependant with the known effect of Ang II and ET-I. They also show that if proliferative action is mediated simultaneously by both, SlP and either Ang II or ET-I, a synergistically increased proliferation is obtained.

Also the inverse can be deduced, that is, blocking proliferation by suppressing in any way the action of SIp on the one hand and Ang II or ET-I on the other hand, a synergistic reduction of proliferation is to be expected.

Example 9: Effect on SlP on Hypertrophy of Cultured Smooth Muscle Cells

Above MTS is used as a labeler of VSMC proliferation. As in Fabry disease the cardiovascular manifestations described was hypethrophy and not the hyperplasia (proliferation), it was determined whether the observed effect is exclusively a proliferative effect or there is an additional hypertrophic effect. This was assessed with the measurement of cell surface area determined by image analysis. After 48 h incubation in serum free medium with sphingosine- 1 phosphate mean planar cell surface area showed an increase tendency for 10 and 100 nM, decrease tendency for 1 μM and and significant decrase for 10 μM p= 0.73, p= 0.15 , p= 0.17 and p=0.005 respectively. These data indicate that sphingosine-1 phosphate has a hypertrophic effect tendency at low concentrations the major effect at 100 nM. This phenomenon is lost at 1 μM and 10 μM. Figure 12 represents the mean of mean planar cell surface area determined by image analysis with control values at 358±3 μm ² , their highest values at 100 nM was 378±10 μm ² and lowest values at 10 μM was 305±8 μm ² . The analysis with planar surface area was performed in 100 cells for each treatment condition. The image analysis (photographs of Figure 11) showed that at 1 μM and 10 μM cells are smaller but more numerous.

This confirms that SlP has a hypertrophic effect, which is concentration dependent.

In a similar experimental setting the precise VSMC proliferation was assessed with fluorescence activating cell sorting, allowing differentiating live cells, cell debris and dead cells. Statistical analysis shows that both total and live cells numbers increase significantly under sphingosine-1 phosphate at 100 nM, 1 μM and 10 μM. The sphingosine-1 phosphate induces mainly cell survival at low concentration (1 nM and 10 nM) and neo-proliferation at higher concentration 100 nM, 1 μM and 10 μM. Interestingly, at high concentrations of 1 μM and 10 μM, cell number increases and cell size decreases, confirming microscopy image analysis above.

Example 10: Sphingosine-1 -phospharte and Analogs Against Atherosclerosis

Careful examination of male and female Fabry patients (mean age 50.9 1.6) revealed the absence of atheroschlerotic plaque at the carotid bifurcation, compared to 34% of plaque in the control population, indicating a protective effect of Fabry disease against atheroschlerosis. This is substantiated by the fact that a lower prevalence of atherosclerotic coronary artery leasions has been reported in Fabry patients compared to non-Fabry subject.

Therefore, SlP, or its analogs protect Fabry patients from development of atherosclerotic plaque.

Example 11 : S IP-Antagonists for Prevention of Hypertrophy and Hyperplasia In order to verify if S IP-antagonists can alleviate S IP-mediated effects of hypertrophy and/or hyperplasia, an S IP-antagonist known to bind at the S1P2 receptor, JTEOl 3, is tested. Accordingly, in the same experimental setting as described in Example 1, vascular smooth muscle cells are exposed to 10 μM SlP, a concentration that was shown to induce hyperplasia in these cells. For verifying the hypothesis, cells are exposed simultaneously to 100 nM JTEO 13. If compared to control cells not treated with the antagonist, it can be seen that JTE013, an S IP-antagonist blocking the S1P2 receptor, substantially decreased the hypertrophic effects of SlP.

Therefore, these results indicate that SlP-anagonists, preferably those blocking receptors that are expressed in such cells, notably S1P2 and S1P5, are useful for alleviating symptoms of vascular hypertrophy and/or hyperplasia.

Example 12: Effects of Administered SlP in Mice on Aortic IMT, Cardiac Weight and Arterial Blood pressure

In order to verify positively that circulating SlP is responsible for the cardiac and aortic implications of Fabry disease, Fabry mice were compared to healthy mice receiving SlP per injection.

Materials and Methods

Mice: Fabry mice B6; 129-Gla ^tmlkul strain was obtained from the Jackson Laboratory.

Control and SlP injected mice used in our experiments are wild type Fabry mice litermate.

Mice were fed regular rodent chow and water ad libitum throughout the study. Only males were used for the study. Each group was constituted by 8 mice (n=8). The local institutional animal committee approved all of the experiments.

SlP mice treatment Blood pressure measurement: 10 week-old mice were injected intraperotoneal with SlP, 0.4 mg/kg/day for 4 weeks. Subsequently, mean arterial blood pressure was measured in three groups of mice control, SlP treated and Fabry. Briefly, mice were weighted and their left carotid artery was catheterized. After mice fully recovered from anesthesia, the arterial line was connected to a pressure transducer, and 30 minutes after, mean blood pressure was recorded.

Histological analysis: After blood pressure measurement mice were killed by intravenous injection of pentobarbital 100 mg/kg. Hearts and aorta were rapidly excised and hearts weighed. Organs were immersion-fixed in 4% paraformaldehyde in PBS embedded in paraffin, and serial sections were cut for analysis by hematoxylin-eosin staining. Serial sections collected every 4 μm were used for analysis.

Results and Conclusions

The results are shown in Figures 14-17. From Figure 14, it can be seen that mice treated with SlP had the same increase aortic intima-media thickness as Fabry-mice.

The Photograph Figures 15A-C and respective magnifications Figures 15 D-F provide a visual impression of the results of Figure 14. Invasion by other cells, such as monocytes into the aortic vessel could not be detected.

Figure 16 A shows that SlP treatment induces statistically significant cardiac hypertrophy and Fabry mice showed a clear tendency for cardiac hypertrophy without reaching statistic significance (P<0.05, n=8). Figure 16 B shows that no statistical significance for differences in arterial blood pressure is observed in the different groups. A tendency for lower blood pressure is observed in both groups SlP treated and fabry mice.

Figure 17 A-C shows cardiac hypertrophy in SlP treated (D) and Fabry (E) mice as compared to control (C). The cardiac hypertrophy is less pronounced in Fabry mice. Administration of SlP results in cardiac and vascular complications identical or similar to those observed in Fabry mice. This confirms that SlP is the causative agent of these symptoms.