THE CHOLESTEROL-SYNTESIS INTERMEDIATES FOR TREATMENT DEMYELINATING DISORDERS

Abstract

The present invention relates to a cholesterol-synthesis intermediate as pharmaceutically active agent and/or pharmaceutical composition comprising the cholesterol-synthesis intermediate optionally together with one or two further pharmaceutically active agent(s) for use in the prophylaxis and/or treatment of demyelinating disorders/diseases, in particular multiple sclerosis.

Claims

1. A method for treatment or prophylaxis of a demyelinating disorder or a demyelinating disease selected from the group consisting of: lysosomes and lysosomal disorders, metachromatic leukodystrophy, multiple sulfatase deficiency, globoid cell leukodystrophy, GM1 gangliosidosis, GM2 gangliosidosis, fabry disease, fucosidosis, mucopolysaccharidoses, free sialic acid storage disorder, neuronal ceroid lipofuscinoses, adult polyglucosan body disease, peroxisomes and peroxisomal disorders, peroxisome biogenesis defects, peroxisomal D-bifunctional protein deficiency, peroxisomal acyl-CoA oxidase deficiency, X-linked adrenoleukodystrophy, refsum disease, mitochondria and mitochondrial disorders, mitochondrial encephalopathy with lactic acidosis and stroke-like episodes, leber hereditary optic neuropathy, Kearns-Sayre syndrome, mitochondrial neurogastrointestinal encephalomyopathy, Leigh syndrome and mitochondrial leukoencephalopathies, pyruvate carboxylase deficiency, multiple carboxylase deficiency, cerebrotendinous xanthomatosis, Cockayne syndrome, trichothiodystrophy with photosensitivity, Pelizaeus-Merzbacher disease and X-linked spastic paraplegia type 2, 18 q-syndrome, phenylketonuria, glutaric aciduria type 1, propionic acidemia, nonketotic hyperglycinemia, maple syrup urine disease, 3-hydroxy 3-methylglutaryl-CoA lyase deficiency, canavan disease, L-2-hydroxyglutaric aciduria, D-2-hydroxyglutaric aciduria, hyperhomocysteinemias, urea cycle defects, serine synthesis defect caused by 3-phosphoglycerate dehydrogenase deficiency, molybdenum cofactor deficiency and isolated sulfite oxidase deficiency, galactosemia, Sjögren-Larsson syndrome, Lowe syndrome, Wilson disease, Menkes disease, fragile X premutation, hypomelanosis of Ito, incontinentia pigmenti, Alexander Disease, giant axonal neuropathy, megalencephalic leukoencephalopathy with subcortical cysts, congenital muscular dystrophies, myotonic dystrophy type, myotonic dystrophy type 2, X-linked Charcot-Marie-Tooth disease, oculodentodigital dysplasia, leukoencephalopathy with vanishing white matter, Aicardi-Goutiéres syndrome, leukoencephalopathy with calcifications and cysts, leukoencephalopathy with brain stem and spinal cord involvement and elevated white matter lactate, hypomyelination with atrophy of the basal ganglia and cerebellum, hereditary diffuse leukoencephalopathy with neuroaxonal spheroids, dentatorubropallidoluysian atrophy, cerebral amyloid angiopathy, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy, polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy, pigmentary orthochromatic leukodystrophy, adult-Onset autosomal dominant leukoencephalopathies, inflammatory and infectious disorders, multiple sclerosis, clinically isolated syndrome, optic neuritis, neuromyelitis optica, transverse myelitis, acute disseminated encephalomyelitis, acute hemorrhagic leucoencephalitis, adrenoleukodystrophy and adrenomyeloneuropathy, progressive multifocal leukoencephalopathy, central pontine and extrapontine myelinolysis, hypoxic-ischemic demyelination, Alzheimer's disease, acute hemorrhagic encephalomyelitis, acquired immunodeficiency syndrome, brucellosis, subacute sclerosing panencephalitis, congenital and perinatal cytomegalovirus infection, Whipple disease, toxic encephalopathies, latrogenic toxic encephalopathies, hypernatremia, Marchiafava-Bignami syndrome, posterior reversible encephalopathy syndrome, langerhans cell histiocytosis, post-hypoxic-ischemic damage, post-hypoxic-ischemic leukoencephalopathy of neonates, neonatal hypoglycemia, delayed posthypoxic leukoencephalopathy, white matter lesions of the elderly subcortical arteriosclerotic encephalopathy, vasculitis, leukoencephalopathy and dural venous fistula, leukoencephalopathy after chemotherapy and/or radiotherapy, gliomatosis cerebri, diffuse axonal injury, wallerian degeneration and myelin loss secondary to neuronal and axonal degeneration, by administering to a patient a cholesterol-synthesis intermediate.

2. The method according to claim 1, wherein the demyelinating disease is associated with microglial activation/inflammation and is selected from multiple sclerosis, optic neuritis, neuromyelitis optica, transverse myelitis, acute disseminated encephalomyelitis, adrenoleukodystrophy, adrenomyeloneuropathy, acute hemorrhagic leucoencephalitis, progressive multifocal leukoencephalopathy, central pontine myelinolysis, extrapontine myelinolysis, hypoxic-ischemic demyelination, Alzheimer's disease and clinically isolated syndrome.

3. The method according to claim 1, wherein the cholesterol-synthesis intermediate is selected from the group consisting of: squalene, 2,3-oxidosqualene, lanosterol, 14-demethyl-lanosterol, 24,25-dihydrolanosterol, 4,4-dimethyl-5α-cholesta-8(9),14,24-trien-3β-ol, 4,4-dimethyl-5α-cholesta-8(9),14-dien-3β-ol, 4,4-dimethyl-5α-cholesta-8(9),24-dien-3β-ol, 4,4-dimethyl-5α-cholest-8(9)-en-3β-ol, 5α-cholesta-8(9),24-dien-3β-ol, 4α-methyl-5α-cholesta-8(9),24-dien-3β-ol, 5α-cholest-8(9)-en-3β-ol, 5α-cholesta-7,24-dien-3β-ol, 5α-cholest-7-en-3β-ol, 7-dehydrodesmosterol, 7-dehydrocholesterol, desmosterol, and N,N-dimethyl-3β-hydroxycholenamide, 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, 24(S),25-epoxycholesterol, hyodeoxycholic acid, ouabagenin, ATI-111, ATI-829, 7-ketocholesterol, 7α-hydroxycholestenone, and olesoxime, methylpiperidinyl-3β-hydroxycholenamide.

4. The method according to claim 1, wherein the cholesterol-synthesis intermediate is used together with one further cholesterol-synthesis intermediate or with two further cholesterol-synthesis intermediates selected from the group consisting of: squalene, 2,3-oxidosqualene, lanosterol, 14-demethyl-lanosterol, 24,25-dihydrolanosterol, 4,4-dimethyl-5α-cholesta-8(9),14,24-trien-3β-ol, 4,4-dimethyl-5α-cholesta-8(9),14-dien-3β-ol, 4,4-dimethyl-5α-cholesta-8(9),24-dien-3β-ol, 4,4-dimethyl-5α-cholest-8(9)-en-3β-ol, 5α-cholesta-8(9),24-dien-3β-ol (zymosterol), 4α-methyl-5α-cholesta-8(9),24-dien-3β-ol, 5α-cholest-8(9)-en-3β-ol, 5α-cholesta-7,24-dien-3β-ol, 5α-cholest-7-en-3β-ol, 7-dehydrodesmosterol, 7-dehydrocholesterol, desmosterol, and N,N-dimethyl-3β-hydroxycholenamide, 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, 24(S),25-epoxycholesterol, hyodeoxycholic acid, ouabagenin, ATI-111, ATI-829, 7-ketocholesterol, 7α-hydroxycholestenone, olesoxime, and methylpiperidinyl-3β-hydroxycholenamide.

5. The method according to claim 1, wherein the cholesterol-synthesis intermediate is squalene.

6. The method according to claim 1, wherein the cholesterol-synthesis intermediate is squalene which is used together with one further active agent selected from the group consisting of alemtuzumab, cladribine, daclizumab, dimethyl fumarate, fingolimod, glatiramer acetate, interferon beta-1a, interferon beta-1b, laquinimod, peginterferon beta-1, mitoxantrone, natalizumab, ocrelizumab, siponimod, prednisone teriflunomide, opicinumab, olesoxime, and/or N,N-dimethyl-3ß-hydroxycholenamide.

7. The method according to claim 6, wherein the cholesterol-synthesis intermediate is squalene which is used together with interferon beta-1a, interferon beta-1b, peginterferon beta-1a, and/or N,N-dimethyl-3ß-hydroxycholenamide.

8. The method according to claim 6, wherein the cholesterol-synthesis intermediate is squalene which is used together with interferon beta-1a and N,N-dimethyl-3ß-hydroxycholenamide or interferon beta-1b and N,N-dimethyl-3ß-hydroxycholenamide.

9. The cholesterol-synthesis intermediate for use method according to claim 6, wherein a weight ratio of the squalene and the one further active agent is in a range of 100,000:1 to 10:1.

10. The method according to claim 1, wherein 0.1-1000 mg/kg squalene is administered per body weight in one day.

11. The method according to claim 1, wherein the cholesterol-synthesis intermediate is used together with a ketogenic diet.

12. A pharmaceutical composition comprising squalene or a squalene extract together with one or two further cholesterol-synthesis intermediates selected from the group consisting of squalene, 2,3-oxidosqualene, lanosterol, 14-demethyl-lanosterol, 24,25-dihydrolanosterol, 4,4-dimethyl-5α-cholesta-8(9),14,24-trien-3β-ol, 4,4-dimethyl-5α-cholesta-8(9),14-dien-3β-ol, 4,4-dimethyl-5α-cholesta-8(9),24-dien-3β-ol, 4,4-dimethyl-5α-cholest-8(9)-en-3β-ol, 5α-cholesta-8(9),24-dien-3β-ol, 4α-methyl-5α-cholesta-8(9),24-dien-3β-ol, 5α-cholest-8(9)-en-3β-ol, 5α-cholesta-7,24-dien-3β-ol, 5α-cholest-7-en-3β-ol, 7-dehydrodesmosterol, 7-dehydrocholesterol, desmosterol, and N,N-dimethyl-3β-hydroxycholenamide, 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, 24(S),25-epoxycholesterol, hyodeoxycholic acid, ouabagenin, ATI-111, ATI-829, 7-ketocholesterol, 7α-hydroxycholestenone, olesoxime, and methylpiperidinyl-3β-hydroxycholenamide or together with one or two further active agent(s) selected from the group consisting of alemtuzumab, cladribine, daclizumab, dimethyl fumarate, fingolimod, glatiramer acetate, interferon beta-1a, interferon beta-1b, laquinimod, peginterferon beta-1, mitoxantrone, natalizumab, ocrelizumab, siponimod, prednisone teriflunomide, opicinumab, and/or N,N-dimethyl-3ß-hydroxycholenamide.

13. The pharmaceutical composition according to claim 12, consisting of squalene or a squalene extract together with one or two further cholesterol-synthesis intermediates selected from the group consisting of squalene, 2,3-oxidosqualene, lanosterol, 14-demethyl-lanosterol, 24,25-dihydrolanosterol, 4,4-dimethyl-5α-cholesta-8(9),14,24-trien-3β-ol, 4,4-dimethyl-5α-cholesta-8(9),14-dien-3β-ol, 4,4-dimethyl-5α-cholesta-8(9),24-dien-3β-ol, 4,4-dimethyl-5α-cholest-8(9)-en-3β-ol, 5α-cholesta-8(9),24-dien-3β-ol, 5α-cholest-8(9)-en-3β-ol, 4α-methyl-5α-cholesta-8(9),24-dien-3β-ol, 5α-cholesta-7,24-dien-3β-ol, 5α-cholest-7-en-3β-ol, 7-dehydrodesmosterol, 7-dehydrocholesterol, desmosterol, N,N-dimethyl-3β-hydroxycholenamide, 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, 24(S),25-epoxycholesterol, hyodeoxycholic acid, ouabagenin, ATI-111, ATI-829, 7-ketocholesterol, 7α-hydroxycholestenone, olesoxime, and methylpiperidinyl-3β-hydroxycholenamide or together with one or two further active agent(s) selected from the group consisting of alemtuzumab, cladribine, daclizumab, dimethyl fumarate, fingolimod, glatiramer acetate, interferon beta-1a, interferon beta-1b, laquinimod, peginterferon beta-1, mitoxantrone, natalizumab, ocrelizumab, siponimod, prednisone teriflunomide, opicinumab, and/or N,N-dimethyl-3ß-hydroxycholenamide, and at least one pharmaceutically acceptable carrier, excipient and/or solvent.

14. The pharmaceutical composition according to claim 13, consisting of squalene or a squalene extract together with one or two further active agent(s) selected from the group consisting of alemtuzumab, cladribine, daclizumab, dimethyl fumarate, fingolimod, glatiramer acetate, interferon beta-1a, interferon beta-1b, laquinimod, peginterferon beta-1, mitoxantrone, natalizumab, ocrelizumab, siponimod, prednisone teriflunomide, opicinumab, olesoxime, and/or N,N-dimethyl-3ß-hydroxycholenamide, and at least one pharmaceutically acceptable carrier, excipient and/or solvent.

15. The pharmaceutical composition according to claim 13, consisting of squalene or a squalene extract together with one or two further active agent(s) selected from interferon beta-1a, interferon beta-1b, peginterferon beta-1a, and N,N-dimethyl-3ß-hydroxycholenamide, and at least one pharmaceutically acceptable carrier, excipient and/or solvent.

16. The pharmaceutical composition according to claim 13, consisting of squalene and interferon beta-1a and N,N-dimethyl-3ß-hydroxycholenamide or of squalene and interferon beta-1b and N,N-dimethyl-3ß-hydroxycholenamide, and at least one pharmaceutically acceptable carrier, excipient and/or solvent.

17. The pharmaceutical composition according to claim 12, wherein the amount of squalene is in a range of 10% to 99% by weight.

18. The pharmaceutical composition according to claim 12, wherein a weight ratio of the squalene and the at least one further active agent is in a range of 100,000:1 to 10:1.

19. The pharmaceutical composition according to claim 12, wherein 0.1-1000 mg/kg squalene is administered per body weight in one day.

20. (canceled)

Description

DESCRIPTION OF THE FIGURES

[0284] FIG. 1: Clinical score of MOG35-55 induced EAE animals [0285] a) Mean clinical score±SEM of mice in the prophylactic squalene treatment paradigm. Mice received squalene diet commencing at 14 days before immunization. [0286] b) Mean clinical score±SEM of mice in the therapeutic squalene treatment paradigm. Mice received squalene diet commencing at the day of disease onset. [0287] c) Mean clinical score±SEM of mice in the prophylactic squalene treatment paradigm in combination with IFNβ1b. IFNβ1b was given as daily i.p. injections starting from day 3 after EAE induction. The number of mice in each experimental group is given in parentheses.

[0288] FIG. 2: Squalene induced reduction of inflammatory cells in spinal cord tissue [0289] a) Serum squalene concentration measured by FPLC 28 days post immunization (Prophylactic squalene treatment) measured by FPLC. [0290] b-d) Number of b) CD45+/CD11 b+ myeloid cells c) abTCR/CD4+, d) abTCR/CD8+ and per gram spinal cord measured by flow cytometry at the peak of EAE disease (day 16) after therapeutic squalene administration in comparison to mice that received normal chow (Ctrl). Bars represent means with individual data points (n=6 mice per group, Student's t-test, *P<0.05).

[0291] FIG. 3: No impact of squalene on peripheral inflammatory cell number [0292] a-c) Number of a) abTCR/CD4+, b) abTCR/CD8+ and c) CD45+/CD11 b+ myeloid cells per ml collected blood measured by flow cytometry at peak of EAE disease (day 16) after therapeutic squalene administration as described in FIG. 2 were comparable in both treatment groups.

[0293] FIG. 4: Squalene induced spinal cord tissue expression profile [0294] a-b) RT-qPCR expression profile of a) cellular marker genes and cholesterol synthesis/metabolism genes and b) inflammatory genes in spinal cord lysates of mice fed normal chow or chow supplemented with squalene in remission phase (prophylactic treatment paradigm). Bars represent normalized means (n=6 animals) of fold expression±SEM (set to 1) (Student's t-test, *P<0.05, **P<0.01, ***P<0.001).

[0295] FIG. 5: Squalene induced expression profile in isolated CD11b+ myeloid cells [0296] a) RT-qPCR expression profile of inflammatory genes in MACS isolated CD11 b* myeloid cells during acute phase (15 days post immunization) phase after prophylactic squalene administration. Bars represent normalized means (n=6 animals)±SEM (set to 1) (Student's t-test, *P<0.05, **P<0.01, ***P<0.001). [0297] b) Expression of cholesterol export genes Abca1 and Apoe in MACS isolated CD11 b* myeloid cells as in a) during acute phase. Bars represent means normalized to untreated control mice (Student's t-test, *P<0.05)

[0298] FIG. 6: Squalene induced remyelination in lysolecithin induced lesions [0299] a) Representative images of spinal cord sections 14 days post lesion (dpl) with 1 μl 1% lysolecithin stained for the oligodendrocyte marker CAll. Squalene treatment started at day of lesion induction. Dotted line marks lesion area with increased cellular density (DAPI). Scale=100 μm [0300] b) Quantification of CAII+ oligodendrocytes within lesions shown in a). Bars represent means of control (n=5) and squalene (n=6) treated animals. Significance evaluated by Student's t-test, **P<0.01. [0301] c) Quantification of lesion area depicted in a). Bars represent means of control (n=5) and squalene (n=6) treated animals. [0302] d) Serum squalene concentration measured by FPLC of animals (n=3) analyzed in a-c). [0303] e) Representative images of spinal cord sections as in a) stained for MBP labeling myelin. [0304] f) Quantification of MBP+myelin within lesions shown in e).

[0305] FIG. 7: Squalene support myelination in vitro [0306] a) Representative images of myelinating co-culture at 21 days in vitro (DIV) and 30 DIV treated with or without 100 μM squalene stained for myelinated axon segments (MBP). [0307] b) Quantification of MBP positive area normalized to SM131 positive axons at 21 DIV and 30 DIV. Bars represent means of individual control and squalene treated cultures (n=4 biological replicates). Significance evaluated by 1 way ANOVA following multiple comparison correction with Holm-Sidak method, **P<0.01.

[0308] FIG. 8: Clinical score of EAE induced animals treated with squalene combination drug therapy [0309] a) Mean clinical score±SEM of mice treated according to the therapeutic treatment paradigm with or without squalene, the synthetic LXR agonist DMHCA, and interferon. Mice received squalene and DMHCA supplemented diet commencing from the day of disease onset. IFNβ1b was given daily by i.p. injections starting from day 3 after induction of the EAE. [0310] b) Expression of cholesterol export genes in MACS isolated CD11 b* myeloid cells during chronic phase from DMHCA treated animals a). Bars represent means normalized to untreated control mice (Student's t-test, **P<0.01, ***P<0.001).

[0311] FIG. 9: Squalene induced reduction of inflammatory cells in combination with anti-inflammatory therapy [0312] a-d) Number of a) abTCR/CD4+, b) abTCR/CD8+c) CD11 b+/CD45.sup.high macrophages and d) CD11 b+/CD45.sup.low microglia per gram spinal cord from mice treated as in FIG. 7, measured by flow cytometry. Bars represent means with individual data points (Student's t-test, *P<0.05, **P<0.01).

[0313] FIG. 10: Clinical score of EAE induced animals treated with different dosages of squalene, IFNβ1b and D1, a pharmaceutical composition comprising tocotrienols, tocopherols, squalene and B vitamins. [0314] a) Mean clinical score±SEM of mice (n=5-6) treated according to the therapeutic treatment paradigm with or without squalene (0.03% [v/w], 0.5% [v/w], 5% [v/w]). Mice received squalene commencing from the day of disease onset. [0315] b) Mean clinical score±SEM of mice (n=4-6) treated according to the therapeutic treatment paradigm with squalene (0.03% [v/w]), D1 (WO 2017/204 618 A1) Composition (tocotrienols, tocopherols, squalene and B vitamins as in see c) and interferon-squalene combination therapy (Squalene 0.03% [v/w], IFNβ1b 30.000 U). Mice received squalene and D1 composition commencing from the day of disease onset. IFNβ1b was given daily by i.p. injections starting from day 3 after induction of the EAE. [0316] c) Animal equivalent dose (AED) of Squalene and D1 (WO 2017/204 618 A1) composition for therapeutic treatment in a) and b) (Nair A B, Jacob S. A simple practice guide for dose conversion between animals and human. J Basic Clin Pharm. 2016 March; 7(2): pp. 27-31).

[0317] FIG. 11: Clinical score of EAE induced animals treated with different squalene doses in combination with IFNβ1b. [0318] a) Mean clinical score±SEM of mice (n=4-6) treated according to the therapeutic treatment paradigm interferon-squalene combination therapy (Squalene 0.03% [v/w], Squalene 0.5% [v/w]; IFNβ1b 30.000 U). Mice received squalene from the day of disease onset. IFNβ1b was given daily by i.p. injections starting from day 3 after induction of the EAE.

[0319] FIG. 12: Clinical score of EAE induced animals treated with squalene and ketogenic combination drug therapy [0320] a) Mean clinical score±SEM of mice (n=9-10) treated according to the therapeutic treatment paradigm with or without squalene (0.5%), a ketogenic diet, and interferon. Mice received squalene and ketogenic diet commencing from the day of disease onset. IFNβ1b was given daily by i.p. injections starting from day 3 after induction of the EAE. [0321] b) Serum β-Hydroxybutyrate (POHB) and glucose concentration in animals with EAE at 28 days post immunization (One-way Anova with Tukeys post-posttest, ***P<0.001).

[0322] FIG. 13: Squalene induced reduction of inflammatory cells in combination with anti-inflammatory and ketogenic therapy [0323] a-c) Number of a) abTCR/CD4+ T cells, b) abTCR/CD8+ T cells and c) CD11 b+/CD45+ microglia/macrophages per gram spinal cord from mice treated as in FIG. 12, measured by flow cytometry. Bars represent means with individual data points (One-way Anova with Tukeys post-posttest, *P<0.05, **P<0.01, ***P<0.01).

[0324] FIG. 14: Squalene induced reduction of inflammatory cells in Plp1-tg mice. [0325] a) Elevated beam testing of control (wildtype), Plp1-tg mice, squalene-treated (0.5% [v/w] Squalene) Plp1-tg. Plp1tg mice were fed therapeutic diet or standard chow between 2 and 12 weeks of age. Bars represent the means with individual data points of three testing sessions. Asterisks mark significant changes (One-way Anova with Tukeys post-posttest). ***p<0.001, **p<0.01, *p<0.05 [0326] b) Number of a) abTCR/CD4+ T cells, b) abTCR/CD8+ T cells and c) CD11 b+/CD45+ microglia/macrophages per gram brain (left) and spinal cord (right) from mice treated as in a), measured by flow cytometry. Bars represent means with individual data points (One-way Anova with Tukeys post-posttest, *P<0.05

[0327] FIG. 15: Clinical score of EAE induced animals treated with squalene and ketogenic combination drug therapy [0328] a) Mean clinical score±SEM of mice (n=19-22) treated according to the prophylactic treatment paradigm with or without cholesterol (2% cholesterol) in combination with interferon. Mice received cholesterol commencing 14 days before immunization. IFNβ1b was given daily by i.p. injections starting from day 3 after induction of the EAE.

[0329] FIG. 16: Clinical score of EAE induced animals treated with squalene and ketogenic combination drug therapy

[0330] Mean clinical score±SEM of mice (n=19-22) treated according to the prophylactic treatment paradigm with or without cholesterol (2% cholesterol) in combination with interferon. Mice received cholesterol commencing 14 days before immunization. IFNβ1b was given daily by i.p. injections starting from day 3 after induction of the EAE.

[0331] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0332] Further modifications and alternative embodiments of various aspects of the invention will be apparent to those skilled in the art in view of this description. Accordingly, this description is to be construed as illustrative only and is for the purpose of teaching those skilled in the art the general manner of carrying out the invention. It is to be understood that the forms of the invention shown and described herein are to be taken as examples of embodiments. Elements and materials may be substituted for those illustrated and described herein, parts and processes may be reversed, and certain features of the invention may be utilized independently, all as would be apparent to one skilled in the art after having the benefit of this description of the invention. Changes may be made in the elements described herein without departing from the spirit and scope of the invention as described in the following claims.

EXAMPLES

Example 1

[0333] Methods

[0334] Mice

[0335] For MOG-EAE, mice purchased from Charles River were immunized subcutaneously with 200 mg myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) in complete Freund's adjuvant (M. tuberculosis at 3.75 mg ml.sup.−1) and i.p. injected twice with 500 ng pertussis toxin. Animals were examined daily and scored for clinical signs of the disease. If disease did not start within 15 days after induction or the clinical score rose above 4, animals were excluded from the analysis. The clinical score was: 0 normal; 0.5 loss of tail tip tone; 1 loss of tail tone; 1.5 ataxia, mild walking deficits (slip off the grid); 2 mild hind limb weakness, severe gait ataxia, twist of the tail causes rotation of the whole body; 2.5 moderate hind limb weakness, cannot grip the grid with hind paw, but able to stay on a upright tilted grid; 3 mild paraparesis, falls down from a upright tiled grid; 3.5 paraparesis of hind limbs (legs strongly affected, but move clearly); 4 paralysis of hind limbs, weakness in forelimbs; 4.5 forelimbs paralyzed; 5 moribund/dead. Mice received 0.5% [v/w] squalene (Sigma) chow commencing either two weeks before immunization defined as prophylactic regimen or at the first appearance of EAE symptoms defined as therapeutic regimen and continued until day 28. DMHCA (Avanti) was dissolved in corn oil (Sigma) and given therapeutically in chow at a dosage of 8 mg kg.sup.−1. IFNβ1b (Bayer) was administrated by daily intraperitoneal (i.p.) injections 30.000 U per animal.

[0336] Focal spinal cord demyelinating lesions were induced under MMF anesthesia (1.0 mg kg.sup.−1 midazolam, 0.05 mg kg.sup.−1 medetomidine and 0.02 mg kg.sup.−1 fentanyl) by stereotactic injection of 1 μl lysolecithin (1%, from egg yolk, alpha-lysophosphatidyl-choline, Sigma) into the left and right ventro-lateral funiculus at Th10 of 8-week old animals. The injection was performed at a rate of ˜1 μl min.sup.−1. This procedure created fusiform demyelinating lesions, 5-6 mm in length. At the day of injection, mice were randomly assigned to normal or 0.5% [v/w] squalene supplemented chow for 14 days, after which the animals were killed, and the spinal cord processed for histology. For serum isolation, blood was collected by cardiac puncture, and serum was prepared after 4 h clotting by centrifugation.

[0337] Cell Isolation and Flow Cytometry.

[0338] Single-cell suspensions from spinal cords were obtained via mechanical dissociation on a cell strainer. Immune cells were separated over a two-phase Percoll-density gradient (70%/30%). Blood was collected by cardiac puncture in EDTA (80 mM) and single-cell suspension was obtained by centrifugation over lymphocyte separation medium (LSM 1077, PAA). Staining of abTCR/CD4 T cells, abTCR/CD8 T cells and CD45/CD11 b cells (macrophages/microglia) was performed using the following antibodies in a 1:200 dilution:Anti-CD3e (clone 145-2C11), BioLegend; anti-CD4 (clone GK 1.5), BD; anti-CD8 (clone 53-6.7), BD; anti-CD8 (clone 53-6.7), BD; anti-CD11b (clone M1/70), BioLegend; anti-CD45.2 (clone 104), BioLegend. The addition of Calibrite APC beads (BD) allowed for cell quantification. Flow cytometry was performed using a FACSCalibur operated by Cell Quest software (Becton Dickinson).

[0339] Magnetic Cell Isolation.

[0340] Microglia/Macrophages were isolated according to the adult brain dissociation protocol (Miltenyi biotec). Spinal cord was isolated on ice followed by removal of meninges. Antibody labeling steps were done according to the respective antibody Microbead kit protocol (Miltenyi biotec), oligodendrocytes (04, 130-096-670) and microglia (CD11 b, 130-093-636). Purity of cell populations was routinely determined by qPCR on extracted and reverse transcribed RNA (see below) and revealed only minimal contamination by other cell types.

[0341] Expression Analyses.

[0342] For tissue expression analyses, spinal cord tissue was dissected from ˜Th10-˜L5. RNA was isolated using QIAshredder and RNeasy protocols (Qiagen). Concentration and quality of RNA was evaluated using a NanoDrop spectrophotometer and RNA Nano (Agilent). cDNA was synthesized with Superscript III (Invitrogen) and quantitative PCRs were done in triplicates with the GoTaq qPCR Master Mix (Promega) on a 7500 Fast Real-Time PCR System (Applied Biosystems). Expression values were normalized to the mean of three—five housekeeping genes, HPRT (Hypoxanthin-Phosphoribosyl-Transferase 1) and Rplp0 (60S acidic ribosomal protein P), Rps13 (Ribosomal Protein S13), Gapdh (Glyceraldehyde-3-Phosphate Dehydrogenase), 18S (18S ribosomal RNA) and quantification was done by applying the ΔΔCt method, normalized to age matched untreated controls (set to 1). All primers were intron-spanning.

[0343] Histochemistry.

[0344] Mice were perfused with 4% paraformaldehyde (PFA). Spinal cord tissue was postfixed overnight, embedded in paraffin and cut into 5 mm sections (HMP 110, MICROM). For immunohistological analyses, sections were deparaffinized followed by antigen-retrieval in sodium citrate buffer (0.01 M, pH 6.0). For immunofluorescence, sections were blocked with serum free protein block (Dako). Primary antibodies were diluted in 2% bovine serum albumin (BSA)/PBS and incubated for 48 h followed by fluorophor coupled secondary antibodies. Lesion area (DAPI clustered nuclei), oligodendrocyte cell number (CAll, carbonic anhydrase 2) and myelin positive area (MBP, myelin basic protein) were evaluated.

[0345] Fast-Protein Liquid Chromatography

[0346] Lipids were extracted from serum samples according to Bligh and Dyer (cit. Can. J. Biochem, 1959). The chloroform phase containing the lipids was evaporated at 40° C. in a vacuum concentrator (Qiagen) and extracted lipids dissolved and saponified in glass tubes with 0.5 M KOH in EtOH for 30 min. Non-saponifiable lipids were extracted with n-hexane. After evaporation of the n-hexane phase at 40° C. in a vacuum concentrator, non-saponifiable lipids were dissolved in MetOH and subjected to reverse phase HPLC (250/4 Nucleoshell RP C18 (Macherey and Nagel) connected to an Äkta FPLC (GE Healthcare), flow rate 0.25 ml/min, liquid phase acetonitril:EtOH 70:30 (v/v), online UV detection at 205. Squalene retention and peak amplitude were related to injections of a range of concentration standards in MetOH. Amplitudes were normalized to the neutral lipid recovery as assessed via online detection of fluorescence of dehydroergosterol (excitation 325 nm, emission 375 nm, Quantamaster (PTI/Horiba) that was spiked into the serum sample.

[0347] Cell Culture

[0348] Myelinating co-cultures were established as described (Thomson, C. E. et al. 2008) with minor modifications. Briefly, E13 embryonic spinal cords were digested in 0.125% Trypsin solution in HBSS (without Ca.sup.+2 and Mg.sup.+2) at 37° C. for 20 min. After stopping the digestion with 1 ml plating media (DMEM, 25% horse serum, 25% HBSS, 50 mg ml.sup.−1 DNAse) the tissue was homogenized by gentle trituration and centrifuged for 5 min. 150,000 cells were plated per poly-L-lysine coated coverslip; 3 coverslips per 35 mm Petri dish. After cell attachment in plating media, differentiation media was added (low glucose DMEM, 10 mg ml.sup.−1 insulin, 10 ng ml.sup.−1 biotin, 50 nM hydrocortisone, 0.5% N1-mix). N1 mix was 1 mg ml.sup.−1 apo-transferrin, 20 mM putrescine, 4 mM progesterone, and 6 mM sodium selenite. 50% media change was performed every 24-48 h with differentiation media. Squalene (100 μM) was added at day 3 to cultures. After 12 days, insulin was removed from differentiation media. Coverslips were fixed with PFA after 21 and 30 days in culture and permeabilized with −20° C. methanol for 10 min. Axonal (SM131) area and the area with myelin sheaths (MBP) of seven randomly chosen visual fields of myelinating co-cultures (×20 magnification) was measured by automated threshold with Fiji Software (SM131 Otsu, MBP Triangle). Specimens were analyzed on an Axiophot observer.Z1 (Zeiss) equipped with an AxioCam MRm and the ZEN 2012 blue edition software and evaluated with Image J software.

[0349] Statistical Analyses.

[0350] Statistical evaluation was done by unpaired Student's t-test for pairwise comparisons or by ANOVA for comparisons of more than two groups. For all statistical tests, significance was measured against an alpha value of 0.05. All error bars show s.e.m. P values are shown as *P<0.05; **P<0.01; ***P<0.001. Data analysis was performed blind to the experimental groups.

[0351] Results

[0352] Here, the applicant could show that the natural triterpene squalene that is an intermediate of cholesterol biosynthesis has the potential to ameliorate disease severity in a mouse model of immune mediated myelin disease (EAE) in a prophylactic (FIG. 1a) and therapeutic (FIG. 1b) treatment paradigm. Animals showed lower clinical scores and did not reach clinical symptoms/scores even at peak of disease (˜day 18), suggesting robust anti-inflammatory effects. Importantly, in addition to the already marked single compound effect (FIG. 1a-b), squalene administration further reduced disease severity in a combined treatment with daily injections of the first line standard drug in Multiple Sclerosis, interferon beta-1b (FIG. 1c). These data suggest that squalene and interferon target different disease processes. Because of this add-on benefit, there is a clear rationale to consider squalene supplementation or a derivative as a future therapy for inflammatory myelin diseases.

[0353] Dietary squalene administration leads to a marked increase in serum squalene in the two disease models tested (FIG. 2a, 6e). Under physiological conditions, squalene is not transported from the circulation across the blood-brain barrier to the CNS. Considering blood-brain barrier breach EAE already before the onset of clinical symptoms (Davalos et al., Annals of Neurology, 2014, 75, pp. 303-308; Schellenberg et al., Magnetic Resonance in Medicine, 2007, 58, pp. 298-305) and locally in lysolecithin-induced lesions, our data is compatible with the possibility of increased compound availability in the CNS parenchyma.

[0354] By FACS analysis of spinal cord tissue from squalene treated animals and untreated controls, we did not observe differences in the number of peripheral immune cells (FIG. 3a-c) suggesting comparable T cell priming in peripheral lymphoid organs. However, we cannot exclude anti-inflammatory effects of squalene on peripheral inflammatory cells e.g. macrophages. In contrast, we found reduced numbers of inflammatory CD4+ and CD8+ T cells as well as CD11 b+/CD45+ myeloid cells at peak of disease (FIG. 2b-d). These data indicate that squalene therapy leads to a reduction of recruited inflammatory cells into the spinal cord parenchyma and likely an amelioration of the inflammatory milieu within the CNS.

[0355] By targeted expression profiling in spinal cord of EAE treated mice, we found significantly increased expression of genes related to myelination and oligodendrocyte differentiation (Plp1, Car2, Olig2, Pdgfra, Cspg4), as well as cholesterol synthesis (Hmgc1, Hmgcr, Fdft1, Dhcr24, Cyp51 al, Mvk), cholesterol export (Abca1, Abcg1, Abcg4, Apoe, Clu) and cholesterol uptake (Ldlr, Vldlr, Apobr, Lrp1, Lrp2) in squalene supplemented mice compared to chow fed controls (FIG. 4a). As cholesterol is rate limiting for myelin membrane synthesis (Saher et al., (2005) Nat. Neuro 8 (4): 468-475), together these data show that squalene facilitates remyelination. We speculate that increased squalene availability induced remyelination in EAE lesions by supporting endogenous cholesterol synthesis and secondarily transport. In addition, we detected reduced expression of pro-inflammatory mediator genes while gene expression of the anti-inflammatory molecules 1110 and Tgfb1 was significantly increased (FIG. 4b). This shift of inflammatory mediators towards an anti-inflammatory milieu suggests an environment permissive for remyelination.

[0356] To directly test the inflammatory status of microglia/macrophages, we performed targeted expression profiling on acutely isolated CD11 b+ cells (FIG. 5). Indeed, isolated microglia/macrophages from squalene treated mice showed reduced expression levels of pro-inflammatory mediator genes such as TNF and IL1β and increased expression of the anti-inflammatory molecules such as TGFβ1 compared to the same cell fraction from chow fed mice (FIG. 5a). In addition, microglia/macrophages from squalene treated mice upregulated the major cholesterol export genes Abca1 and Apoe (FIG. 5b) implying increased delivery of microglial cholesterol to oligodendrocytes for remyelination. Together, these data demonstrate that dietary squalene supplementation has a direct impact on tissue microglia/macrophages during EAE, inducing a shift to an anti-inflammatory phenotype and increased cholesterol efflux that both supports tissue repair. We speculate that mechanistically squalene particularly increases LXR signaling directly or via desmosterol that facilitated the export of cholesterol.

[0357] Oligodendrocyte cholesterol is rate-limiting for myelin biogenesis. Squalene as an intermediate of cholesterol biosynthesis could fuel into the cholesterol biosynthesis pathway in oligodendrocytes to support remyelination. Therefore, we used the lysolecithin demyelination model that lacks an inflammatory component and evaluated oligodendrocyte differentiation and remyelination in squalene supplemented mice. The number of CAll positive mature oligodendrocytes and the degree of MBP positive myelinated axons within lesion area was strongly increased in squalene supplemented mice compared to control mice (FIG. 6a-f) while the size of the lesion area determined by DAPI positive increased nuclear density was comparable (FIG. 6c). To test, weather squalene directly supports oligodendrocyte differentiation and myelination, in vitro studies were performed. Myelinating cultures showing spontaneous oligodendrocyte myelination, starting around 18 days in vitro were treated with or without squalene and myelin area (MBP) was quantified in relation to axonal density (SM131). We observed increased MBP positive area (FIG. 7a-b) at 21 days in vitro (DIV) and 30 DIV indicating a direct effect of squalene availability on myelination.

[0358] In addition to mediating an anti-inflammatory phenotype in microglia/macrophages in EAE (FIG. 4b, 5a) and to facilitating myelination by oligodendrocytes (FIG. 6, FIG. 7), squalene treatment enhance the expression of cholesterol efflux genes that likely facilitated remyelination by oligodendrocytes (FIG. 5b). Cholesterol efflux and attenuated inflammation is the major outcome of LXR signaling (J. R. Secor McVoy et al., Journal of Neuroinflammation, 2015, 12:27; D. G. Thomas et al., Cell Reports, 2018, 25, pp. 3774-3785). By inducing LXR signaling with the LXR agonist N,N-dimethyl-3ß-hydroxycholenamide (DMHCA) in microglia/macrophages, EAE disease severity could be reduced (FIG. 8a, FIG. 9a-d) paralleled by upregulation of cholesterol efflux genes (FIG. 8b). To test, whether squalene administration could potentiate the efficiency of anti-inflammatory therapy due to its pro-remyelinating on oligodendrocytes and inflammation dampening effect on microglia/macrophages, we combined squalene therapy with DMHCA. In agreement with our hypothesis, squalene supplementation combined with the administration of DMHCA and interferon beta-1b, strongly reduced EAE disease severity compared to untreated controls and to single and double compound treated animals (FIG. 8). This was further supported by reduced number of inflammatory cells (CD4 and CD8 T-cells, CD11 b+/CD45 high macrophages, CD11 b+/CD45 low microglia) within spinal cord tissue (FIG. 9 a-d).

Example 2

[0359] Methods

[0360] Mice

[0361] Plp1 transgenic Plp1-tg, line #72 mice (Readhead et al., 1994, Stumpf, Berghoff et al., 2019) harbor three copies of the murine Plp1 gene. Dietary treatment was applied between 2 and 12 weeks of age.

[0362] Treatment

[0363] Dietary supplementation with squalene (0.5% [v/w]).

[0364] Beam Test

[0365] To assess bilateral sensorimotor coordination that involves connecting white matter structures, elevated beam test (in-house made, MPI of experimental medicine) was applied. Mice were trained to run toward a hiding box on a beam (width 1.5 cm) one week before testing. In one testing session number of slips in a defined 55 cm distance over three repeats was counted. Mean number of slips after three testing sessions (8 weeks, 10 weeks and 12 weeks) was quantified.

[0366] Readhead C, Schneider A, Griffiths I, Nave K A (1994) Premature arrest of myelin formation in transgenic mice with increased proteolipid protein gene dosage. Neuron 12:583-595. https://doi.org/10.1016/0896-6273(94)90214-3

[0367] Results

[0368] To further evaluate therapeutic single compound squalene application, different dosing was tested in EAE mice. Here, the applicant could show that the potential to ameliorate disease severity in EAE was dose depend comparing doses of 0.03% [v/w], 0.5% [v/w] and 5% [v/w] Squalene (FIG. 10a). Disease amelioration was already present at low 0.03% [v/w] squalene dosing (FIG. 10a, 10b). Comparison of a pharmaceutical composition comprising interferon beta-1b (30.000 U) and low squalene dose of 0.03% [v/w] shows higher potential to ameliorate disease severity in EAE than therapeutic intervention with D1 composition or single compound application (0.03% [v/w] Squalene)(FIG. 10b). Applied compound concentration was calculated according to the animal equivalent dose (AED) providing proposed doses for clinical application (FIG. 10c). In an additional experiment, pharmaceutical composition comprising interferon beta-1b (30.000 U) and a higher squalene dose of 0.5% [v/w] shows a stronger disease ameliorating effect then lower dosing of squalene (0.03% [v/w] Squalene) in combination with interferon beta-1b (30.000 U) (FIG. 11).

[0369] The applicant further explored therapeutic squalene strategies by combination of a pharmaceutical composition comprising interferon beta-1b (30.000 U) and squalene (0.5% [v/w]) and a ketogenic diet. Here, combination therapy with interferon beta-1b (30.000 U), squalene (0.5% [v/w]) and ketogenic diet shows higher potential to ameliorate disease severity in EAE than single or double compound therapy (FIG. 12a). Increased serum concentration of ketone bodies (β-Hydroxybutyrate) and decreased Glucose concentration was not affected by combination therapy (FIG. 12b). Amelioration of clinical disease severity was also reflected by reduced number of inflammatory cells in spinal cord tissue (FIG. 13a-c).

[0370] To test therapeutic efficiency of squalene in other neurodegenerative diseases with demyelinating pathology, squalene administration was tested in a mouse model of a hereditary leukodystrophy, Pelizaeus-Merzbacher disease. Here, squalene single compound treatment shows high potential to ameliorate clinical disease severity indicated by elevated beam testing (FIG. 14a). In addition, amelioration of clinical disease severity was also reflected by reduced number of inflammatory cells in brain and spinal cord tissue (FIG. 14b).

Example 3

[0371] Methods

[0372] Sterol abundance was quantified by lipid gas chromatography coupled to mass spectrometry (GC-MS). Samples were lyophilized at a shelf temperature of −56° C. for 24 h under vacuum of 0.2 mBar (Christ LMC-1 BETA 1-16) and weighed for calculation of water content and normalization as described53. For the sterol analysis by GC-MS analysis, lyophilized tissue was ground to a fine powder using a shaking mill and glass balls (5 mm). Metabolites were extracted in a two-phase system of Methyl-tert-butyl ether:Methanol 3:1 (v/v) and H2O, and pentadecanoic acid was added as an internal standard. 10-200 μl of organic phase was dried under a stream of nitrogen, dissolved in 10-15 μl pyridine and derivatised with twice the volume of N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) to transform the sterols and the standard to their trimethylsilyl (TMS)-derivatives. Each sample was analyzed twice, with a higher split to quantify cholesterol and with a lower split to measure all other sterols. The samples were analyzed on an Agilent 5977N mass selective detector connected to an Agilent 7890B gas chromatograph equipped with a capillary HP5-MS column (30 m×0.25 mm; 0.25 μm coating thickness; J&W Scientific, Agilent). Helium was used as carrier gas (1 ml/min). The inlet temperature was set to 280° C. and the temperature gradient applied was 180° C. for 1 min, 180-320° C. at 5 K/min and 320° C. for 5 min. Electron energy of 70 eV, an ion source temperature of 230° C., and a transfer line temperature of 280° C. was used. Spectra were recorded in the range of 70-600 Da/e. Sterols were identified by the use of external standards. (FIG. 16)