WO1993002685A1 - Plasmalopsychosines and plasmalocerebrosides and methods of treating neuronal diseases employing the same - Google Patents

Plasmalopsychosines and plasmalocerebrosides and methods of treating neuronal diseases employing the same Download PDF

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Publication number
WO1993002685A1
WO1993002685A1 PCT/US1992/005853 US9205853W WO9302685A1 WO 1993002685 A1 WO1993002685 A1 WO 1993002685A1 US 9205853 W US9205853 W US 9205853W WO 9302685 A1 WO9302685 A1 WO 9302685A1
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Prior art keywords
compound
acetal
lane
plasmalopsychosine
chirality
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PCT/US1992/005853
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French (fr)
Inventor
Edward Nudelman
Sen-Itiroh Hakomori
B. Steven Levery
Yasuyuki Igarashi
Khalid Sadozai
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The Biomembrane Institute
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Publication date
Application filed by The Biomembrane Institute filed Critical The Biomembrane Institute
Priority to DE69225712T priority Critical patent/DE69225712D1/en
Priority to JP5503579A priority patent/JPH06509354A/en
Priority to EP92915984A priority patent/EP0596937B1/en
Publication of WO1993002685A1 publication Critical patent/WO1993002685A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
    • C07H15/10—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical containing unsaturated carbon-to-carbon bonds

Definitions

  • the present invention relates to two newly isolated compounds
  • a and B collectively termed "plasmalopsychosines.”
  • Compound A is psychosine with a 3,4 cyclic acetal (C16 or C18).
  • Compound B is psychosine with a 3,4 cyclic acetal (C16 or C18).
  • psychosine galactosylsphi ⁇ gosine
  • the present invention also relates to two newly isolated com ⁇ pounds C and D, collectively termed "plasmalocerebrosides".
  • Com ⁇ pound C has an aliphatic aldehyde conjugated through a 3,4 cyclic
  • Compound D has an aliphatic aldehyde (plasmal) conjugated through
  • the fatty aldehyde can be, among others, paimital
  • Lipid components of cells are generally either acidic or neutral.
  • Acidic lipids include gangliosides, sulfatide, phosphoinositide, and phosphatidic acid.
  • Neutral lipids include neutral gi ⁇ colipids and neutral glycerides.
  • Anionic (basic) lipids such as sphingosine, N,N- dimeth ⁇ l-sphingosine and lyso-glycosphingolipids are assumed to be present as minor components modulating cellular functions, such as transmembrane signaling (1-4).
  • Kotchetkov et al. (13) described "sphingoplasmalogen" as a minor component of chromatographically fast-migrating cerebroside in brain.
  • the compound was assumed to have a structure with fatty aldehyde linked to the C3 hydroxyl group of galactosyl cerebroside through an unsaturated ether bond, based on infrared spectroscopy (absence of absorption at 1750 cm '1 for ester linkage); fatty alde ⁇
  • hydes were identified as p-nitro-phenylhydrazide under Wittenberg's conditions (1 ). The structure was claimed to be as shown below and termed "sphingo-plasmalogen".
  • sychosines of the present invention have much slower mobility and have two aliphatic chains (one sphingosine, one plasmal); also, the orientation of the aliphatic chains linked to the galactopyranosyl
  • a class of cerebrosides containing a fatty acid ester group A class of cerebrosides containing a fatty acid ester group
  • ester cerebrosides have also been isolated from brain. These compounds were shown to have much higher thin-layer chroma- tography (TLC) mobility than regular cerebroside (39,40).
  • TLC thin-layer chroma- tography
  • gangliosides and synthetic sialosyl compounds are potent stimulators of neuritogenesis, particularly in the presence of nerve growth factor.
  • NGF ganglio- side/nerve growth factor
  • tissue is of great interest.
  • one object of the invention is to provide four isolated or synthetic compounds that show remarkable neuritogenic
  • Another object of the invention is to provide compositions and methods for treatment of neuronal diseases and tissue damage.
  • n is a number greater than 0, and pharmaceutically accept ⁇ able salts thereof.
  • n 2 and n 3 each is a number greater than 0, and pharmaceuti ⁇
  • the present invention also provides a composition for treating neuronal diseases and tissue damage comprising one or more of the above-described plasmalopsychosines and/or plasmalocerebrosides and pharmaceutically acceptable salts thereof; and a pharmaceutically acceptable carrier, diluent or excipient.
  • the present invention further provides a method forming
  • neurites from nerve cells comprising contacting the cells with an
  • Figures 1 A and 1 B are high-performance thin-layer chromatog ⁇ raphy (HPTLC) patterns of anionic lipids adsorbed on carboxymethyl SEPHADEX and eluted with triethylamine in chloroform-methanol
  • Fig 1A thin-layer chromatograph was developed in chloroform-methanol- 28% NH 4 OH (80:20:2). Bands were detected
  • Lane 1 total eluate from carboxymethyl SEPHADEX column with 0.5 M triethylamine; lane 2, purified
  • Fig. 1B The same chromatogram as in Fig. 1A. Bands were detected by spraying with 0.01 % PRIMULINE and viewed under UV light.
  • Figure 2 is an HPTLC pattern of anionic lipid from various
  • FIG. 3 is an HPTLC pattern of purified plasmalopsychosine
  • Lane 1 compound A; lane 2, compound A treated in 0.3 N HCI in MeOH 80°C 30 minutes; lane 3, compound A treated with 0.3 N NaOH in MeOH, 80°C 40 minutes; lane 4, standard psychosine; lane 5, compound B; lane 6, compound B treated in 0.3 N HCI in MeOH
  • Figures 4A and 4B are the data from gas chromatography- chemical ionization/mass spectrometry (GC-CI/MS) of long chain
  • Figures 5A to 5D are the data from fast atom bombardment-
  • Fig. 5A + FAB mass spectrum of upper band lipid in 3-nitrobenzyl alcohol (NBA) matrix
  • Fig. 5B + FAB mass spectrum of middle band lipid in NBA matrix
  • Fig. 5C + FAB mass spectrum of middle band lipid in NBA/sodium acetate
  • Figures 6A-6D are data from + FAB-MS of products of treat ⁇
  • Figures 7A-7D are data from + FAB-MS of products of acetyla- tion/deacetylation: Fig. 7A, peracetylated upper band lipid in NBA
  • Fig. 7B peracetylated middle band lipid in NBA matrix
  • Fig. 7C peracetylated middle band lipid in NBA/sodium acetate matrix
  • Fig. 7D peracetylated and de-O-acetylated middle band lipid in NBA matrix (inset: same product in NBA/sodium acetate matrix, showing no change in masses of pseudomolecular ions).
  • Figures 8A-8D are data from ' GC-MS analysis of partially methylated alditol acetates (PMAAs) from permethylation, hydrolysis, reduction, and acetylation of lipids: Fig. 8A, PMAA from upper band
  • Fig. 8B PMAA from middle band lipid
  • Fig. 8C PMAA from upper band lipid following brief acid treatment
  • Fig. 8D standard
  • Figures 9A and 9B are neuritogenesis patterns of Neuro-2A
  • FIG. 5B show different areas of the culture dish.
  • Figure 10 is a graph showing the effect of plasmalopsychosine on neurite formation in Neuro-2A cells: Abscissa: concentration of plasmalopsychosine ( g/ml). Ordinate: percentage of Neuro-2A cells developing neurites ( > 50 ⁇ m in length).
  • the circles (open and closed) represent results for a mixture of the upper and middle bands of plasmalopsychosine, + and - nerve growth factor (NGF); the open
  • triangles represent results in the presence of NGF for a mixture of
  • bovine brain gangliosides containing the gangliosids GM1 ,
  • Figure 1 1 is a high-performance thin-layer chromatography (HPTLC) pattern of various non-polar glycosphingolipids from Folch's
  • Lane 1 standard cerebroside (CMH); lane 2,
  • FIG 12 is an HPTLC pattern of cerebroside (CMH), plasma ⁇
  • Figure 13 is a gas chromatography-electron impact/mass spectrometry (GC-EI/MS) pattern of long chain fatty acid methyl
  • Peaks marked by an asterisk are unidentified impurities.
  • Figure 14 is a positive ion fast atom bombardment ( + FAB) mass spectrum of unknown lipid component in a 3-nitrobenzyl alcohol (NBA) matrix. The peaks are labelled with nominal, monoisotopic
  • Figure 15 is a gas chromatography-mass spectrometry (GC-MS)
  • Peaks are identified as PMAAs of 1: 2,3,4,6-tetra-
  • Figure 16 is a scheme for synthesizing plasmalopsychosine
  • Figure 17 is a HPTLC pattern of fractions obtained on an lATROBEADS column of the plasmalopsychosine synthetic products
  • Figures 18A-18C are data from GS-MS analysis of partially
  • anionic lipid through cation exchange chromatography in chloroform- methanol followed by a series of chromatographies on a FLORISIL and lATROBEADS column has been developed.
  • the major anionic lipids
  • n is a number greater than 0 and preferably 14 or 16.
  • Plasmalopsychosines regardless of the position of the acetal linkage, have strong neuritogenic effects on neuroblastoma cells, particularly in the presence of nerve growth factor (NGF).
  • NGF nerve growth factor
  • compounds A and B can be isolated by
  • anionic lipids and anionic glycosphingolipids from human brain.
  • the lipids are extracted and a lower layer is prepared. This is
  • HPLC high-performance thin-layer chromatography
  • brum is dissected and separated into gray and white matter with a razor blade.
  • a razor blade to scrape the outer layer of cortex, it is possible to obtain a near-pure gray matter fraction weighing about 50 g from adult human brain.
  • White matter is considerably easier to prepare by cutting the brain into vertical sections and separating large areas of white matter.
  • tissue is homogenized in about five volumes (i.e., five times volume/weight of wet tissue) of isopropanol/hexane/water (IHW)
  • CM SEPHADEX chromatography The anionic lipid fraction is prepared from the total lower layer lipid by CM SEPHADEX chromatography. CM SEPHADEX is carefully washed and equilibrated using the following protocol. It is crucial that the SEPHADEX is equilibrated
  • TEA is removed from the SEPHADEX by extensive washing in MW 1 :1.
  • the equilibrated CM SEPHADEX is then washed with 100% methanol followed by CMW 40:60:5 (hereinafter "sol A").
  • sol A is added until
  • the brain extract is completely dissolved.
  • about 1 L solvent is required for 500 gm of tissue. This solution is passed over a bed of
  • CM SEPHADEX having a volume of 50-200 ml (about 100 ml per kg wet tissue) and allowed to elute by gravity filtration. An additional amount of sol A is washed through the column and the
  • CM SEPHADEX 0.5 M TEA fraction from CM SEPHADEX is evaporated to dryness several times using absolute ethanol to rid the sample of TEA. The fraction is then transferred to a test tube and dissolved in
  • HPTLC chromatography
  • HPLC high performance liquid chromatography
  • a long column e.g., about 0.4x60 cm
  • lATROBEADS sica gel; 10 ⁇ M
  • the 0.5 M TEA fraction is prepared for injection by evaporating to dryness and dissolving in 100% hexane in the following manner. For a 2 ml injection, 100 ⁇ of chloroform/methanol (2:1 ) is added,
  • the cap is screwed on tightly, and the sample is slightly warmed to about 50°C and sonicated to form a thick oil. In most cases, this
  • the sample is loaded onto the column and subjected to a
  • Carbohydrate analysis can be performed by gas chromatogra- phy-mass spectrometry (GC-MS) employing trimethysilyl derivatives
  • FAB-MS Fast atom bombard ⁇ ment-mass spectrometry
  • n 2 and n 3 each is a number greater than 0, and pharma ⁇
  • n 2 is 14 or 16 and n 3 is 12, 14, 16, 18, 20, 21, 22, 23 or 24.
  • the human brain cerebroside fraction can be
  • magnesium oxide and silicic acid gel (from Sigma; mesh 60-100) is
  • the presence of acid-labile glycolipids can be detected by hydrolysis of samples in methanol/aqueous 0.1 N HCI (1 :1 , v/v) heated at about 90°C for about 10 minutes, followed by Folch partitioning and thin-layer chromatography (TLC) examination of the
  • labile fast-migrating glycolipid is a consistent component of brain extract, and is found in the unabsorbed fraction on carboxymethyl-
  • HPLC high-performance liquid chromatography
  • fraction VI contains most of the acid-labile glycolipid and is free of cerebroside and ester cerebrosides. The fraction VI is further
  • fraction VI A is further purified on a long lATROBEADS column (e.g., about 0.5x100 cm) with a shallow lATROBEADS column (e.g., about 0.5x100 cm) with a shallow lATROBEADS column (e.g., about 0.5x100 cm) with a shallow lATROBEADS column (e.g., about 0.5x100 cm) with a shallow lATROBEADS column (e.g., about 0.5x100 cm) with a shallow
  • the compounds can be purified by preparative TLC. A homogeneous band is obtained as shown in Fig. 11, lane 6. On TLC, the compounds migrate faster than cholesterol which migrates faster than ester cerebrosides. The compounds do not contain sulfate or sialic acid which are known to be acid-labile.
  • the structure of compounds C and D can be determined after methanolysis by identifying enol methyl ethers derived from fatty aldehydes by gas chromatography-mass spectrometry (GC-MS) analysis and by fast atom bombardment mass spectrometry (FAB-MS)
  • the acetal linkage can be determined by methylation analysis. Following permethyiation, acid hydrolysis, reduction, and acetylation of the native lipid, the resulting partially methylated hexitol acetates are analyzed by GC-MS as described in detail in Example 7.
  • neurites can be any suitable material. According to a further aspect of this invention, neurites can be any suitable material.
  • the nerve cells such as from neuroblastoma cell lines, are cultured in gelatin-coated plates by known methods (11 ,12).
  • the effective dose is determined by adding various concentrations (e.g., 5-150 ⁇ M) of one or more of compounds A, B, C and D, and the cells are cultured for the observation of neurite formation, as described in more
  • neurogenic activity in a variety of neuroblastoma cells is often used as a criterion to evaluate ability of candidate reagents to repair neuronal
  • Both plasmalocerebroside compounds C and D also display remarkable neurogenic activity in a variety of neuroblastoma cells. No clear effect in the early stages of cell culture is observed, but neurite formation, i.e. neurites > 50 ⁇ m long, becomes increasingly apparent
  • the present invention provides a composition for treating neuronal diseases and tissue damage comprising one or more of compounds A, B, C and D and pharmaceutically acceptable salts thereof; and a pharmaceutically acceptable carrier, diluent or
  • the present invention also provides a method for treating
  • neuronal diseases and tissue damage comprising administering to a host in need of treatment a biologically effective amount of one or more of the compounds A, B, C and D, and pharmaceutically acceptable salts thereof.
  • Specific cases include treatment of Alzheimer's disease, spinal
  • the effective amount of compounds A, B, C, and D can be determined using art-recognized methods, such as by establishing
  • Suitable doses of medicaments of the instant invention depend upon the particular medical application, such as the severity of the
  • the number of doses, daily dosage and course of treatment may vary from individual to individual.
  • the compounds A, B, C and/or D can be administered in a
  • Suitable pharmaceutically acceptable carriers, diluents, or excipients for the medicament of the instant invention depend upon the particular medical use of the medicament and can be determined readily by the skilled artisan.
  • the medicament can be formulated into solutions, emulsions, or suspensions.
  • the medicament is likely to contain any of a variety
  • Such subsidiary ingredients include disintegrants, binders (including liposomes), surfactants, emulsifiers, buffers, soiubilizers and preservatives.
  • disintegrants include disintegrants, binders (including liposomes), surfactants, emulsifiers, buffers, soiubilizers and preservatives.
  • binders including liposomes
  • surfactants include emulsifiers, buffers, soiubilizers and preservatives.
  • tissue was also used as a source of extraction. In all cases, tissue was homogenized in five volumes (i.e., five times vol ⁇ ume/weight of wet tissue) of isopropanol/hexane/water (IHW) (55:25:20 v/v/v, upper phase removed), filtered over a B ⁇ chner
  • CM SEPHADEX carboxymethyl
  • the dry resin was washed extensively over a B ⁇ chner funnel in 0.2 N HCI and allowed to soak for several hours in the acid. The resin is then washed extensively with deionized water with intermit ⁇ tent soaking, followed by stepwise washing with methanol/water
  • TEA triethylamine
  • sol A was added until the solution became totally soluble. For 500 gm of tissue, this
  • Figs. 1A and 1 B The results are shown in Figs. 1A and 1 B.
  • Fig. 1 A TLC was developed in chloroform-methanol-28% NH 4 OH (80:20:2). Bands were detected by orcinol-sulfuric acid.
  • Lane 1 total eluate from carboxymethyl SEPHADEX column with 0.5 M TEA; lane 2, purified compound A; lane 3, purified compound B; lane 4, purified compound C; lane 5, sphingosine.
  • Fig. 1 B is the same chromatogram as in Fig. 1 B, but the bands were detected by spraying with 0.01 % PRIMULINE and viewed under UV light.
  • Fig. 1A which shows the pattern with orcinol-sulfuric acid
  • CAMINE (Sigma). It was also possible to detect compounds A and B with 0.5% orcinol (Sigma) in 10% sulfuric acid followed by baking in a thin-layer chromatography (TLC) oven.
  • the 0.5 M TEA fraction was prepared for injection by evaporat ⁇ ing to dryness and redissolving in 100% hexane in the following manner. For a 2 ml injection, 100 ⁇ of chloroform/methanol (2:1 ) was added, the cap was screwed on tightly, and the sample was
  • the sample was loaded onto the column and subjected to gradient eluting at 0.5 ml/min. Gradient elution was started from the
  • sphingosine was also conveniently purified, as well as a slower migrating sphingosine analog (Fig. 1B, lanes 4,5).
  • cerebrum were collected from the same human brain and processed side by side to obtain lower phases. Equal weights of cerebellum and brainstem were also obtained. These samples were passed over CM
  • Fig. 2 shows the orcinol staining of various fractions from gray and white matter, cerebellum, and brainstem.
  • the lanes are as follows: lanes 1 and 10, standard CMH; lane 2, lower phase from white matter; lane 3, lower phase
  • Fig. 3 is the HPTLC pattern of purified plasmalopsychosine and degradation product by weak acid
  • the lanes in Fig. 3 are as follows: lane 1 , compound A; lane
  • n-hexadecanol and n-octadecanol purchased from
  • Aledhydes were converted to enol methyl ethers (EMEs) by treatment with 0.5 N HCI/5 M H 2 O in methanol at 80°C for 5.5 hr.
  • the methanolysate was cooled and extracted 3x with hexane.
  • the combined hexane extracts were evaporated under N 2 stream at 37°C
  • Extrel ELQ 400 quadrupole mass spectrometer Gas chromatoraphy was performed using a 30 m DB-5 (J & W Scientific, Collins Cordova, CA) bonded-phase fused silica capillary column (0.25 mm o.d., 0.25 ⁇ film thickness; splitless injection; temperature program, 140-
  • Lipid samples 50-100 ⁇ g were methanolyzed in 1.0 ml 0.5 N HCI in anhydrous MeOH for 24 hr at 80°C. The methanolysate was cooled and extracted 3x with hexane. The acidic MeOH lower layer was neutralized by addition of Ag 2 CO 3 (approximately 10 mg) and treated with acetic anhydride (100 ⁇ l) for 6 hr at room temperature. Following centrifugation and removal of
  • Methylation/linkaoe anal y sis Linkage positions of substituents on glycosyl residues were determined by permethylation of approxi ⁇ mately 50 ⁇ g of each sample (see previous section), followed by hydrolysis, reduction, peracetylation and GC-MS as described in detail
  • N,Q-acetylated de-Q-acetylated lipids were analyzed by + FAB-MS using NBA matrix, with and without addition of sodium acetate.
  • lipids could be stained with orcinol, indicating the presence of some carbohydrate component, they were subjected to monosaccharide analysis, by GC-MS of trimethysilyl methyl glycosides produced
  • esters were detected in any of the lipid fractions analyzed. A number of unknown peaks were observed. Following evaluation of the results of FAB-MS analysis of the intact lipids (described below), the identity of these peaks was carefully determined, and several major compo ⁇ nents found to correspond to long chain enol methyl ethers. Two components were found to be identical in retention times and mass spectra to enol methyl ethers prepared by acidic methanolysis of authentic 16:0 and 18:0 long chain aldehydes. Two other compo ⁇
  • Figures 4A and 4B are the results of gas chromatography- chemical ionization/mass spectrometry (GC-CI/MS) of long chain methyl enol ethers.
  • Fig. 4A shows the results from methanolysis of
  • Fig. 4B shows the results from methanolysis of standard n-16:0 and -18:0 aldehydes. Peaks were identified as 1 :
  • Figure 5 shows FAB-MS of native lipids: Fig. 5A, + FAB mass
  • Fig. 5D FAB mass spectrum of middle band lipid in TEA/15- crown-5 matrix.
  • Figs. 5A, 5B and 5C may correspond to loss of a portion of one molecule in the dimeric species, although the exact nature of this loss is not clear at this time. Since they represent the loss of an odd mass fragment (257 u), one may assume that it is a portion of a sphingosine chain including the nitrogen atom that is
  • Figure 6 shows + FAB-MS of products of treatment
  • HCI/HgCI 2 Matrix: NBA.
  • A upper band lipid, following brief acid
  • C lower band lipid following extended acid treatment
  • D d18: 1 galactopsychosine standard.
  • lipids Peracetylation of the native lipids with acetic anhydride/pyri- dine resulted in incorporation of four acetate groups, as illustrated in Figs. 7A, 7B and 7C.
  • Fig. 7A is peracetylated upper band lipid in NBA matrix
  • Fig. 7B is peracetylated middle band lipid in NBA matrix
  • Fig. 7C is peracetyl ⁇ ated middle band lipid in NBA/sodium acetate matrix
  • Fig. 7D is peracetylated and de-O-acetylated middle band lipid in NBA matrix
  • the sphingosine fragment can eliminate one and two molecules of HOAc, to yield the ions at m/z 324 and 264, respectively. Elimination of HOAc from the fragment at m/z 366 yields the ion at m/z 306.
  • Figs. 8 A and 8B are PMAA from upper band lipid; Fig. 8B is PMAA from middle band lipid; Fig. 8C is PMAA from upper band lipid following brief acid treatment; Fig. 8D is standard galactose PMAAs.
  • Peaks are identified as PMAAs of 1 : 2,3,6-tri-O-; 2: 3,4,6 + 2,4,6-tri-O-; 3: 2,3 4-tri-O-; 4: 2,6-di-O-; 5: 4,6-di-O-; 6: 3,6-di-O-; 7: 2,3-di-O-; 8: 6-mono-O-; 9: 3,4-di-O-; 10: 2-mono-O-; and 11 : 3 (or 4)-mono-O-
  • Human brain cerebroside fraction was obtained by homogeniza ⁇ tion of brain tissue with five volumes (i.e., five times volume/weight
  • FLORISIL a mixture of magnesium oxide and silicic acid gel
  • the dried lower phase was suspended in hexane (1 L per 200 g original tissue), passed over the FLORISIL column, and
  • Acid-labile glycolipids were detected by hydrolysis of samples in methanol-aqueous ⁇ .1 N HCI (1 :1 , v/v) heated at 90°C for 10 min, followed by Folch partitioning and TLC examination of lower phase.
  • glycolipid with high TLC mobility converting to the same mobility as normal cerebroside by this treatment was regarded as the acid-
  • Cerebroside and ester cerebrosides did not show altered TLC mobility under these conditions.
  • the acid-labile fast-migrating glycolipid was found in the unab-
  • fraction VI The pooled fraction (called fraction VI) was considered to contain most of the
  • fraction VI was further purified by lATROBEADS chromatog- raphy, loaded on the columns in pure hexane and subjected to a gradient up to isopropanol/hexane (IH) 30:70.
  • the fraction VI A ( Figure 11 , lane 5), thus obtained, was further purified on a long
  • Figure 11 is a high-performance thin layer chromatography
  • HPTLC HPLC pattern of various non-polar glycosphingolipids from Folch's lower phase prepared from human brain. The chromatogram was developed in a solvent mixture of chloroform/methanol/28% NH 4 OH (80:20:2). Lane 1 is standard CMH (cerebroside); lane 2 is lower
  • lane 6 is purified plasmal cerebroside from Fraction 47-58; lane 7 is purified ester-cerebrosides.
  • Lane 1 CMH; lane 2, CMH degraded by 0.3 N HCI in MeOH;
  • Fatty acids were estimated as methylesters (FAMEs) liberated by methanolysis (1.0 ml 0.5 N HCI in anhydrous methanol, 80°C, 24 hr) of about 30-
  • glycosyl residues were determined by permethylation of approxi ⁇ mately 50 ⁇ g of each sample, followed by hydrolysis, reduction, peracetylation and GC-MS as described in detail elsewhere (22), except that the analysis was performed on the Extrel ELQ 400 GC-MS system described above (DB-5 column; splitless injection; temperature
  • EMEs enol methyl ethers
  • Fig. 13 is a CG-EI/MS of long chain FAMEs and EMEs from methanolysis of the unknown lipid component. The peaks were
  • Peaks marked by an asterisk are unidentified
  • Ceramide ions (Y 0 ) were found most abundantly at m/z 520, 546, 548, and 630, corresponding to compositions having d18:1 sphingosine N-acylated primarily with 16:0, 18:1 , 18:0, and 24:1 fatty acids. These would be expected on the basis of the
  • olecular ion at m/z 956 would correspond to a galactocerebroside
  • the peaks are identified as PMAAs of 1 : 2,3,4,6- tetra-O-; 2: 2,6-di-O-; and 3: 4,6-di-O-Me-Gal.
  • the primary component detected was 2,6-di- ⁇ -Me-Gal, along
  • the di-O_-Me- peaks represent 3,4- and 4,6-linked substituents, respectively, on galactose and show that the lipid fraction must be comprised of isomeric cyclic acetals derived from cerebroside, mostly in a five-membered 3,4-linked ring, with some
  • Gal is consistent with the low abundance pseudo-molecular ion
  • toma cell lines in which neurite formation is dependent either on nerve growth factor (NGF) or gangiioside.
  • NGF nerve growth factor
  • Neuroblastoma cell lines were cultured in gelatin-coated plates as described previously (11,12).
  • bovine brain ganglioside required at least 100-150 g/ml.
  • Other types of cells including mouse and human neuroblasto ⁇ ma, showed similar degrees of neuritogensis induced by plasmalopsy- chosine.
  • Psychion by itself showed a strong cytotoxic effect on various neuroblastoma cell lines; cell growth was inhibited, morpholo ⁇
  • Figures 9A and 9B show a neuritogenesis pattern of Neuro-2A cells in the presence of 50 //g/ml plasmalopsychosine compounds A and B at different areas on the culture dish.
  • Figure 10 is a graph showing the effect of plasmalopsychosine on neurite formation in Neuro-2A cells, wherein the abscissa represents the concentration of plasmalopsychosine (//g/ml) and the ordinate represents the percentage of Neuro-2A cells developing neurites (> 50//m in length).
  • the circles (open and closed) represent results for a mixture of the upper and middle bands of plasmolo- psychosine, + and - nerve growth factor (NGF).
  • NTF nerve growth factor
  • BCG bovine brain gangliosides
  • the neurite formation is due to the plasmalopsychosine.
  • Plasmalocerebroside had no clear effect in early stages of cell culture. However, neurite formation, i.e., > 50 //m long, became
  • plasmalocerebroside possesses neuritogenic activity. With 50 //g/ml concentration, after
  • Plasmalopsychosine A and B were chemically synthesized from
  • Cyclic acetals were prepared as follows: To a solution of FMOC-psychosine in N,N-dimethylformamide, ⁇ - ⁇ -dimethoxy hexadecane and p-toluene sulfonic acid were added and the reaction mixture was stirred at room temperature for 19 hours. Then the
  • the sample was prepared for injection by adding 100 ⁇ of chloroform/methanol 2: 1 and slightly warning while sonicating.
  • Lane 1 is crude synthetic preparation of psychosine acetals
  • Lanes 2- 5 are pooled fractions of synthetic product from HPLC on an IATROBEAD column
  • Lane 6 is total eluate of anionic lipid

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Abstract

An isolated or synthetic plasmalopsychosine selected from the group consisting of compound A and compound B, wherein n1 is a number greater than 0, and pharmaceutically acceptable salts thereof. An isolated or synthetic plasmalocerebroside selected from the group consisting of compound C and compound D, wherein n2 and n3 each is a number greater than 0; and pharmaceutically acceptable salts thereof. The plasmalopsychosines and plasmalocerebrosides are useful to treat neuronal diseases and tissue damage.

Description

PLASMALOPSYCHOSINES AND PLASMALOCEREBROSIDES AND
METHODS OF TREATING NEURONAL
DISEASES EMPLOYING THE SAME
FIELD OF THE INVENTION
i '•' 5 The present invention relates to two newly isolated compounds
A and B, collectively termed "plasmalopsychosines." Compound A is psychosine with a 3,4 cyclic acetal (C16 or C18). Compound B
is psychosine (galactosylsphiπgosine) with a 4,6 cyclic acetal of a
C16 or C18 aldehyde. These compounds display remarkable neurito-
10 genie activities in a variety of neuroblastoma cells.
The present invention also relates to two newly isolated com¬ pounds C and D, collectively termed "plasmalocerebrosides". Com¬ pound C has an aliphatic aldehyde conjugated through a 3,4 cyclic
acetal linkage at the galactopyranosyl moiety of a cerebroside.
15 Compound D has an aliphatic aldehyde (plasmal) conjugated through
a 4,6 cyclic acetal linkage at the galactopyranosyl moiety of a cerebroside. The fatty aldehyde can be, among others, paimital
(C16:0), stearal (C18:0) or one having an olefinic double base
(C18:1 ).
20 BACKGROUND OF THE INVENTION
Lipid components of cells are generally either acidic or neutral. Acidic lipids include gangliosides, sulfatide, phosphoinositide, and phosphatidic acid. Neutral lipids include neutral giγcolipids and neutral glycerides. Anionic (basic) lipids such as sphingosine, N,N- dimethγl-sphingosine and lyso-glycosphingolipids are assumed to be present as minor components modulating cellular functions, such as transmembrane signaling (1-4).
Kotchetkov et al. (13) described "sphingoplasmalogen" as a minor component of chromatographically fast-migrating cerebroside in brain. The compound was assumed to have a structure with fatty aldehyde linked to the C3 hydroxyl group of galactosyl cerebroside through an unsaturated ether bond, based on infrared spectroscopy (absence of absorption at 1750 cm'1 for ester linkage); fatty alde¬
hydes were identified as p-nitro-phenylhydrazide under Wittenberg's conditions (1 ). The structure was claimed to be as shown below and termed "sphingo-plasmalogen".
Figure imgf000004_0001
However, the presence of sphingoplasmalogen or any bound aliphatic (fatty) aldehyde (piasmal) in glycosphingolipid was denied in four subsequent investigations (18, 42, 43, 44). Further, extensive studies of multiple fast migrating cerebroside extensively studied b
Klenk and Lδhr (15), Tamai et al. (16,17), and Kishimoto et al. (18) concluded that all these fast-migrating glycosphingolipids are cerebrosides esterified at different positions of the hydroxyl groups with fatty acid. These previously-reported compounds, whether sphingoplasmalogen or fast-migrating ester cerebrosides, showed very
different thin-layer chroma-tography mobility compared to the plasmalopsychosines of the present invention. That is, the plasmalop-
sychosines of the present invention have much slower mobility and have two aliphatic chains (one sphingosine, one plasmal); also, the orientation of the aliphatic chains linked to the galactopyranosyl
moiety appears to be in an entirely opposite direction.
The fatty aldehyde (or long-chain aliphatic aldehyde), termed "plasmal," was originally discovered by Feulgen & Voit in 1924 (19), and was recognized as a component of a giycerophospholipid termed piasmalogens in 1929 (6). The structure of plasmalogen, originally
claimed to be a 1 ,2-cyclic acetal linkage (37), was eventually identified as 1-alkenyl-2-acyl-3-phosphorylcholine (20).
A class of cerebrosides containing a fatty acid ester group and
termed ester cerebrosides have also been isolated from brain. These compounds were shown to have much higher thin-layer chroma- tography (TLC) mobility than regular cerebroside (39,40). The
locations of the fatty acid were identified to be the C3 hydroxyl group of sph.ngosine and the C3 or C6 hydroxyl group of galactose (15, 16, 17, 18).
Neuroblastoma cell lines have been widely used to screen substances having possible promoting effects on neuritogenesis in
vivo. Some gangliosides and synthetic sialosyl compounds are potent stimulators of neuritogenesis, particularly in the presence of nerve growth factor. In a recent study, administration of a ganglio- side/nerve growth factor (NGF) mixture to patients with Alzheimer's syndrome was claimed to improve clinical symptoms (21 ). Similarly, following neural tissue damage provoked by various factors, adminis¬ tration of a gangiioside mixture has been claimed to produce partial recovery.
In view of the possible involvement of sphingosine, N, ,N- dimethylsphingosine and lyso-glycosphingolipids in modulation of transmembrane signaling ( 1 -4) chemical identification, purification and
characterization of these compounds occurring naturally in neural
tissue is of great interest.
SUMMARY OF THE INVENTION
Accordingly, one object of the invention is to provide four isolated or synthetic compounds that show remarkable neuritogenic
activity. Another object of the invention is to provide compositions and methods for treatment of neuronal diseases and tissue damage.
These and other objects have been achieved by providing an isolated or synthetic plasmalopsychosine selected from the group consisting of compound A and compound B:
( « )
Figure imgf000007_0001
CH3- C H , ( B >
Figure imgf000007_0002
wherein n, is a number greater than 0, and pharmaceutically accept¬ able salts thereof.
The objects of the present invention have also been achieved by providing an isolated plasmalocerebroside selected from the group consisting of compound C and compound D:
Figure imgf000008_0001
j ) -CH 3 < D )
Figure imgf000008_0002
wherein n2 and n3 each is a number greater than 0, and pharmaceuti¬
cally acceptable salts thereof.
The present invention also provides a composition for treating neuronal diseases and tissue damage comprising one or more of the above-described plasmalopsychosines and/or plasmalocerebrosides and pharmaceutically acceptable salts thereof; and a pharmaceutically acceptable carrier, diluent or excipient.
The present invention further provides a method forming
neurites from nerve cells comprising contacting the cells with an
effective amount of one or more of the above-identified plasmalo¬ psychosines and/or plasmalocerebrosides.
The present invention additionally provides a method of treating neuronal diseases and tissue damage comprising administering to a host in need of treatment a biologically effective amount of one or
moreofthe above-describedplasmalopsychosinesand/orplasmalocer- ebrosides.
BRIEF DESCRIPTION OF THE FIGURES
Figures 1 A and 1 B are high-performance thin-layer chromatog¬ raphy (HPTLC) patterns of anionic lipids adsorbed on carboxymethyl SEPHADEX and eluted with triethylamine in chloroform-methanol
mixture: Fig 1A: thin-layer chromatograph was developed in chloroform-methanol- 28% NH4OH (80:20:2). Bands were detected
by orcinol-sulfuric acid. Lane 1 , total eluate from carboxymethyl SEPHADEX column with 0.5 M triethylamine; lane 2, purified
compound A; lane 3, purified compound B; lane 4, purified compound
E; lane 5, sphingosine. Fig. 1B: The same chromatogram as in Fig. 1A. Bands were detected by spraying with 0.01 % PRIMULINE and viewed under UV light.
Figure 2 is an HPTLC pattern of anionic lipid from various
regions of human brain. Anionic lipids were isolated by chromatogra-
phy on carboxymethyl SEPHADEX and eluated with 0.5 M triethyl¬ amine and the thin layer chromatograph was developed in chloro- form/methanol/NH4OH (80:20:2). Lanes 1 and 10, standard ceramide monohexoside (CMH); lane 2, lower phase from white matter; lane 3, lower phase from cerebellum; lane 4, lower phase from brain stem; lane 5, lower phase from gray matter; lane 6, 0.5 M triethylamine
eluate from carboxymethyl SEPHADEX column of white matter; lane
7, the same fraction as in lane 6 but prepared from cerebellum; lane
8, the same fraction as in lane 6 but prepared from brain stem; lane
9, the same fraction as in lane 6 but prepared from gray matter.
Figure 3 is an HPTLC pattern of purified plasmalopsychosine
and degradation products by weak acid and alkaline treatment: Lane 1 , compound A; lane 2, compound A treated in 0.3 N HCI in MeOH 80°C 30 minutes; lane 3, compound A treated with 0.3 N NaOH in MeOH, 80°C 40 minutes; lane 4, standard psychosine; lane 5, compound B; lane 6, compound B treated in 0.3 N HCI in MeOH
80°C 30 minutes; lane 7, compound B treated in 0.3 N NaOH in MeOH 80°C 40 minutes; lane 8, CMH. Figures 4A and 4B are the data from gas chromatography- chemical ionization/mass spectrometry (GC-CI/MS) of long chain
methyl enol ethers: Fig. 4A, from methanolysis of the middle band
lipid; Fig 4B, from methanolysis of standard n-16:0 and -18:0
aldehydes. Peaks were identified as 1 , 16:0; 2a and 2b, isomeric
18:1 ; and 3, 18:0 methyl enol ethers, having pseudomolecular ion
masses of 255, 281 , and 283 u, respectively. Peaks marked by asterisk are impurities common to both samples, probably arising from
the derivatization reagents. Figures 5A to 5D are the data from fast atom bombardment-
mass spectrometry (FAB-MS) of native lipids: Fig. 5A, +FAB mass spectrum of upper band lipid in 3-nitrobenzyl alcohol (NBA) matrix; Fig. 5B, +FAB mass spectrum of middle band lipid in NBA matrix; Fig. 5C, +FAB mass spectrum of middle band lipid in NBA/sodium acetate
matrix; Fig. 5D, FAB mass spectrum of middle band lipid in triethyl¬
amine (TEA) 15-crown-5 matrix.
Figures 6A-6D are data from + FAB-MS of products of treat¬
ment with HCI/HgCI2: Matrix: NBA. Fig. 6A, upper band lipid, following brief acid treatment, resulting in conversion to middle band
lipid; Fig. 6B, upper band lipid following extended acid treatment; Fig.
6C, lower band lipid following extended acid treatment; Fig. 6D,
d18:1 galactopsychosine standard. Figures 7A-7D are data from +FAB-MS of products of acetyla- tion/deacetylation: Fig. 7A, peracetylated upper band lipid in NBA
matrix; Fig. 7B, peracetylated middle band lipid in NBA matrix; Fig. 7C, peracetylated middle band lipid in NBA/sodium acetate matrix; Fig. 7D, peracetylated and de-O-acetylated middle band lipid in NBA matrix (inset: same product in NBA/sodium acetate matrix, showing no change in masses of pseudomolecular ions).
Figures 8A-8D are data from' GC-MS analysis of partially methylated alditol acetates (PMAAs) from permethylation, hydrolysis, reduction, and acetylation of lipids: Fig. 8A, PMAA from upper band
lipid; Fig. 8B, PMAA from middle band lipid; Fig. 8C, PMAA from upper band lipid following brief acid treatment; Fig. 8D, standard
galactose PMAAs. Peaks are identified as PMAAs of 1: 2,3,6-tri-O; 2: 3,4,6 + 2,4,6-tri-O; 3: 2,3,4-tri-O; 4: 2,6-di-O; 5: 4,6-di-O; 6: 3,6- di-O; 7: 2,3-di-O; 8: 6-mono-O-; 9: 3,4-di-O; 10: 2-mono-O-; and 11 :
3 (or 4)-mono-O-Me-Gal.
Figures 9A and 9B are neuritogenesis patterns of Neuro-2A
cells in the presence of 50/yg/ml plasmalopsychosine. Figs. 5A and
5B show different areas of the culture dish. Figure 10 is a graph showing the effect of plasmalopsychosine on neurite formation in Neuro-2A cells: Abscissa: concentration of plasmalopsychosine ( g/ml). Ordinate: percentage of Neuro-2A cells developing neurites ( > 50 μm in length). The circles (open and closed) represent results for a mixture of the upper and middle bands of plasmalopsychosine, + and - nerve growth factor (NGF); the open
triangles represent results in the presence of NGF for a mixture of
bovine brain gangliosides (BBG) containing the gangliosids GM1 ,
GD1 a, GDI b and GT.
Figure 1 1 is a high-performance thin-layer chromatography (HPTLC) pattern of various non-polar glycosphingolipids from Folch's
lower phase prepared from human brain. The chromatograph was
developed in a solvent mixture of chloroform-methanol-28% NH4OH
(80:20:2 by volume). Lane 1 , standard cerebroside (CMH); lane 2,
lower phase obtained on Folch's partition; lane 3, unabsorbed pass through of total lower phase by carboxy-methyl SEPHADEX; lane 4,
Fr. VI obtained from FLORISIL column eluted by dichloroethane-
acetone (1 :1 , by volume); lane 5, Fraction 47-58 eluate from
IATROBEAD chromatography; lane 6, purified plasmal cerebroside from Fraction 47-58; lane 7, purified compounds C and D.
Figure 12 is an HPTLC pattern of cerebroside (CMH), plasma¬
locerebrosides C and D and ester cerebroside and their degradation pattern with weak acid and weak base: Lane 1 , CMH; lane 2, CMH
degraded bγ 0.3 N HCI MeOH; lane 3, CMH treated with 0.3 N NaOH; lane 4, plasmalocerebroside; lane 5, plasmalocerebroside treated with 0.3 N HCI in MeOH; lane 6, plasmalocerebroside treated with 0.3 N NaOH in MeOH; lane 7, ester cerebroside 1; lane 8, ester cerebroside 1 treated with 0.3 N HCI in MeOH; lane 9, ester cerebro¬
side 1 treated with 0.3 N NaOH in MeOH; lane 10, ester cerebroside
2; lane 11 , ester cerebroside 2 treated with 0.3 N HCI in MeOH; lane
12, ester cerebroside 2 treated with 0.3 N NaOH in MeOH.
Figure 13 is a gas chromatography-electron impact/mass spectrometry (GC-EI/MS) pattern of long chain fatty acid methyl
esters (FAMEs) and enol methyl ethers (EMEs) from methanolysis of unknown lipid component: The peaks were identified as marked.
Peaks marked by an asterisk are unidentified impurities.
Figure 14 is a positive ion fast atom bombardment ( + FAB) mass spectrum of unknown lipid component in a 3-nitrobenzyl alcohol (NBA) matrix. The peaks are labelled with nominal, monoisotopic
masses.
Figure 15 is a gas chromatography-mass spectrometry (GC-MS)
analysis of partially methylated alditol acetates (PMAAs) from permethylation, hydrolysis, reduction, and acetylation of unknown
lipid component. Peaks are identified as PMAAs of 1: 2,3,4,6-tetra-
0-; 2: 2,6-di-O-; and 3: 4,6-di-O-Me-Gal.
Figure 16 is a scheme for synthesizing plasmalopsychosine
compounds A and B. Figure 17 is a HPTLC pattern of fractions obtained on an lATROBEADS column of the plasmalopsychosine synthetic products
(Lanes 2-5) compared with crude synthetic product (Lane 1 ) and
anionic lipids obtained from CM-sephadex column chromatography of human brain extract (Lane 6). The HPTLC was developed in chloro- form/ methanol/ 28% NH4OH (80:20:2) and visualized by spraying with
orcinal-sulfuric acid and baking on a hot plate.
Figures 18A-18C are data from GS-MS analysis of partially
methylated alditols/ acetals from permethylation, hydrolysis, reduction
and acetylation of the plasmalopsychosine synthetic products. Figure
18A: data from fraction of Figure 17 Lane 5 product; Figure 18B: data from fraction of Figure 17 Lane 4 product; Figure C: standard PMAAs.
DETAILED DESCRIPTION OF THE INVENTION ISOLATION AND PURIFICATION OF PLASMALOPSYCHOSINE
COMPOUNDS A AND B A procedure for systematic isolation and characterization of
anionic lipid through cation exchange chromatography in chloroform- methanol followed by a series of chromatographies on a FLORISIL and lATROBEADS column has been developed. The major anionic lipids,
compounds A and B, present exclusively in the extract of white matter, have been identified as cyclic plasmal linked at different hydroxyl groups of the galactosyl residue of psychosine. Isolation, chemical characterization, and biological properties of these com¬
pounds are hereby described.
CHs (fl>
Figure imgf000016_0001
CH .3.-- -CH-CH-(CH,)-CH- (B)
Figure imgf000016_0002
n, is a number greater than 0 and preferably 14 or 16.
According to the present invention, white and gray matter of human brain were carefully separated and subjected to systematic chemical analysis. As a result, two major anionic glγcolipids, termed
compounds A and B, were identified as plasmal (fatty aldehyde)
conjugated with psychosine through 3,4-cyclic acetal and 6,4-cyclic
acetal linkage, respectively, at the galactosyl residue of psychosine. A relatively minor compound E was identified as 4,6-cyclic plasmal conjugate of psychosine but had 3-hydroxysphingosine. These three compounds are hereby collectively termed "plasmalopsychosines." Plasmalopsychosines, regardless of the position of the acetal linkage, have strong neuritogenic effects on neuroblastoma cells, particularly in the presence of nerve growth factor (NGF).
More specifically, compounds A and B can be isolated by
preparing anionic lipids and anionic glycosphingolipids from human brain. The lipids are extracted and a lower layer is prepared. This is
followed by separation of the anionic lipid fraction by carboxymethyl (CM) SEPHADEX chromatography. The isolated compounds A and B
can be further purified by high-performance thin-layer chromatography (HPTLC), followed by high-performance liquid chromatography (HPLC)
(lATROBEADS).
The isolation and purification procedure is described in more detail below and exemplified in Example 1.
Preparation of anionic lipids and anionic αlvcosDhinQolioids Extraction and preparation of lower laver. Human brain (cere¬
brum) is dissected and separated into gray and white matter with a razor blade. With careful practice, using a razor blade to scrape the outer layer of cortex, it is possible to obtain a near-pure gray matter fraction weighing about 50 g from adult human brain. White matter is considerably easier to prepare by cutting the brain into vertical sections and separating large areas of white matter. In both these cases, tissue is homogenized in about five volumes (i.e., five times volume/weight of wet tissue) of isopropanol/hexane/water (IHW)
(55:25:20 v/v/v, upper phase removed: when this solvent solution is prepared, two phases form and the upper phase which is predomi- nantly hexane is removed), filtered over a Bϋchner funnel, and the residue is re-homogenized in the same solvent. (Hereinafter, all
references to ratios of solvents are by volume.) After the first filtration over the Bϋchner funnel, the residue is re-homogenized twice in chloroform/methanol/water (CMW) (2:1 :0.1). The filtrates are pooled, evaporated to dryness, and brought up in chloro- form/methanol (2:1 ) to a suitable volume (for example, about 0.5-3 L for 500 gm starting tissue) for Folch's partition (5). For Folch
partition, one-sixth volume deionized water is added to the chloro-
form/methanol (2:1 ) extract solution in a leak-proof container and the contents are mixed by repeated inversions (about 20). After the phases resolve (usually 2 to 3 hours), the upper phase is drawn off and replaced with an equal volume of chloroform/methanol/water with 0.2% KCI (1 :10:10). This is repeated two additional times, and the
resulting lower phase is evaporated to dryness by rotary evaporator. Separation of anionic Moid fraction by carboxymethyl (CM)
SEPHADEX chromatography. The anionic lipid fraction is prepared from the total lower layer lipid by CM SEPHADEX chromatography. CM SEPHADEX is carefully washed and equilibrated using the following protocol. It is crucial that the SEPHADEX is equilibrated
properly in order to achieve effective binding of anionic lipids. The dry resin is washed extensively over a Bϋchner funnel in 0.2 N HCI
and allowed to soak for several hours in the acid. The resin is then washed extensively with deionized water with intermittent soaking,
followed by stepwise washing of methanol/water (MW) 20:80, 50:50, 70:30, and 90:10. Subsequently, the SEPHADEX column is
soaked in a solution of 2.0 M aqueous triethylamine (TEA)-MW (1 :1 :1 ) and allowed to sit at room temperature overnight. Excess
TEA is removed from the SEPHADEX by extensive washing in MW 1 :1. The equilibrated CM SEPHADEX is then washed with 100% methanol followed by CMW 40:60:5 (hereinafter "sol A").
To the dried lower phase of brain extract, sol A is added until
the brain extract is completely dissolved. For 500 gm of tissue, about 1 L solvent is required. This solution is passed over a bed of
equilibrated CM SEPHADEX having a volume of 50-200 ml (about 100 ml per kg wet tissue) and allowed to elute by gravity filtration. An additional amount of sol A is washed through the column and the
total pass-through fraction is collected and saved. The column is
then washed with MW 90:10 until the bed volume is equilibrated (the SEPHADEX will shrink slightly). Anionic lipids are eluted using a solution of 0.5 M TEA in MW 90:10 (titrated to a pH of 9.25 by gently bubbling CO2 gas through the solvent). For 500 gm starting tissue, about 500 ml of about 0.5 M TEA is sufficient to quantitative¬ ly elute compounds A and B, as well as compound E and sphingosine. This concentration of TEA also quantitatively elutes standard psychosine, although psychosine is absent in brain extract. Further, increasing the TEA concentration up to 2.0 M does not result in
elution of any other detectable species.
Further purification of compounds A. B and E using HPLC IATROBEAD chromatography
The 0.5 M TEA fraction from CM SEPHADEX is evaporated to dryness several times using absolute ethanol to rid the sample of TEA. The fraction is then transferred to a test tube and dissolved in
a suitable volume of chloroform/methanol (about 2-10 ml). Ten μl of the sample is chromatographed on high-performance thin-layer
chromatography (HPTLC) plates in chloroform/methanol-NH4OH 80:20:2. Viewing can be accomplished with a hand-held UV light
using either 0.8% PRIMULIN in 80% acetone or 30% FLUORES- CAMINE. It is also possible to detect compounds A, B and E with 0.5% orcinol in 10% sulfuric acid followed by baking in a thin layer
chromatography (TLC) oven. To separate compounds A, B and E from the more polar spήingosines and contaminating neutral glycolipids, it is necessary to
perform several high performance liquid chromatography (HPLC)
gradient runs. This is accomplished using a very nonpolar IHW
gradient. A long column (e.g., about 0.4x60 cm) packed with lATROBEADS (silica gel; 10 μM) is first equilibrated by washing the
column as follows: at about 2.0 ml/min the starting concentrations are IHW 55:40:5; the gradient is increased to IHW 55:25:20 over
about the next 30 minutes, followed by decreases to IHW 55:40:5
for about 30 minutes, IH 60:40 for about 30 minutes, and finally washing with hexane 100% for about 30 minutes.
The 0.5 M TEA fraction is prepared for injection by evaporating to dryness and dissolving in 100% hexane in the following manner. For a 2 ml injection, 100 μ\ of chloroform/methanol (2:1 ) is added,
the cap is screwed on tightly, and the sample is slightly warmed to about 50°C and sonicated to form a thick oil. In most cases, this
almost completely solubilizes the lipid. To this thick oil, 2 ml of 100% hexane is added while sonicating. In some cases, a very fine, opalescent precipitate forms, but this does not interfere with the
injection.
The sample is loaded onto the column and subjected to a
gradient eluting at about 0.5 ml/min. Gradient elution is started from the hexane to IHW 10:89:1 (about 25 to 150 minutes), and continues to IHW 24:74:2 (about 150 to 400 minutes), to IHW 55:40:5 (about 400 to 500 minutes), and to IHW 55:25:20 (about 500 to 600
minutes). Effluent (about 3 ml/tube) is collected over a fraction
collector in 100 tubes, and the tubes are streaked for HPTLC analysis
(chloroform/methanol/NH4OH, 80:20:2). Fractions are pooled based on separation of three detectable bands corresponding to compounds A, B and E. However, due to sphingosine overlap, several HPTLC runs are necessary in order to purify the compounds A, B and E to homogeneity. In this manner, sphingosine is also conveniently
purified, as well as a slower migrating sphingosine analog.
Chemical charapterization of plasmalopsychosine compounds A and
S
Carbohydrate analysis can be performed by gas chromatogra- phy-mass spectrometry (GC-MS) employing trimethysilyl derivatives
of methyl glycosides produced by methanolysis. Fast atom bombard¬ ment-mass spectrometry (FAB-MS) analysis of native lipid can be
obtained in both positive and negative ion modes (7-9). Preliminary analysis of fatty aldehydes can be made using fatty acid methyl ester fraction yielded on methanolysis of lipids. However, a number of unknown peaks will be identified as enol methyl ether of C16-C18 fatty aldehyde, in addition to fatty acid methyl esters. These peaks are carefully identified using GC-MS in conjunction with FAB-MS analysis. Structural information can also be obtained by FAB-MS of
per-N-O-acetγlated and de-O-acetylated lipids, and by classical methylation analysis with GC-MS.
ISOLATION AND PURIFICATION OF PLASMID CEREBROSIDE COMPOUNDS C AND D
In the present study, during investigation of fast-migrating (on
thin-layer chromatography) glycolipids from human brain, an acid- labeled minor component was detected and separated by successive chromatographies on columns of FLORISIL and lATROBEADS (silica
gel) in an isopropanol/hexane/water system, and preparative high- performance thin-layer chromatography. In contrast to the majority of fast-migrating glycolipids, which were identified as fatty acid esters of cerebroside, the acid-labile minor component was isolated and
characterized as a plasmal conjugate of cerebroside, through 3,4- or 4,6-cyclic acetal linkage at the galactopyranosyl residue of cerebro¬
side. isolation, chemical characterization, and biological activity of these compounds are hereby described. Isolation of plasmalocerebrosides
According to the present invention, the two newly isolated
plasmalocerebrosides which are designated compound C and com¬ pound D have the structures shown below:
CHj (C>
Figure imgf000025_0001
CH (CH2 )n3-CH3 -CH-CH-(CH2)-CH3 <D)
Figure imgf000025_0002
wherein n2 and n3 each is a number greater than 0, and pharma¬
ceutically acceptable salts thereof. Preferably n2 is 14 or 16 and n3 is 12, 14, 16, 18, 20, 21, 22, 23 or 24.
In order to isolate compounds C and D, a fast-migrating compo- nent from column chromatography of a human brain cerebroside extract is first isolated. The human brain cerebroside fraction can be
obtained by homogenization of brain tissue with about five volumes
(i.e., five times volume/weight of wet tissue) of isopropa¬ nol/hexane/water (IHW) 55:25:20 (v/v/v) and filtration through a Bϋchner funnel. The residue is re-homogenized in the same volume of the same solvent. The extracts are pooled, evaporated to dryness,
and subjected to Folch partition using about 1 L of chloroform/metha- nol (CMe) 2:1 and about 166 ml water per 10 g original wet weight of tissue. The lower phase is subjected to Folch partitioning three more times and then repartitioned with "theoretical upper phase" (chloroform/methanol/water with 0.2% KG 10:10:1 ). The resulting lower phase is evaporated to complete dryness. A large column (bed volume about 1 L per 1 kg original tissue) of FLORISIL (a mixture of
magnesium oxide and silicic acid gel) (from Sigma; mesh 60-100) is
prepared and equilibrated in pure hexane. The dried lower phase is suspended in hexane (about 1 L per 200 g original tissue), passed over the FLORISIL column, and exhaustively washed with about 4 L of hexane. The FLORISIL column is then eluted with 2 L of hexane/d-
ϊchloroethane (DCE) 2:1 , then with 2 L of DCE, and finally with 1 L of DCE/acetone 1 :1. The final eluate contains the desired acid-labile
fast-migrating component.
The presence of acid-labile glycolipids can be detected by hydrolysis of samples in methanol/aqueous 0.1 N HCI (1 :1 , v/v) heated at about 90°C for about 10 minutes, followed by Folch partitioning and thin-layer chromatography (TLC) examination of the
lower phase. The glycolipid with high TLC mobility which is converted
to the same mobility as cerebrosides by this treatment is regarded as the acid-labile cerebroside. Cerebroside and ester cerebroside do not show altered TLC mobility under these conditions. Isolation and preliminary characterization of acid-labile olvcosphingo-
liods present in the fast-migrating fraction
According to the present invention, the presence of an acid-
labile fast-migrating glycolipid is a consistent component of brain extract, and is found in the unabsorbed fraction on carboxymethyl-
SEPHADEX and diethylaminoethyl-SEPHADEX of the Folch's lower phase as well as in the DCE-acetone 1 : 1 eluate fraction on chroma¬ tography over FLORISIL. This fast-migrating glycolipid fraction is
further purified by high-performance liquid chromatography (HPLC) on an lATROBEADS column loaded in pure hexane and eluted with a
gradient to IHW 55:50:5 at about 1 ml/min for about 3 hours. Fractions are collected into 200 tubes. The acid-labile glycolipid component is eluted in tube Nos. 130-154. The pooled fraction
(called fraction VI) contains most of the acid-labile glycolipid and is free of cerebroside and ester cerebrosides. The fraction VI is further
purified by chromatography on lATROBEADS, loading on the columns in pure hexane, and subjected to a gradient up to isoproponal/hexane (IH) 30:70. The fraction VI A thus obtained is further purified on a long lATROBEADS column (e.g., about 0.5x100 cm) with a shallow
gradient, loaded with pure hexane, and gradient eluted to IHW
50:40:5 for 3 hours. Alternatively, the compounds can be purified by preparative TLC. A homogeneous band is obtained as shown in Fig. 11, lane 6. On TLC, the compounds migrate faster than cholesterol which migrates faster than ester cerebrosides. The compounds do not contain sulfate or sialic acid which are known to be acid-labile.
Chemical characterization of compounds C and D
The structure of compounds C and D can be determined after methanolysis by identifying enol methyl ethers derived from fatty aldehydes by gas chromatography-mass spectrometry (GC-MS) analysis and by fast atom bombardment mass spectrometry (FAB-MS)
as described in detail in the Example 7.
The acetal linkage can be determined by methylation analysis. Following permethyiation, acid hydrolysis, reduction, and acetylation of the native lipid, the resulting partially methylated hexitol acetates are analyzed by GC-MS as described in detail in Example 7.
METHOD OF FORMING NEURITES FROM NERVE CELLS
According to a further aspect of this invention, neurites can be
formed from nerve cells by contacting the nerve cells with an effective amount of one or more of the compounds A, B, C and D. The nerve cells, such as from neuroblastoma cell lines, are cultured in gelatin-coated plates by known methods (11 ,12). The effective dose is determined by adding various concentrations (e.g., 5-150 μM) of one or more of compounds A, B, C and D, and the cells are cultured for the observation of neurite formation, as described in more
detail in Example 8.
COMPOSITION AND METHOD FOR TREATING NEURONAL DISEASES
AND TISSUE DAMAGE
Both psychosine compounds A and B display remarkable
neurogenic activity in a variety of neuroblastoma cells. Neurite formation in neuroblastoma and retinoblastoma cells is often used as a criterion to evaluate ability of candidate reagents to repair neuronal
tissue damage.
The effect of compounds A and B on neurite formation in neuroblastoma cells, in comparison to existing gangliosides, is
presented in detail in Example 8. Thus, whereas psychosine is highly hemolytic and assumed to be highly cytotoxic, it is virtually absent in normal brain tissue (either white or gray matter). In contrast,
plasmalopsychosine, a major component of white matter, shows
strong neuritogenic activity in neuroblastoma cells. Psychosine used as a control in these experiments showed cytotoxic effects and inhibited cell growth even at very low doses. Plasmalopsychosine does not inhibit PKC, in contrast to the strong inhibitory effect of psychosine. While not wanting to be bound by the following
hypothesis, it is possible that plasmalopsychosine is uniquely
incorporated into cells and is slowly converted to psychosine and thereby regulates activity of PKC and other protein kinases essential for cell growth regulation. Growth inhibition subsequently induces differentiation. The quantity of psychosine generated could be minimal but yet optimal for stimulation of differentiation and neurite
formation.
Both plasmalocerebroside compounds C and D also display remarkable neurogenic activity in a variety of neuroblastoma cells. No clear effect in the early stages of cell culture is observed, but neurite formation, i.e. neurites > 50 μm long, becomes increasingly apparent
by 1 week. After 2 weeks of culture, neurite formation in Neuro-2A
cell culture is very pronounced. Accordingly, the present invention provides a composition for treating neuronal diseases and tissue damage comprising one or more of compounds A, B, C and D and pharmaceutically acceptable salts thereof; and a pharmaceutically acceptable carrier, diluent or
excipient. The present invention also provides a method for treating
neuronal diseases and tissue damage comprising administering to a host in need of treatment a biologically effective amount of one or more of the compounds A, B, C and D, and pharmaceutically acceptable salts thereof.
Specific cases include treatment of Alzheimer's disease, spinal
injury such as paralysis, cerebral vascular accidents where there is
loss of neural tissue, brain trauma, Parkinson's disease, amyotropic lateral sclerosis and multiple schlerosis.
The effective amount of compounds A, B, C, and D can be determined using art-recognized methods, such as by establishing
dose-response curves in suitable animal models or in non-human primates, and extrapolating to human; extrapolating from suitable m vitro data, for example as described herein; or by determining effectiveness in clinical trials.
Suitable doses of medicaments of the instant invention depend upon the particular medical application, such as the severity of the
disease, the weight of the individual, the age of the individual, alf- life in circulation, etc., and can be determined readily by the skilled
artisan. The number of doses, daily dosage and course of treatment may vary from individual to individual.
The compounds A, B, C and/or D can be administered in a
variety of ways such as intravenously or by direct subdural injections.
Suitable pharmaceutically acceptable carriers, diluents, or excipients for the medicament of the instant invention depend upon the particular medical use of the medicament and can be determined readily by the skilled artisan.
The medicament can be formulated into solutions, emulsions, or suspensions. The medicament is likely to contain any of a variety
of art-recognized excipients, diluents, fillers, etc. Such subsidiary ingredients include disintegrants, binders (including liposomes), surfactants, emulsifiers, buffers, soiubilizers and preservatives. The artisan can configure the appropriate formulation comprising com¬ pounds A, B, C and/or D by seeking guidance from numerous authorities and references such as "Goodman & Gilman's The
Pharmaceutical Basis of Therapeutics" (6th ed., Goodman et al.. eds., MacMillan Publ. Co., NY, 1980).
CHEMICAL SYNTHESIS OF COMPOUNDS A. B. C. AND D
Plasmalopsychosine compounds A and B and plasmalocerebro-
sides compounds C and D can be readily synthesized chemically by
a method such as that described for the synthesis of plasmalopsy¬
chosine compounds in Example 9.
E AMPLES
The invention will now be described by reference to specific
examples which are not intended to be limiting. EXAMPLE 1
ISOLATION AND PURIFICATION OF COMPOUNDS A AND B
Preparation of Anionic Lioids and Anionic Glycosphingolipids
Extraction and preparation of lower laver. Adult human brain
(cerebrum) was dissected and separated into gray and white matter with a razor blade. With careful practice, using a razor blade to
scrape the outer layer of cortex, it was possible to obtain a near-pure gray matter fraction weighing 50 g. White matter was considerably easier to prepare, by cutting the brain into vertical sections and
separating large areas of white matter. An entire small brain
(cerebellum) was also used as a source of extraction. In all cases, tissue was homogenized in five volumes (i.e., five times vol¬ ume/weight of wet tissue) of isopropanol/hexane/water (IHW) (55:25:20 v/v/v, upper phase removed), filtered over a Bϋchner
funnel, and the residue was re-homogenized in the same solvent.
(Hereinafter, all references to ratios of solvents are by volume unless
otherwise indicated.) After the first filtration over the Bϋchner funnel, the residue was re-homogenized twice in chloroform/methanol/water
(CMW) (2:1 :0.1 ). All filtrates were pooled, evaporated to dryness,
and resuspended in chloroform/methanol (2:1 ) to a suitable volume
(0.5-3 L) for Folch's partition (5). For Folch partition, one-sixth volume deionized water was added to a chloroform/methanol (2:1 ) extract solution in a screw-cap container and the contents were inverted 20 times. After the phases had resolved (usually 2 to 3 hours), the upper phase was drawn off and replaced with an equal volume of "theoretical upper phase" (CMW-0.2% KCI, 1 :10:10). This was repeated two additional times, and the resulting lower phase was evaporated to dryness in a rotary evaporator.
Separation of anionic Moid fraction bv CM SEPHADEX chroma¬
tography. The anionic lipid fraction was prepared from the total lower layer lipid by carboxymethyl (CM) SEPHADEX chromatography. CM SEPHADEX (Sigma, C-25) was carefully washed and equilibrated
using the following protocol. It was crucial that the SEPHADEX was equilibrated properly in order to achieve effective binding of anionic
lipids. The dry resin was washed extensively over a Bϋchner funnel in 0.2 N HCI and allowed to soak for several hours in the acid. The resin is then washed extensively with deionized water with intermit¬ tent soaking, followed by stepwise washing with methanol/water
(MW) (20:80, 50:50, 70:30, and 90:10). Subsequently, the SEPHADEX column was soaked in a solution of 2.0 M aqueous
triethylamine (TEA) (Mallinckrodt)-MW (1 :1 :1 ) and allowed to sit at room temperature overnight. Excess TEA was removed from the
SEPHADEX by extensive washing in MW (1 :1). The equilibrated CM SEPHADEX was then washed with 100% methanol followed by CMW (40:60:5) (hereinafter "sol A").
To the dried lower phase of brain extract, sol A was added until the solution became totally soluble. For 500 gm of tissue, this
usually was about 1 L solvent. This was passed over a bed of equilibrated CM SEPHADEX having a volume of 50-200 ml (about
100 ml per kg wet tissue) and allowed to elute by gravity filtration. An additional 2 L of sol A was washed through the column and the total pass-through fraction was collected and saved. The column was
then washed with MW (90:10) until the bed volume equilibrated
(SEPHADEX would shrink slightly). Anionic lipids were eluted using a solution of 0.5 M TEA in MW (90:10, titrated to a pH of 9.25 by gently bubbling CO2 gas through the solvent). For 500 gm starting tissue, 500 ml of 0.5 M TEA was sufficient to quantitatively elute
compounds A and B, as well as sphingosine (SPN) and a compound designated "compound E." In separate tests, this concentration of TEA also quantitatively eluted standard psychosine, although psychosine was absent in brain extract. Increasing the TEA concen¬
tration up to 2.0 M did not result in elution of any other detectable
species.
The results are shown in Figs. 1A and 1 B. In Fig. 1 A, TLC was developed in chloroform-methanol-28% NH4OH (80:20:2). Bands were detected by orcinol-sulfuric acid. Lane 1, total eluate from carboxymethyl SEPHADEX column with 0.5 M TEA; lane 2, purified compound A; lane 3, purified compound B; lane 4, purified compound C; lane 5, sphingosine. Fig. 1 B is the same chromatogram as in Fig. 1 B, but the bands were detected by spraying with 0.01 % PRIMULINE and viewed under UV light.
Fig. 1A, which shows the pattern with orcinol-sulfuric acid,
with compounds A, B and E stained purple, indicates the presence of carbohydrate. Other bands were different in coloration with orcinol- sulfuric acid reaction.
Fgrther Pyrificg 'pn Qf CQ ppgnςl? A gn<j β using HPLC lATRQBgAQ Chromatography
The 0.5 M TEA fraction from CM SEPHADEX was evaporated
to dryness several times using absolute ethanol to rid the sample of
TEA. The fraction was then transferred to a screw-cap tube and diluted to a final volume of 2-1 Oml in chloroform/methanol 2:1 , and 10//I was chromatographed on high-performance thin-layer chroma¬ tography (HPTLC) (Merck) plates in chloroform/methanol-NH4OH
80:20:2. Viewing was accomplished with hand-held UV light using either 0.8% PRIMULIN (Sigma) in 80% acetone or 30% FLUORES-
CAMINE (Sigma). It was also possible to detect compounds A and B with 0.5% orcinol (Sigma) in 10% sulfuric acid followed by baking in a thin-layer chromatography (TLC) oven.
To separate compounds A and B, as well as E, from the more
polar sphingosines and contaminating neutral glycolipids, it was
necessary to perform several HPLC gradient runs. This was accom¬ plished using a very nonpolar IHW gradient. A long column (0.4x60
cm) packed with lATROBEADS (10 μM) was first equilibrated by washing the column according to the following scheme: at 2.0
ml/min the starting concentrations were IHW (55:40:5); the gradient
was increased to IHW (55:25:20) over the next 30 minutes, followed by decreases to IHW (55:40:5) for 30 minutes, IH (60:40) for 30 minutes, and finally washing with hexane (100%) for 30 minutes.
The 0.5 M TEA fraction was prepared for injection by evaporat¬ ing to dryness and redissolving in 100% hexane in the following manner. For a 2 ml injection, 100 μ\ of chloroform/methanol (2:1 ) was added, the cap was screwed on tightly, and the sample was
slightly warmed under hot tap water to about 50°C and sonicated. In most cases, this almost completely solubilized the lipid. To this thick oil, 2 ml of 100% hexane was added during sonication. In some cases, a very fine, opalescent precipitate formed, but this never
interfered with the injection. The sample was loaded onto the column and subjected to gradient eluting at 0.5 ml/min. Gradient elution was started from the
hexane to IHW 10:89:1 from 25 to 150 minutes, and continued from this solvent to IHW 24:74:2 (150 to 400 minutes), to IHW 55:40:5
(400 to 500 minutes), and to IHW 55:25:20 (500 to 600 minutes).
Effluent (3 ml/tube) was collected over a fraction collector in 100 tubes, and the tubes were streaked for HPTLC analysis (chloro- form/methanol/NH4OH, 80:20:2). Fractions were pooled based on
separation of three detectable compound A, B and E bands. Howev- er, sphingosine overlap made several HPTLC runs necessary in order
to purify compounds A and B, as well as E, to homogeneity. In this manner, sphingosine was also conveniently purified, as well as a slower migrating sphingosine analog (Fig. 1B, lanes 4,5).
EXAMP ξ 2 COMPARISON OF GRAY VS. WHITE MATTER AND
CEREBELLUM VS. BRAINSTEM
Equal weights (100 gm) of white and gray matter from
cerebrum were collected from the same human brain and processed side by side to obtain lower phases. Equal weights of cerebellum and brainstem were also obtained. These samples were passed over CM
SEPHADEX as described above and eluted with 0.5 M TEA.. Fig. 2 shows the orcinol staining of various fractions from gray and white matter, cerebellum, and brainstem.
In Fig. 2, the lanes are as follows: lanes 1 and 10, standard CMH; lane 2, lower phase from white matter; lane 3, lower phase
from cerebellum; lane 4, lower phase from brain stem; lane 5, lower phase from gray matter; lane 6, 0.5 triethylamine eluate from
carboxymethyl SEPHADEX column of white matter; lane 7, the same fraction as in lane 6 but prepared from cerebellum; lane 8, the same fraction as in lane 6 but prepared from brain stem; lane 9, the same fraction as in lane 6 but prepared from gray matter.
Lane 6 clearly shows that the major source of compounds A
and B is the white matter of the cerebrum. There was no detectable
amount of compounds A, B or E in cerebral gray matter, trace amounts in the cerebellum, and 10-15% (relative to cerebral white matter) in the brainstem (Fig. 2, lanes 6-9).
Further, compounds A, B and E were present in human brain
white matter but undetectable in gray matter. The composition of
compounds A, B and E in six different brains with different ages was
measured quantitatively as described above. EXAMPLE 3 CHEMICAL DEGRADATION OF COMPOUNDS A. B AND E
Compounds A, B and E separated on HPTLC were all stained
by orcinol-sulfuric acid reaction with a color typical for neutral glysophingolipid (GLS), but were all negative with resorcinol-HCl reaction specific for gangliosides. Preliminary chemical degradation with weak acid/base treatment was performed. Weak acid treatment as catalyzed by mercuric chloride (0.1 % HgCI2 in 0.1 N HCI) was performed according to the original method of Feulgen et al. (6); alternatively, glycolipid was treated in 0.3 N HCI in MeOH at 80°C for
30 minutes. Weak base hydrolysis was carried out in 0.3 N NaOH in
MeOH at 80 °C for 40 minutes.
The results are shown in Fig. 3, which is the HPTLC pattern of purified plasmalopsychosine and degradation product by weak acid
and alkaline.
The lanes in Fig. 3 are as follows: lane 1 , compound A; lane
2, compound A treated in 0.3 N HCI in MeOH 80° C 30 minutes; lane
3, compound A treated with 0.3 N NaOH in MeOH, 80°C 40 minutes; lane 4, standard psychosine; lane 5, compound B; lane 6, compound B treated in 0.3 N HCI in MeOH 80°C 30 minutes; lane 7, compound B treated in 0.3 N NaOH in MeOH 80°C 40 minutes; lane 8, ceramide monohexoside (CMH).
The results show that compounds A, B and E (results not shown for compound E) could be degraded to the same position as
psychosine after weak acid hydrolysis catalyzed by HgCI2 in 0.1 N
HCI or 0.3 N HCI in MeOH, but were resistant to base hydrolysis (Fig.
3, lanes 5-8).
EXAMPLE 4
STRUCTURAL CHARACTERIZATION OF COMPOUNDS A. B AND E AS PLASMALOPSYCHOSINE
Preparation of long chain enol methyl ether standards. Long
chain alcohols (n-hexadecanol and n-octadecanol), purchased from
Aldrich (Milwaukee, Wl), were oxidized to aldehydes using pyridinium
dichromate in CH2CI2, according to the method of Corey and Schmidt (25). Identity and purity of products were verified by GC-MS.
Aledhydes were converted to enol methyl ethers (EMEs) by treatment with 0.5 N HCI/5 M H2O in methanol at 80°C for 5.5 hr. The methanolysate was cooled and extracted 3x with hexane. The combined hexane extracts were evaporated under N2 stream at 37°C
to approximately 10 μ\, then diluted with hexane for analysis by GC-
MS as described below. Under these conditions, production of EME derivatives was favored over conversion to long chain dimethylace- tals.
Long chain aldehyde analysis. Lipid samples (400-500 μ) were
methanolyzed in 2.0 ml 0.5 N HCI/5 M H20 in MeOH for 5.5 hr at
80°C. The methanolysate was cooled and extracted 3x with hexane.
The combined hexane extracts were evaporated under N2 stream at 37°C to approximately 10 μ\, then taken up in a volume of hexane (10-50 //I) providing a suitable dilution for analysis by GC-MS. GC-
MS of aiiquots of the hexane extractable material were performed using a Hewlett-Packard 5890A gas chromatograph interfaced to an
Extrel ELQ 400 quadrupole mass spectrometer. Gas chromatoraphy was performed using a 30 m DB-5 (J & W Scientific, Ranch Cordova, CA) bonded-phase fused silica capillary column (0.25 mm o.d., 0.25 μ film thickness; splitless injection; temperature program, 140-
250°C at 4°C/min). The mass spectrometer was operated in either
Cl (isobutane; mass range, 150-500 u, scanned once per second) or El (mass range 50-500 u, scanned once per second) mode. EME derivatives were identified by characteristic ions and retention times compared with synthetic standards (see previous section), verified by
co-injection when necessary.
Monosaccharide analysis. Lipid samples (50-100 μg) were methanolyzed in 1.0 ml 0.5 N HCI in anhydrous MeOH for 24 hr at 80°C. The methanolysate was cooled and extracted 3x with hexane. The acidic MeOH lower layer was neutralized by addition of Ag2CO3 (approximately 10 mg) and treated with acetic anhydride (100 μl) for 6 hr at room temperature. Following centrifugation and removal of
the MeOH, the precipitate was washed 2x with 1 ml portions of
MeOH. The combined MeOH extracts were dried under N2 stream. The resulting monosaccharide methyl glycosides were analyzed as their per-O-trimethylsilyl ethers (26, 27) by GC-MS using the Extrel ELQ 400 system described above (DB-5 column; splitless injection;
temperature program, 140-270°C at 4°C/min; CI-MS (isobutane)
mode). The combined hexane extracts were evaporated under N2 stream at 37°C to approximately 10 μ\, then diluted with hexane for
anlaysis by GC-MS under the conditions described in the previous section. Chemical derivatizations of intact lipids. Lipid samples
(approximately 50 μg) were permethylated by the method of Ciukanu and Kerek (28), as modified by Larson et al (29), except that equal
volumes of Mel and DMSO were used (100 //I each). The reaction time was 30 min, and Mel was removed by flushing with N2 for 25 min at 37°C prior to partitioning between CHCI3 and H2O. After
washing 3x with H2O, the CHCl3 was evaporated to dryness under N„ Lipid samples were per-N,£-acetylated with 2:1 pyridine-acetic anhydride (0.5 ml, 20 hr, room temperature). The reagents were removed by flushing under N2 stream at 37°C, with addition of anhy¬ drous toluene as co-distallant. A portion of each sample was subse-
quently de-fi-acetylated by the Zemplέn procedure (brief treatment with NaOMe in anhydrous MeOH) (30).
Methylation/linkaoe analysis. Linkage positions of substituents on glycosyl residues were determined by permethylation of approxi¬ mately 50 μg of each sample (see previous section), followed by hydrolysis, reduction, peracetylation and GC-MS as described in detail
elsewhere (22), except that the analysis was performed on the Extrel ELQ 400 GC-MS system described above (DB-5 column; splitless injection; temperature program, 140-250 °C at 4°C/min; EI-MS mode), with identification of partially methylated alditol acetate (PMAA) derivatives made by retention time and characteristic electron-impact mass spectra (31 , 32). Identifications were con¬ firmed by comparison with PMAAs in known standard mixtures.
Fast atom bombardment mass spectrometry. FAB-MS was
performed on a JEOL (Tokyo, Japan) HX-110/DA-5000 mass spectrometer/data system, operated in the accumulation mode at full acceleration voltage (10 kV); xenon beam, 6 kV; resolution, 3000.
Aliquots of sample (approximately 20 μg) in MeOH were transferred to a FAB target and suspended in an appropriate matrix. For native lipid samples analyzed by FAB-MS the matrix was TEA/15-crown-5 (33, 34) and the mass range was 100-2000 u. Three scans were accumulated for each spectrum. Sodium iodide in glycerol was used
as the calibration standard.
Samples of native, acid treated, per-N,_ -acetylated, and per-
N,Q-acetylated de-Q-acetylated lipids were analyzed by + FAB-MS using NBA matrix, with and without addition of sodium acetate.
Other conditions were the same as above. Kl/Csl was used as the calibration standard.
Methanolysis: monosaccharide analysis. Since the unknown
lipids could be stained with orcinol, indicating the presence of some carbohydrate component, they were subjected to monosaccharide analysis, by GC-MS of trimethysilyl methyl glycosides produced
following acidic methanolysis. In each case, peaks were clearly observed for the usual trimethlyisilyl derivatives of galactose (data not
shown). No other saccharide peaks were observed, except for a trace ( < 1 %) of glucose detected in the methanolysate of the uppermost band. Methanolysis: analysis of fattv aldehydes. GC-MS analysis of the hexane wash, following acidic methanolysis, is normally used for determination, as methyl esters, of the fatty acyl components of glycosphingolipids, in general those attached to sphingosine to make
up' he ceramide moieties. In the present case, no fatty acid methyl
esters were detected in any of the lipid fractions analyzed. A number of unknown peaks were observed. Following evaluation of the results of FAB-MS analysis of the intact lipids (described below), the identity of these peaks was carefully determined, and several major compo¬ nents found to correspond to long chain enol methyl ethers. Two components were found to be identical in retention times and mass spectra to enol methyl ethers prepared by acidic methanolysis of authentic 16:0 and 18:0 long chain aldehydes. Two other compo¬
nents, having molecular weights 2 amu less than those synthesized from the 18:0 aldehydes, and having slightly faster retention times, were assumed to correspond to isomeric unsaturated 18:1 species. These four components are identified in the GC-MS reproduced in
Figs. 4A and 4B.
Figures 4A and 4B are the results of gas chromatography- chemical ionization/mass spectrometry (GC-CI/MS) of long chain methyl enol ethers. Fig. 4A shows the results from methanolysis of
the middle band lipid; Fig. 4B shows the results from methanolysis of standard n-16:0 and -18:0 aldehydes. Peaks were identified as 1 :
16:0; 2a and 2b: isometric 18:1 ; and 3: 18:0 methyl enol ethers, having pseudomolecular ion masses of 255, 281 , and 283 u, respectively. Peaks marked by an asterisk are impurities common to both samples, probably arising from the derivatization reagents.
FAB-MS analysis of native lipids. FAB mass spectra of the
unknown native lipids were obtained in both positive and negative ion
modes (7-9), and the results are shown in Figs. 5A to 5D.
Figure 5 shows FAB-MS of native lipids: Fig. 5A, +FAB mass
spectrum of upper band lipid in 3-nitrobenzyl alcohol (NBA) matrix; Fig. 5B, +FAB mass spectrum of middle band lipid in NBA matrix; Fig. 5C, '''FAB mass spectrum of middle band lipid in NBA/sodium acetate
matrix; Fig. 5D, FAB mass spectrum of middle band lipid in TEA/15- crown-5 matrix.
The positive ion spectra of the upper and middle (HPTLC) bands
are reproduced in Figs. 5A and 5B. Observed in both spectra were prominent ions at m/z 684, 710, and 712 (nominal, monoisotopic masses). That these corresponded to pseudomolecular ions [MH] + was confirmed by obtaining spectra following addition of sodium
acetate to the matrix. Sodiated molecular ions were then observed at m/z 706, 732, and 734 (see Fig. 5C). Further confirmation was
provided by negative ion spectra, in which mode pseudomolecular
ions [M-H] could be observed at m/z 682, 708, and 710 (see Fig.
1 D). Since these ions correspond to the odd molecular weights 683, 709, and 711 Da, it could be concluded that each species contains an odd number of nitrogen atoms. Interestingly, the negative ion spectra was characterized by the presence of extra peaks apparently associated with the pseudomolecular ions. Each pseudomolecular ion is accompanied by an ion at m/z [M-H +42], along with a less
abundant one at m/z [M-H + 26]. Such adduct ions were previously observed in the negative ion spectra of semisynthetic lyso- and de-N- acetyl gangliosides only when TEA was used as the matrix (1,2). They have been observed only with compounds containing a free amino group, and are believed to result from an addition reaction with some component in the matrix, either present as an impurity, or
formed by decomposition of TEA under the conditions of fast atom bombardment (2). In this case, the conclusion that the lipids bear a
primary amino function is consistent with their detection by f luoresca- mine on HPTLC plates. Of some further interest was the observation in the positive ion
spectra, of peaks consistent with dimeric ions. These were found
between 1300 and 1500 u at masses corresponding to the possible combinations of the monomeric species, i.e., at m/z
Figure imgf000048_0001
and [M- +M2-H + 2Na]+ (see Figs. 5A, 5B and 5C). In the negative ion spectra, they were accompanied, again, by adduct ions 26 and 42
u to higher mass (Fig. 5D). Such noncovalent self-associations of
glycolipids in FAB spectra have not been previously reported, although Ballou and Dell (23) studied the interaction between long chain alkyl trimethylammoniun ions and a natural 3-methyl-mannose polymer from Mvcobacterium smeomatitis by +FAB-MS. In the
positive ion spectra, a second set of ions, observed between 1 100
and 1200 u (Figs. 5A, 5B and 5C), may correspond to loss of a portion of one molecule in the dimeric species, although the exact nature of this loss is not clear at this time. Since they represent the loss of an odd mass fragment (257 u), one may assume that it is a portion of a sphingosine chain including the nitrogen atom that is
cleaved off.
The differences in mass between the observed pseudomole¬
cular ions (26 and 28 u) suggested a difference in structure corre¬ sponding to a two-carbon alkyl chain, with predominant monounsatur- ation in the heavier homolog. Since the upper and middle bands
yielded qualitatively similar spectra, it was further inferred that a structural isomerism was responsible for the difference in Rf between them. In both cases, the major fragment ion in the positive mode
was observed at m/z 282, associated with less abundant ions at m/z 250, 264, 300, and 310. The ions at m/z 300, 282, and 264 were previously observed by Hara and Taketomi (24) to be characteristic fragments of unsaturated d18:1 sphingosine in positive mode FAB spectra of psychosines (representing, for gaiactopsychosine, for example, [M + H-Gal]+, [M + H-Gal-H20]+, and [M + H-Gal-2H20]+, respectively). Confirmation of the unknown lipids as derivatives of psychosine, and of the possible isomeric relationship between them was provided by degradative experiments monitored by FAB-MS. FAB-MS of the products of mild acid hydrolysis. Brief treatment of the upper band lipid with 0.1 N HCI/HgCI2 yielded a
product whose Rf was identical to that of the middle band on HPTLC. The +FAB mass spectrum of this product (Fig. 6A) was virtually identical to those of the native untreated upper or middle band lipids, demonstrating an acid catalyzed transformation of the upper to the
middle band lipid. On more extended treatment of the upper band, or treatment of the middle band, a new product was observed, having
an Rf identical to that of authentic galactopsychosine. The +FAB mass spectra of these products were virtually identical to those obtained for galactopsychosine (Figs. 6B, 6C and 6D).
Figure 6 shows +FAB-MS of products of treatment with
HCI/HgCI2: Matrix: NBA. A, upper band lipid, following brief acid
treatment, resulting in conversion to middle band lipid; B, upper band
lipid following extended acid treatment; C, lower band lipid following extended acid treatment; D, d18: 1 galactopsychosine standard.
Because the lipids are newly isolated covalent modifications of psychosine, the following further conclusions can be reached. Given the great relative abundance of the ion at m/z 282 (in Figs. 5A and 5Bi compared with that at m/z 310 (which may represent a homolog containing d20:1 sphingosine), it is apparent that the differences in mass of the pseudomolecular ions must be due largely to differences
in mass of the modifying group(s), rather than to the occurrence of different sphingosine chain lengths. The modifying groups would
have to be such as to add incremental masses of 222, 248, and 250
u to that of the psychosine. Identical differences in mass were also observed in a series of low abundance fragments (m/z 444, 470, 472), found in the spectra of the native lipids (Figs. 5A and 5B),
which could be analogs of the fragment found at m/z 222 in the FAB mass spectrum of psychosines (24) (see Fig. 6D), but which is
concomitantly eliminated from the spectra of the native modified lipids. Interestingly, a pair of fragments found at m/z 250 and 252
in the spectrum of psychosines (24) (see Fig. 6D) were also found in the spectra of the native modified lipids (Figs. 5A and 5B), while there
was no set of ions observed with masses incrementally increased as found for the m/z 222 fragment. Coincidentally, the differences in mass correspond to the differences in chain length of the enol methyl ethers found by GC-MS of the hexane soluble methanolysis products,
suggesting that these might be chemical transformants of the modifying groups in question. Previously, the structure of a plasmalo- gen-like form of glycosphingolipid was proposed by Kochetkov et al. (13), in which the 3-OH group of psychosine was modified by attach¬ ment of a long-chain enol ether. However, in the total absence of any fragments corresponding to loss of the hexose moiety, as commonly
observed in FAB mass spectra of glycosphingolipids (such as psychosine), it seemed more likely that the modifying group(s) must be attached to the galactose residue, rather than to the sphingosine moiety. The idea that these modifications might take the form of enol ethers was also shown to be erroneous by further derivatization
experiments followed by FAB-MS.
FAB-MS ofseouentiallvper-N.O-acetvlated and de-O-acetvlated
lipids. Peracetylation of the native lipids with acetic anhydride/pyri- dine resulted in incorporation of four acetate groups, as illustrated in Figs. 7A, 7B and 7C.
Fig. 7A is peracetylated upper band lipid in NBA matrix; Fig; 7B is peracetylated middle band lipid in NBA matrix; Fig. 7C is peracetyl¬ ated middle band lipid in NBA/sodium acetate matrix; Fig. 7D is peracetylated and de-O-acetylated middle band lipid in NBA matrix
(inset: same product in NBA/sodium acetate matrix, showing no
change in masses of pseudomolecular ions).
For the upper lipid (Fig. 7A), pseudomolecular ions [M + Na]+
at 874, 900, and 902 corresponded to the addition of 4x42 u to each of the native species. In addition, ions at m/z 792, 818, and 820 were observed, representing [MH-60]+, a facile neutral loss of HOAc.
Confirmation of the higher mass group as being the true pseudomole¬
cular ions was confirmed by addition of sodium acetate to the matrix,
as illustrated for the per-acetylated middle band lipid (Fig. 7C). A concomitant suppression of the [MH-60]+ ions was observed under
this condition. At the lower end of the spectra, the triply unsaturated (doubly dehydrated) ion m/z 264 was now the predominant sphingo¬
sine fragment. Also observed was an ion at m/z 366, probably
representing a single dehydration of the N-Ac, O-Ac sphingosine fragment (m/z 384). The sphingosine fragment can eliminate one and two molecules of HOAc, to yield the ions at m/z 324 and 264, respectively. Elimination of HOAc from the fragment at m/z 366 yields the ion at m/z 306. The origin of the group of odd-mass ions,
m/z 469, 495, and 497, is unclear at this time.
As illustrated for the middle band lipid (Fig. 7D), de-O-acetyla- tion with MeONa/MeOH resulted in the loss of three O-Ac groups, and retention of one N-Ac, confirming again the presence of a
reactive amine in the native lipid. Sodiated molecular ions were now observed at m/z 748, 774, and 776. Sphingosine ions were again observed at m/z 324, 306, and 264, representing the singly dehydrat¬ ed, mono-N-acetylated fragment, the doubly dehydrated, mono-N- acetylated fragment, and the elimination of HOAc from the singly dehydrated fragment, respectively. The dehydrated N-Ac, O-Ac ion at m/z 366 was no longer observed. Similar results were obtained for the upper band lipid (not shown).
These results establish that (a) the 3-OH group of sphingosine is free in the native lipids and (b) the modifying group(s) occupy two hydroxyl positions on the galactose moiety. This could not be accom¬ modated by the attachment of two enol ethers in tandem, since the
mass increases relative to free psychosine would have to be twice those observed. The only modification consistent with the FAB-MS
and other data appeared to be attachment of long chain aldehydes as cyclic acetals. Acetylation of d 18:1 sphingosine with 16:0, 18:1 , and 18:0 fatty aldehydes would yield the observed molecular weights for the new lipids. This conclusion was confirmed by methyla-
tion/linkage analysis, as described below.
Methylation analysis bv GC-MS. Following permethylation,
acid hydrolysis, reduction, and acetylation of the native lipids, the
resulting partially methylated hexitol acetates were analyzed by GC-
MS (Figs. 8 A and 8B). Fig. 8A is PMAA from upper band lipid; Fig. 8B is PMAA from middle band lipid; Fig. 8C is PMAA from upper band lipid following brief acid treatment; Fig. 8D is standard galactose PMAAs. Peaks are identified as PMAAs of 1 : 2,3,6-tri-O-; 2: 3,4,6 + 2,4,6-tri-O-; 3: 2,3 4-tri-O-; 4: 2,6-di-O-; 5: 4,6-di-O-; 6: 3,6-di-O-; 7: 2,3-di-O-; 8: 6-mono-O-; 9: 3,4-di-O-; 10: 2-mono-O-; and 11 : 3 (or 4)-mono-O-
Me-Gal.
From the upper band lipid, 2,6-di-£-Me-Gal was obtained, while
2,3-di-Q-Me-Gal was obtained from the middle band lipid. These
represent 3,4- and 4,6-linked galactose moieties, respectively, and clearly show that the lipids must be isomeric cyclic acetals derived
from psychosine, that in the upper band forming a five membered ring, and that in the middle band forming a six membered ring. The
product from limited acid treatment of the upper band also yielded
2,3-di-Q-Me-Gal (Fig. 8C), demonstrating the facile isomerization of the five-membered ring into the more stable six-membered ring. Finally, while the chiralities at the acetal C-1 positions have not
definitively been determined, they are believed to be an equatorial orientation for the long chain in the six-membered acetal ring, and a
pseudoequatorial orientation for this group in the five-membered ring.
EXAMPLE 5 ISOLATION AND PURIFICATION OF COMPOUNDS C AND D
Galactosyl cerebroside and sulfatide used in the examples were
purchased from Sigma Chemical Co.. Fatty aldehyde (plasmal) was purchased from Aldrich.
Isolation of "Fast Migrating Component"
Human brain cerebroside fraction was obtained by homogeniza¬ tion of brain tissue with five volumes (i.e., five times volume/weight
of wet tissue) of isopropanol/hexane/water (IHW) 55:25:20 (v/v/v)
and filtration through a Bϋchner funnel (hereinafter, all solvent ratios are by volume). The residue was subjected to re-homogenization in the same volume of the same solvent. The extract was pooled, evaporated to dryness, and subjected to Folch partition using 1 L of chloroform/methanol (CM) 2:1 and 166 ml water per 100 g original
wet weight of tissue. The lower phase was repartitioned two additional times with "theoretical upper phase" (chloro¬ form/methanol/water with 0.2% KCI 10:10:1 ). The resulting lower phase was evaporated to complete dryness. A large column (bed
volume 1 L per 1 kg original tissue) of FLORISIL (a mixture of magnesium oxide and silicic acid gel) (from Sigma; mesh 60-100) was prepared and equilibrated in pure hexane (Burdiack & Jackson
Chemical Co.). The dried lower phase was suspended in hexane (1 L per 200 g original tissue), passed over the FLORISIL column, and
exhaustively washed with 4 L of hexane. The FLORISIL column was
then eluted with 2 L of hexane/dichloroethane (DCE) 2:1 , then with
2 L of DCE, and finally with 1 L of DCE/acetone 1 :1. The final eluate
contained acid-labile fast-migrating component.
Detection of acid-labile olvcolipids
Acid-labile glycolipids were detected by hydrolysis of samples in methanol-aqueous θ.1 N HCI (1 :1 , v/v) heated at 90°C for 10 min, followed by Folch partitioning and TLC examination of lower phase.
The glycolipid with high TLC mobility converting to the same mobility as normal cerebroside by this treatment was regarded as the acid-
labile cerebroside. Cerebroside and ester cerebrosides did not show altered TLC mobility under these conditions.
Isolation and preliminary characterization of acid-labile glycosphingo¬
lipids present in the fast-miorating fraction
The acid-labile fast-migrating glycolipid was found in the unab-
sorbed fraction of brain extract on carboxymethyl-SEPHADEX and diethylaminoethyl-SEPHADEX of the Folch's lower phase as well as in the DCE-acetone 1 :1 eluate fraction on chromatography over
FLORISIL. This component was further purified by high-performance liquid chromatography (HPLC) on an IATROBEAD column loaded in
pure hexane and eluted with a gradient to IHW 55:40:5 at 1 ml/min for 3 hours. Fractions were collected into 200 tubes. The acid-labile glycolipid component was eluted in tube Nos. 130-154. The pooled fraction (called fraction VI) was considered to contain most of the
acid-labile glycolipid and was free of cerebroside and ester cerebrosid¬ es. The fraction VI was further purified by lATROBEADS chromatog- raphy, loaded on the columns in pure hexane and subjected to a gradient up to isopropanol/hexane (IH) 30:70. The fraction VI A (Figure 11 , lane 5), thus obtained, was further purified on a long
IATROBEAD column (0.5x100 cm) with a gradient, loaded with pure hexane, and gradient eluted to IHW 50:40:5 for 3 hours. Alternative- ly, the compound was purified by preparative thin layer chromatogra¬ phy (TLC). The homogeneous band obtained is shown in Figure 1 1 ,
lane 6. On TLC, the compound migrated faster than cholesterol which migrated faster than ester cerebrosides. The compound did
not contain sulfate or sialic acid which are known to be acid-labile. Figure 11 is a high-performance thin layer chromatography
(HPTLC) pattern of various non-polar glycosphingolipids from Folch's lower phase prepared from human brain. The chromatogram was developed in a solvent mixture of chloroform/methanol/28% NH4OH (80:20:2). Lane 1 is standard CMH (cerebroside); lane 2 is lower
phase obtained on Folch's partition; lane 3 is unabsorbed pass
through of total lower phase by carboxymethyl-SEPHADEX; lane 4 is
Fr. VI obtained by FLORISIL column (eluted by dichloroethane/actone
(1 : 1 , by volume); lane 5 is Fraction 47-58 eluate on IATROBEAD
chromatography; lane 6 is purified plasmal cerebroside from Fraction 47-58; lane 7 is purified ester-cerebrosides.
EXAMPLE 6 CHEMICAL DEGRADATION OF COMPOUNDS C AND D
Compounds C and D, as well as cerebroside (CMH), were chemically degraded with an acid or base treatment. Acid treatment was in 0.3 N HCI in MeOH at 80°C for 30 minutes. Weak base
hydrolysis was carried out in 0.3N NaOH in MeOH at 80°C for 40
minutes. The compounds and their degradation products were then separated by high-performance thin-layer chromatography. The results are shown in Fig. 12.
In Fig. 12, the lanes are as follows:
Lane 1 , CMH; lane 2, CMH degraded by 0.3 N HCI in MeOH;
lane 3, CMH 0.3 N NaOH; lane 4, plasmalocerebroside; lane 5, plasmalocerebroside treated with 0.3 N HCI in MeOH; lane 6, plasmalocerebroside treated with 0.3 N NaOH in MeOH; lane 7, ester cerebroside 1 ; lane 8, ester cerebroside 1 treated with 0.3 N HCI in MeOH; lane 9, ester cerebroside 1 treated with 0.3 N NaOH in MeOH; lane 10, ester cerebroside 2; lane 11 , ester cerebroside 2 treated with
0.3 N HCI in MeOH; lane 12, ester cerebroside 2 in 0.3 N NaOH in
MeOH.
The results show that the plasmalocerebrosides are acid-labile and base stable, whereas the cerebroside esters in lanes 7 and 10 are essentially acid resistant.
EXAMPLE 7
STRUCTURAL CHARACTERIZATION OF COMPOUNDS C AND D
Fattv acid, aledhvde. and monosaccharide analysis. Fatty acids were estimated as methylesters (FAMEs) liberated by methanolysis (1.0 ml 0.5 N HCI in anhydrous methanol, 80°C, 24 hr) of about 30-
50 μg of lipid. Fatty aldehydes released during the same procedure were converted to long chain enol methyl ethers (EMEs) as described for the psychosine fatty acetals). Both of these components were extracted from the methanolysate, prior to neutralization, by partition¬ ing 3x with approximately equal volumes of hexane. The combined hexane extracts were reduced in volume under N2 stream at 35-40°C to approximately 1-2 μ\, then taken up in a volume of hexane (10-50 μ\) providing a suitable dilution for analysis by GC-MS. GC-MS of aliquots of the hexane extractable material were performed using a Hewlett-Packard 5890A gas chromatograph interfaced to an Extrel
ELQ 400 quadrupole mass spectrometer. Gas chromatography was performed using a 30 m DB-5 (J & W Scientific, Ranch Cordova, CA) bonded-phase fused silica capillary column (0.25 mm o.d., 0.25 μm
film thickness; splitless injection; temperature program, 150-290°C at 4°C/min). The mass spectrometer was operated in either Cl
(isobutane; mass range, 150-500 u, scanned once per second) or El
(mass range 50-500 u, scanned once per second) mode. Derivatives
were identified by characteristic ions and retention times, verified by co-injection with standards when necessary.
The remaining acidic MeOH lower layer was neutralized by
addition of Ag2C03 (approximately 10 mg) and treated with acetic anhydride (100 μ\) for 6 hr at room temperature. Following centrifu¬ gation and removal of the MeOH, the precipitate was washed 2x with
1 ml portions of MeOH. The combined MeOH extracts were dried under N2 stream. The resulting monosaccharide methyl glycosides
were anlayzed as their per-O-trimethylsilyl ethers (26, 27) by GC-MS using the Extrel ELQ 400 system described above (DB-5 column; splitless injection; temperature program, 140-270°C at 4°C/min; Cl-
MS (isobutane) mode). Methylation/linkaoe analysis. Linkage positions of substituents
on glycosyl residues were determined by permethylation of approxi¬ mately 50 μg of each sample, followed by hydrolysis, reduction, peracetylation and GC-MS as described in detail elsewhere (22), except that the analysis was performed on the Extrel ELQ 400 GC-MS system described above (DB-5 column; splitless injection; temperature
program, 140-250°C at 4°C/min; Ei-MS mode), with identification of partially methylated alditol acetate (PMAA) derivatives made by
retention time and characteristic electron-impact mass spectra (31,32). Identifications were confirmed by comparison with PMAAs in known standard mixtures.
Fast atom bombardment mass spectrometry. + FAB-MS was
performed on a JEOL (Tokyo, Japan) HX-110/DA-5000 mass spectrometer/data system, operated in the accumulation mode at full acceleration voltage (10 kV); xenon beam, 6 kV; mass range, 3000; resolution, 3000. Aliquots of sample (approximately 20 μg) in MeOH were transferred to a FAB target and suspended in NBA matrix. Three scans were accumulated for each spectrum. Kl/Csl was used
as the calibration standard. Identification of fattv aldehyde and fattv acid bv GC-MS after
methanolysis. The acid-labile compounds gave a "plasmal reaction" under classical conditions indicating the presence of plasmal. This was confirmed by GC-MS analysis after methanolysis.
GC-MS analysis of hexane extract of HCI-methanolysate revealed the presence of multiple peaks which were not detected in
the methanolysate of normal cerebroside or ester cerebroside in addition to those peaks corresponding to fatty acid methyl esters
(FAMEs) 16:0, 18:1 , 18:0, and 24:1. These peaks were determined by comparison of retention times along with electron impact (El) and
chemical ionization (Cl) mass spectra to authentic compounds. They
were thus identified as enol methyl ethers (EMEs) derived from fatty aldehyde, i.e., as EMEs of 16:0, 18:0 and 18: 1 (Fig. 13).
Fig. 13 is a CG-EI/MS of long chain FAMEs and EMEs from methanolysis of the unknown lipid component. The peaks were
identified as marked. Peaks marked by an asterisk are unidentified
impurities.
+ FAB-MS analysis of native lipid. A FAB mass spectrum of the unknown native lipid was obtained in the positive ion mode. The spectrum is reproduced in Fig. 14. In Fig. 14, the peaks are labeled
with nominal, monoisotopic masses. The spectrum was characterized in the lower mass end by
fragments at m/z 282 and 264, which correspond in both mass and relative abundance to the sphingosine-related ions derived by de-N- acylation and dehydration of ceramide (W and W", respectively, in the nomenclature of Domon and Costello (35), as commonly found in positive ion FAB and FAB-CID (fast atom bombardment - collision
induced dissociation) spectra of cerebrosides having d18:1 sphingo-
sine (36, 38, 41 ). Ceramide ions (Y0) were found most abundantly at m/z 520, 546, 548, and 630, corresponding to compositions having d18:1 sphingosine N-acylated primarily with 16:0, 18:1 , 18:0, and 24:1 fatty acids. These would be expected on the basis of the
FAME analysis (Fig. 13). A small peak consistent with a cerebroside [MH]+ was observed at m/z 792 (corresponding to Hex»Cer with d18:1 sphingosine and 24:1 fatty acid). The primary group of psuedomolecular ions [MH]+ were found at m/z 930, 932, 956, 958, and 960. The even mass numbers observed correspond to odd molecular weights, and therefore to compounds containing an odd number of nitrogen atoms. In analogy to the psychosine acetal
structures determined previously, these pseudomolecular ion species
were hypothesized to correspond to cerebrosides which have been modified by long chain fatty aldehydes attached in cyclic acetal linkages to vicinal hydroxy groups of the galactose moiety. As
determined by analysis of the GC-MS peaks corresponding to long- chain EMEs (Fig. 13), these aldehydes would be primarily 16:0, 18:1, and 18:0 species. The observed pseudomolecular ion abundances would therefore reflect a complex distribution according to the proportions of both fatty acid and fatty aldehyde moieties of different
lengths found in the lipid. For example, the most abundant pseudom¬
olecular ion at m/z 956 would correspond to a galactocerebroside
acetal having d18:1 sphingosine, 18:1 fatty acid and 18:1 fatty aldehyde. The ion at m/z 930 could correspond to either d18:1
sphingosine, 18: 1 fatty acid, and 16:0 aldehyde, or d18: 1 sphingo¬ sine, 16:0 fatty acid, and 18: 1 aldehyde. Other ions in the cluster
represent other possible combinations (all with di 8: 1 sphingosine) of the most abundant fatty acid and aldehyde species. The conclusion that the compounds are cerebrosides modified by acetal linkage to
vicinal hydroxγ groups of galactose was confirmed by methyla- tion/linkage analysis, as described below.
Methylation analysis by GC-MS. Following permethylation, acid hydrolysis, reduction, and acetylation of the native lipid, the resulting partially methylated hexitol acetates were analyzed by GC-
MS.
The results are shown in Fig. 15.
In Fig. 15, the peaks are identified as PMAAs of 1 : 2,3,4,6- tetra-O-; 2: 2,6-di-O-; and 3: 4,6-di-O-Me-Gal.
The primary component detected was 2,6-di-φ-Me-Gal, along
with smaller peaks corresponding to 2,3-di-Q-Me-Gal and 2,3,4,6- tetra-Q-Me-Gal. The di-O_-Me- peaks represent 3,4- and 4,6-linked substituents, respectively, on galactose and show that the lipid fraction must be comprised of isomeric cyclic acetals derived from cerebroside, mostly in a five-membered 3,4-linked ring, with some
six-membered 4,6-linked ring. The small trace of 2,3,4,6-tetra-_2-Me-
Gal is consistent with the low abundance pseudo-molecular ion
detected for unsubstituted cerebroside. These linkages were also found in separate components of the psychosine acetals previously determined. The chiralities of the acetal C-1 positions have not been definitively determined. However, they are believed to be an
equatorial orientation for the long chain in the six-membered acetal ring, and a pseudo-equatorial orientation for this group in the five- membered ring is assumed.
EXAMPLE 8 DETERMINATION OF NEURITOGENIC ACTIVITY
Neuritogenic activity of compounds A, B, C, D and E was determined as previously described (10), employing various neuroblas¬
toma cell lines in which neurite formation is dependent either on nerve growth factor (NGF) or gangiioside. Neuroblastoma cell lines were cultured in gelatin-coated plates as described previously (11,12).
Various concentrations (5-150 μM) of glycolipid were added and cultured for observation of neurite formation. Incidence of cells forming neurites > 50 μm long was counted as a percent of total population. Photographs for cells treated with compounds A and B were taken at 24 hour intervals.
Striking neurite formation was observed in mouse neuroblasto¬ ma Neuro-2A cells on addition of plasmalopsychosine compounds A
and B, particularly in the presence of NGF at 50 //g/ml concentration; neurites, i.e., > 50
Figure imgf000067_0001
long, comprised as much as 60-80% of the total cell population. This concentration was much lower than that previously reported for a ganglioside effect. That is, the most effective ganglioside, GT1 b, required a concentration of 200 g/ml.
A mixture of bovine brain ganglioside required at least 100-150 g/ml. Other types of cells, including mouse and human neuroblasto¬ ma, showed similar degrees of neuritogensis induced by plasmalopsy- chosine. Psychosine by itself showed a strong cytotoxic effect on various neuroblastoma cell lines; cell growth was inhibited, morpholo¬
gy changed, and cells eventually died in the presence of 10-20 //g/ml psychosine. No neuritogenesis occurred in the presence of
psychosine. Patterns of neurite formation for plasmalopsychosine
compounds A and B are shown in Figs. 9A, 9B and 10. Figures 9A and 9B show a neuritogenesis pattern of Neuro-2A cells in the presence of 50 //g/ml plasmalopsychosine compounds A and B at different areas on the culture dish.
Figure 10 is a graph showing the effect of plasmalopsychosine on neurite formation in Neuro-2A cells, wherein the abscissa represents the concentration of plasmalopsychosine (//g/ml) and the ordinate represents the percentage of Neuro-2A cells developing neurites (> 50//m in length). The circles (open and closed) represent results for a mixture of the upper and middle bands of plasmolo- psychosine, + and - nerve growth factor (NGF). The open triangles
represent results in the presence of NGF for a mixture of bovine brain gangliosides (BBG) containing the gangliosides GM1 , GD1a, GD1b
and GT.
The results represented in Fig. 10 show that even when NGF
is added to cells treated with plasmolopsychosine, no effect is seen.
Thus, the neurite formation is due to the plasmalopsychosine.
Plasmalocerebroside had no clear effect in early stages of cell culture. However, neurite formation, i.e., > 50 //m long, became
increasingly apparent by 1 week. Thus, plasmalocerebroside possesses neuritogenic activity. With 50 //g/ml concentration, after
2 weeks of incubation, neurite formation in Neuro-2A cell culture was more pronounced in the presence of plasmalocerebroside than plasmalopsy-chosine.
EXAMPLE 9
CHEMICAL SYNTHESIS OF COMPOUNDS A AND B Plasmalopsychosine A and B were chemically synthesized from
psychosine according to the "Synthetic scheme for plasmalopsycho¬ sines A and B" (Fig. 16).
To a solution of psychosine (prepared synthetically or obtained
by the alkaline hydrolysis of CMH extracted from bovine brain) in a mixture of chloroform and water, 9-fiuorenylmethyl chloroformate and potassium carbonate (45) were added and the reaction mixture was stirred at room temperature for 21 hours. After the evaporation of the reaction mixture in vacuo, a small volume of water was added to the residue which formed a white slurry. This slurry was loaded on
a pre-conditioned BOND ELUT C-18 column and rinsed with water ro
remove water-soluble components. The retained lipophilic com¬ pounds were recovered by eluting the column with methanol and the
eluate was evaporated in vacuo to give FMOC-psychosine. Thin layer chromatography of the product in chloroform/methanol 9:1 or
toluene/methanol 3:1 showed the presence of some U.V. positive impurities, which were removed using a silica column and tolu- ene/methanol 3:1 or chloroform/methanol 9:1 as solvent mixture. The purified compound, obtained in 87% yield, [σ]25 D + 5.17 (C 1.42
in CHCI3) was then used for making cyclic acetals of psychosine. The other reactant,σ-σ-dimethoxy hexadecane, required for the formation of cyclic acetals was synthesized in two steps from n- hexadecanol (Aldrich, Milwaukee, Wl): i) Oxidation using pyridinium chlorochromate (46) in dichloromethane to give aldehyde and ii) reaction of the aldehyde with trimethylorthoformate (Aldrich,
Milwaukee, WJ) in the presence of AMBERLITE IR-120 (Rohm & Haas Co., PA) under reflux (47).
Cyclic acetals were prepared as follows: To a solution of FMOC-psychosine in N,N-dimethylformamide, σ-σ-dimethoxy hexadecane and p-toluene sulfonic acid were added and the reaction mixture was stirred at room temperature for 19 hours. Then the
reaction mixture was quenched with triethylamine to neutralize p- toluene sulfonic acid, and evaporated in vacuo. Residue was
transferred to a BOND ELUT C-18 column and rinsed with water. Cyclic acetals of FMOC-psychosine along with other lipophilic
compounds were finally eluted from the column using methanol, and eluate was evaporated in vacuo. Desired cyclic acetals were roughly separated from other compounds by silica column chromatography using toluene/methanol 3:1 solvent. The mixture of cyclic acetals of FMOC-psychosine was then treated with pipyridine for three hours to remove FMOC protecting group (48), and evaporated in vacuo.
Separation of plasmalopsychosine A and B from other products
was accomplished using isopropanol/hexane/water gradient on lATROBEADS (10//M) column, pre-equilibrated as described in Example 1.
The sample was prepared for injection by adding 100 μ\ of chloroform/methanol 2: 1 and slightly warning while sonicating. To
this about 1.5 ml of hexane was added during sonication. Sample was loaded onto the column and eluted with hexane, gradually
changing to isopropanol/hexane/water gradient 30:69: 1 over a period of 200 minutes and eluting with same gradient for 50 minutes (200-
250 minutes). Gradient was finally changed to 55:25:20 (250-400 minutes) and elution was continued for the next 200 minutes (400-
600 minutes) with the same gradient. Eluate (6 mins/tube) was
collected and each fraction was checked by HPTLC (chloro-
form/methanol/NH4OH 80:20:2). Identical fractions on HPTLC were pooled together, concentrated and compared with anionic lipid fractions of human brain obtained from carboxymethyl sephadex column chromatography. The results are shown in Figure 17, where
Lane 1 is crude synthetic preparation of psychosine acetals, Lanes 2- 5 are pooled fractions of synthetic product from HPLC on an IATROBEAD column, and Lane 6 is total eluate of anionic lipid
fractions of human brain (cerebrum) obtained from carboxymethyl
sephadex column with 0.5 M triethylamine.
Fractions identical to upper and middle band lipids plasmalopsy¬ chosine A and plasmalopsychosine B were further characterized by NMR, FAB-MS and ethylation by GC-MS (Figures 18A-18C), which conformed to the assigned structure. Fractions of Lane 2 and Lane 3 of Figure 17 have not yet been characterized.
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While the invention has been described in detail above with reference to a preferred embodiment, various modifications within the scope and spirit of the invention will be apparent to people of working skill in this technological field. Thus, the invention should be consid¬
ered as limited only by the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:
1. An isolated or synthetic plasmalopsychosine selected from the group consisting of compound A and compound B:
CH-CH-CH-(CH_)-CH, (fl)
CHj-tCHjJ OH
Figure imgf000078_0001
-CH-CH-(CH2)-CH3 (B)
Figure imgf000078_0002
wherein n1 is a number greater than 0, and pharmaceutically
acceptable salts thereof.
2. The isolated or synthetic plasmalopsychosine of claim 1 , which is the compound A.
3. The isolated or synthetic plasmalopsychosine of claim 2,
wherein the chiralitγ at the acetal C-1 position of the com- pound A is an equatorial orientation.
1 4. The isolated or synthetic plasmalopsychosine of claim 1 , which
2 is the compound B.
1 5. The isolated or synthetic plasmalopsychosine of claim 4,
2. wherein the chirality at the acetal C-1 position of the com-
3 pound B is a pseudo-equatorial orientation.
1 6. An isolated or synthetic plasmalocerebroside selected from the
2 group consisting of Compound C and Compound D:
,)-CHj (C>
Figure imgf000079_0001
C NHC0-(CH2)n3-CH3 -CH--CH-CH-CH_CH-(CH,)-CH- (D)
Figure imgf000079_0002
wherein n2 and n3 each is a number greater than 0, and
pharmaceutically acceptable salts thereof.
7. The isolated or synthetic plasmalocerebroside of Claim 6,
which is the Compound C.
8. The isolated or synthetic plasmalocerebroside of Claim 7,
wherein the chirality at the acetal C-1 position of the com- pound C is an equatorial orientation.
9. The isolated or synthetic plasmalocerebroside of Claim 6, which is the Compound D.
10. The isolated or synthetic plasmalocerebroside of Claim 9,
wherein the chirality at the acetal C-1 position of the com- pound D is a pseudo-equatorial orientation.
11. A composition for treating neuronal diseases and tissue damage comprising one or more plasmalopsychosines and/or plasmalocerebrosides selected from the group consisting of
compound A, compound B, compound C and compound D:
( A )
Figure imgf000080_0001
CH3-(CH2 )
Figure imgf000080_0002
Figure imgf000081_0001
C H - < D )
Figure imgf000081_0002
wherein n n2 and n3 each is a number greater than 0, and pharma-
ceutically acceptable salts thereof; and a pharmaceutically acceptable carrier, diluent or excipient.
12. The composition of claim 1 1 , wherein the plasmalopsychosine is the compound A.
13. The composition of claim 12, wherein the chirality at the acetal C-1 position of the compound A is an equatorial orientation.
14. The composition of claim 1 1 , wherein the plasmalopsychosine
is the compound B.
15. The composition of claim 14, wherein the chirality at the acetal
C-1 position of the compound B is a pseudo-equatorial orienta- tion.
16. The composition of Claim 11 , wherein the plasmalocerebroside is the Compound C.
17. The composition of Claim 16, wherein the chirality at the
acetal C-1 position of the compound C is an equatorial orienta- tion.
18. The composition of Claim 11 , wherein the plasmalocerebroside is the Compound D.
19. The composition of Claim 18, wherein the chirality at the acetal C-1 position of the compound D is a pseudo-equatorial orientation.
20. A method of forming neurites from nerve cells comprising contacting said cells with an effective amount of one or more plasmalopsychosines and/or plasmalocerebrosides selected from the group consisting of compound A, compound B, compound C and compound D:
Figure imgf000083_0001
-CH ■CH-(CH2)-CHj (B)
Figure imgf000083_0002
Figure imgf000083_0003
(C)
Figure imgf000083_0004
j)-CH3 <D:
Figure imgf000083_0005
wherein n1# n2 and n3 each is a number greater than 0.
21. The method of claim 20, wherein the plasmalopsychosine is
the compound A.
22. The method of claim 21 , wherein the chirality at the acetal C-1 position of the compound A is an equatorial orientation.
23. The method of claim 20, wherein the plasmalopsychosine is the the compound B.
24. The method of claim 23, wherein the chirality at the acetal C-1 position of the compound B is a pseudo-equatorial orientation.
25. The method of Claim 20, wherein the plasmalocerebroside is the Compound C.
26. The method of Claim 25, wherein the chirality at the acetal C-1 position of the compound C is an equatorial orientation.
27. The method of Claim 20, wherein the plasmalocerebroside is the Compound D.
28. The method of Claim 27, wherein the chirality at the acetal C-1 position of the compound D is a pseudo-equatorial orientation.
29. A method for treating neuronal diseases and tissue damage comprising administering to a host in need of treatment a biologically effective amount of one or more plasmalopsycho¬
sines and/or plasmalocerebrosides selected from the group
consisting of compound A, compound B, compound C and compound D:
-CH-CH-(CH2)-CH3 (A)
-CH-CH-(CH2)-CHj (B)
Figure imgf000085_0001
Figure imgf000086_0001
wherein n1 f n2 and n3 each is a number greater than 0; and
pharmaceutically acceptable salts thereof.
30. The method of claim 29, wherein the plasmalopsychosine is the compound A.
31. The method of claim 30, wherein the chirality at the acetal C-1
position of the compound A is an equatorial orientation.
32. The method of claim 29, wherein the plasmalopsychosine is the compound B.
33. The method of claim 32, wherein the chirality at the acetal C-1
position of the compound B is a pseudo-equatorial orientation.
34. The method of Claim 29, wherein the plasmalocerebroside is the compound C.
35. The method of Claim 34, wherein the chirality at the acetal C- 1
position of the compound C is an equatorial orientation.
36. The method of Claim 29, wherein the plasmalocerebroside is the compound D.
37. The method of Claim 36, wherein the chirality at the acetal C-1
position of the compound D is a pseudo-equatorial orientation.
PCT/US1992/005853 1991-07-31 1992-07-20 Plasmalopsychosines and plasmalocerebrosides and methods of treating neuronal diseases employing the same WO1993002685A1 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
DE69225712T DE69225712D1 (en) 1991-07-31 1992-07-20 PLASMALOPSYCHOSINE AND PLASMALOCEREBROSIDE AND METHOD FOR TREATING NEURONAL DISEASES WITH THESE SUBSTANCES
JP5503579A JPH06509354A (en) 1991-07-31 1992-07-20 Plasmablopsychocines and plasmalocerebrosides, and methods for treating neuronal diseases using them
EP92915984A EP0596937B1 (en) 1991-07-31 1992-07-20 Plasmalopsychosines and plasmalocerebrosides and methods of treating neuronal diseases employing the same

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US738,375 1991-07-31

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4544552A (en) * 1982-05-28 1985-10-01 Solco Basel Ag Process for the preparation of cell and tissue regenerating substances

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4544552A (en) * 1982-05-28 1985-10-01 Solco Basel Ag Process for the preparation of cell and tissue regenerating substances

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, Volume 81, issued 1974, VEINBERG et al., "Sphingosine and its natural compounds. IX. Possible selective protection of hydroxyl groups in galactocerebrosides", see entire Abstract. *
Drugs of Today, Volume 22, No. 2, issued 1986, J.C. SAMSON, "Gangliosides (Cronassial)as therapeutic agents in peripheral neuropathies", pages 73-107. *
Japan. Journal of Experimental Medicine, Volume 44, No. 2, issued 1974, M. KUBOTO et al., "Minor Glycolipids Being Less Polar Than Cerebroside in Porcine Spinal Cord", pages 145-150, especially page 149. *
Journal of Lipid Research, Volume 9, issued 1968, KISHIMOTO et al., "6-Acyl galactosyl ceramides of pig brain: structure and fatty acid composition", pages 27-33, entire document. *
See also references of EP0596937A4 *
The Journal of Pharmacology and Experimental Therapeutics, Volume 252, No. 1, issued January 1990, MANEV et al., "Glutamate Induced Neuronal Death in Primary Cultures of Cerebellar Granule Cells: Protection by Synthetic Derivatives of Endogenous Sphingolipids", pages 419-427. *

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