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WO2002000674A1 - Messagers phosphoglycane et leurs applications medicales - Google Patents

Messagers phosphoglycane et leurs applications medicales Download PDF

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Publication number
WO2002000674A1
WO2002000674A1 PCT/GB2001/002775 GB0102775W WO0200674A1 WO 2002000674 A1 WO2002000674 A1 WO 2002000674A1 GB 0102775 W GB0102775 W GB 0102775W WO 0200674 A1 WO0200674 A1 WO 0200674A1
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WIPO (PCT)
Prior art keywords
substance
ipg
phosphatidyl
cyclitol
ipgs
Prior art date
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PCT/GB2001/002775
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English (en)
Inventor
Khalid Elased
Khalid Gumaa
Hassan Rahmoune
Thomas Rademacher
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Rodaris Pharmaceuticals Limited
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Publication date
Application filed by Rodaris Pharmaceuticals Limited filed Critical Rodaris Pharmaceuticals Limited
Priority to EP01940824A priority Critical patent/EP1409496A1/fr
Priority to AU2001274318A priority patent/AU2001274318A1/en
Priority to CA002427811A priority patent/CA2427811A1/fr
Publication of WO2002000674A1 publication Critical patent/WO2002000674A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • C07H1/08—Separation; Purification from natural products
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H11/00—Compounds containing saccharide radicals esterified by inorganic acids; Metal salts thereof
    • C07H11/04—Phosphates; Phosphites; Polyphosphates

Definitions

  • the present invention relates to phosphoglycan messengers (PGMs) and their medical uses, and in particular to PGMs comprising one or more lipid moieties, and in particular to their use in treating dyslipidemia and related conditions.
  • PGMs phosphoglycan messengers
  • Glycosylphosphatidylinositols are essential components in the plasma membrane of cells (Thomas et al, 1990) including malaria parasites, both as membrane anchors for proteins and as the sole class of free glycolipids (Gerold et al,1997), and, in their role of precursors of IPGs, they may also play a role in insulin signal transduction (Saltiel & Cuatrecasas, 1986) . Binding of insulin to its receptor leads to phosphatidylinositol-specific phospholipase cleavage of GPI and generation of two extracellular signals, diacyl glycerol and inositol phosphoglycans (IPGs) .
  • IPGs inositol phosphoglycans
  • IPGs Inositol phosphoglycans
  • GPI-PLD a unique insulin second messenger system
  • IPG-A mediators mimic the lipogenic activity of insulin in adipocytes and inhibit cAMP-dependent protein kinase (Caro et al, 1997) .
  • IPG-P mediators mimic the glycogenic activity of insulin in skeletal muscle and stimulate pyruvate dehydrogenase phosphatase (PDH) .
  • PDH dehydrogenase phosphatase
  • W098/11116 and W098/11117 disclose the purification, isolation and characterisation of IPG-P and IPG-A from human tissue. Prior to these applications, it had not been possible to isolate single components from the tissue derived IPG fractions, much less in sufficient quantities to allow structural characterisation.
  • IPGs have been isolated from different sources including mammals, yeast and Trypanosomes, and have been extensively tested for their insulin mimetic activity in vitro, they have so far only been tested in vivo in streptozotocin (STZ) -diabetic rats (Huang et al, 1993;
  • IPG-P from malaria parasites has been shown to have insulin mimetic activities in vitro (Caro et al, 1996) .
  • No IPGs or IPG-related compounds from any source have been studied in murine models of Type 2 diabetes.
  • GPIs purified from malaria parasites have been tested in vivo only in normal mice pretreated with thioglycollate (Schofield & hackett, 1993) in experiments demonstrating that some components of parasitized erythrocytes trigger the release of toxins such as TNF from activated macrophages.
  • the present invention is based on the finding that phosphoglycan messengers (PGMs) , and in particular PGMs comprising lipid groups, are biologically active and therefore have therapeutic utility in the treatment of conditions characterised by dyslipidemia or lipodystrophy.
  • PGMs of the present invention can be used to treat conditions characterised by abnormal or elevated levels of cholesterol, plasma triglycerides, and/or high density lipoproteins (HDL) , especially elevated levels or amounts of these substances, or an abnormal LDL: HDL ratio.
  • the PGMs of the present invention can be contrasted with the GPI-anchors purified in prior art studies as the latter are protein linked.
  • GPIs are biologically inactive and merely serve as the precursor of biologically active IPGs (Rademacher et al, 1994;
  • PGMs comprising lipid groups, such as GPIs
  • GPIs lipid groups
  • PGM second messengers for example as anti-dyslipidaemic agents for treating lipidic disorders associated with Type 1 or Type 2 diabetes, cardiovascular disorders and their complications such as coronary artery disease (CAD) other than associated with diabetes, hyperlipidemia, the treatment of systemic lupus erythematosis (SLE) , especially atherosclerotic lesions.
  • CAD coronary artery disease
  • SLE systemic lupus erythematosis
  • the present invention provides a substance which is a carbohydrate derivatised phosphatidyl cyclitol.
  • the substance comprises one or more lipid moieties attached to the cyclitol group or the phosphatidyl group via one or more ester and/or ether linkages.
  • the substance has one or more properties selected from modulating plasma cholesterol, plasma triglycerides and/or high density lipoprotein levels or modulating, and in particular lowering, the LDL:HDL ratio.
  • the substance may also have one or more properties selected from reducing blood glucose in a diabetic ob/ob and db/db urine model, regulating lipogenesis, stimulating pyruvate dehydrogenase phosphatase, inhibiting cAMP dependent protein kinase, inhibiting fructose-1, 6- bisphosphatase, and inhibiting glucose-6-phosphatase. These and other properties of the PGMs are discussed below. As mentioned above, preferred substances are devoid of protein.
  • the present invention provides one or more of the substances described above for use in method of medical treatment, e.g. as discussed above.
  • the results show that the PGMs disclosed herein can be used to treat (either prophylactically or therapeutically) conditions characterised by dyslipidemia or lipodystrophy.
  • the results disclosed herein show that PGMs of the present invention can be used to treat conditions characterised by abnormal or elevated levels of cholesterol, plasma triglycerides and/or high density lipoproteins (HDL) , especially elevated levels or amounts of these substances, or an abnormal LDL: HDL ratio.
  • HDL high density lipoproteins
  • PGMs comprising lipids were previously thought to be the precursor of biologically active IPGs or to stimulate the release of toxins, the finding that they can be used in the treatment of medical disorders requiring the administration of PGMs such as GPIs and IPGs is highly surprising.
  • the present invention provides the use of one or more of the above substances for the preparation of a medicament for the treatment of a condition ameliorated by administration of a PGM second messenger. Examples of such conditions are described below.
  • the present invention provides an isolated PGM as obtainable from malaria parasites.
  • the PGMs are obtainable using a sequence of steps comprising:
  • FIG. 1 High pH anion exchange chromatograms of IPG-A, showing separation of sugars in CarbopacTM PA 10 column (a and b) and separation of sugar alditols in CarbopacTM MAI column (c) .
  • FIG. 1 High pH anion exchange chromatograms of IPG-P, showing separation of sugars in CarbopacTM PA 10 column (a and b) and separation of sugar alditols in CarbopacTM MAI column (c) .
  • PGMs Phosphoglycan Messengers
  • PGMs denote a class of substances comprising inositol phosphoglycans (IPGs) and glycosyl phosphatidyl inositols (GPIs) .
  • IPGs are examples of a family of PGMs which may comprise lipidic groups
  • GPIs are examples of a family of PGMs comprising one or more lipidic groups attached to the cyclitol.
  • free IPGs are produced from GPIs by cleavage by enzymes such as GPI-PLD which removes the lipid groups from the parent GPI.
  • the GPI anchors of the prior art are protein linked.
  • the present invention concerns PGM substances which are carbohydrate derivatised phosphatidyl cyclitols.
  • the phosphatidyl groups comprise one or more lipid moieties attached to the cyclitol group or the phosphatidyl group via one or more ester and/or ether linkages.
  • the cyclitol is ⁇ hiro-inositol, iziyo-inositol, or derivatives thereof, such as substituted derivatives thereof, including pinitol (3-O-methyl-c.hiro-inositol) .
  • the lipid moieties are linked to the phosphatidyl group.
  • this group is linked to one or two oxygen linked lipid moieties.
  • the lipid moieties can be selected from the group consisting of diacyl, dialkyl, acyl-alkyl, lyso-acyl, lyso-alkyl, lyso, acyl or alkyl lipids.
  • lipidic moieties include 1-0- (C16: 0) lyso-alkylglycerol; (C16 : 0) ) lyso- acylglycerol; (C18 : 0) ) iyso-acylglycerol; (C20: 0) ) lyso- acylglycerol; (C22 : 0) ) lyso-acylglycerol; ceramide, (C16:0) fatty acid- (C18 : 1) sphingosine; ceramide, (C16:0) fatty acid- (C18 : 0) sphinganine; ceramide, (C24:0) fatty acid- (C18 : 1) sphingosine; ceramide,
  • the carbohydrate group is a hexose, and more preferably is selected from glucosamine, galactosamine, galactose, mannose, glucose, fucose or xylose including substituted derivatives thereof.
  • the substances of the invention have one or more properties selected from lowering blood glucose, lowering plasma cholesterol or plasma triglycerides and/or normalising the ratio of low: high density lipoproteins (LDL: HDL ratio), for example by reducing the level of low density lipoprotein.
  • LDL low: high density lipoproteins
  • these properties can be readily assessed in a suitable animal model such as a diabetic oh/oh and dh/dh mice fed with a high fat diet or in STZ-diabetic mice.
  • the substances may also have one or more of the properties attributed to IPGs in the prior art, such as regulating lipogenesis, stimulating pyruvate dehydrogenase phosphatase, inhibiting cAMP dependent protein kinase, inhibiting fructose-1, 6-bisphosphatase and/or inhibiting glucose- ⁇ -phosphatase.
  • IPG-A mediators modulate the activity of a number of insulin-dependent enzymes such as cAMP dependent protein kinase (inhibits), adenylate cyclase (inhibits) and cAMP phospho-diesterases (stimulates) .
  • IPG-P mediators modulate the activity of insulin-dependent enzymes such as pyruvate dehydrogenase phosphatase (stimulates) and glycogen synthase phosphatase (stimulates) .
  • the IPG-A mediators mimic the lipogenic activity of insulin on adipocytes, whereas the IPG-P mediators mimic the glycogenic activity of insulin on muscle.
  • IPG-A and IPG-P mediators are mitogenic when added to fibroblasts in serum free media. The ability of the mediators to stimulate fibroblast proliferation is enhanced if the cells are transfected with the EGF-receptor. IPG-A mediators can stimulate cell proliferation in the chick cochleovestibular ganglia.
  • Soluble IPG fractions having IPG-A and IPG-P activity have been obtained from a variety of animal tissues including rat tissues (liver, kidney, muscle, brain, adipose, heart) and bovine liver. IPG-A and IPG-P biological activity has also been detected in human liver and placenta, malaria parasitized RBC and mycobacteria.
  • the ability of an anti-inositolglycan antibody to inhibit insulin action on human placental cytotrophoblasts and BC3H1 myocytes or bovine-derived IPG action on rat diaphragm and chick cochleovestibular ganglia suggests cross-species conservation of many structural features. However, it is important to note that although the prior art includes these reports of IPG-A and IPG-P activity in some biological fractions, the purification or characterisation of the agents responsible for the activity is not disclosed.
  • IPG-A substances are cyclitol-containing carbohydrates, also containing Zn 2+ ion and optionally phosphate and having the properties of regulating lipogenic activity and inhibiting cAMP dependent protein kinase. They may also inhibit adenylate cyclase, be mitogenic when added to EGF-transfected fibroblasts in serum free medium, and stimulate lipogenesis in adipocytes.
  • IPG-P substances are cyclitol-containing carbohydrates, also containing Mn 2+ and/or Zn 2+ ions and optionally phosphate and having the properties of regulating glycogen metabolism and activating pyruvate dehydrogenase phosphatase. They may also stimulate the activity of glycogen synthase phosphatase, be mitogenic when added to fibroblasts in serum free medium, and stimulate pyruvate dehydrogenase phosphatase.
  • the PGMs of the invention may be derivatised in various ways .
  • derivatives of the PGMs includes salts, coordination complexes with metal ions such as Mn 2+ and Zn 2+ , esters such as in vivo hydrolysable esters, free acids or bases, hydrates, prodrugs or lipids, coupling partners.
  • Salts of the PGM compounds of the invention are preferably physiologically well tolerated and non toxic. Many examples of salts are known to those skilled in the art.
  • Compounds having acidic groups such as phosphates or sulfates, can form salts with alkaline or alkaline earth metals such as Na, K, Mg and Ca, and with organic amines such as triethylamine and Tris (2- hydroxyethyl) amine.
  • Salts can be formed between compounds with basic groups, e.g. amines, with inorganic acids such as hydrochloric acid, phosphoric acid or sulfuric acid, or organic acids such as acetic acid, citric acid, benzole acid, fumaric acid, or tartaric acid.
  • Compounds having both acidic and basic groups can form internal salts.
  • Esters can be formed between hydroxyl or carboxylic acid groups present in the compound and an appropriate carboxylic acid or alcohol reaction partner, using techniques well known in the art.
  • Derivatives which as prodrugs of the PGM compounds are convertible in vivo or in vitro into one of the active PGMs.
  • at least one of the biological activities of compound will be reduced in the prodrug form of the compound, and can be activated by conversion of the prodrug to release the compound or a metabolite of it.
  • Other derivatives include coupling partners of the compounds in which the compounds is linked to a coupling partner, e.g. by being chemically coupled to the compound or physically associated with it.
  • Examples of coupling partners include a label or reporter molecule, a supporting substrate, a carrier or transport molecule, an effector, a drug, an antibody or an inhibitor.
  • Coupling partners can be covalently linked to compounds of the invention via an appropriate functional group on the compound such as a hydroxyl group, a carboxyl group or an amino group.
  • Other derivatives include formulating the compounds with liposomes.
  • PGMs described herein or their derivatives can be formulated in pharmaceutical compositions, and administered to patients in a variety of forms, in particular to treat conditions which are ameliorated by the administration of inositol phosphoglycan second messengers .
  • compositions for oral administration may be in tablet, capsule, powder or liquid form.
  • a tablet may include a solid carrier such as gelatin or an adjuvant or an inert diluent.
  • Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. Such compositions and preparations generally contain at least 0.1wt% of the compound.
  • Parenteral administration includes administration by the following routes: intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraocular, transepithelial, intraperitoneal and topical (including dermal, ocular, rectal, nasal, inhalation and aerosol) , and rectal systemic routes.
  • routes including dermal, ocular, rectal, nasal, inhalation and aerosol
  • the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • suitable solutions using, for example, solutions of the compounds or a derivative thereof, e.g. in physiological saline, a dispersion prepared with glycerol, liquid polyethylene glycol or oils.
  • compositions can comprise one or more of a pharmaceutically acceptable excipient, carrier, buffer, stabiliser, isotonicizing agent, preservative or anti- oxidant or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • a pharmaceutically acceptable excipient e.g. orally or parenterally.
  • Liquid pharmaceutical compositions are typically formulated to have a pH between about 3.0 and 9.0, more preferably between about 4.5 and 8.5 and still more preferably between about 5.0 and 8.0.
  • the pH of a composition can be maintained by the use of a buffer such as acetate, citrate, phosphate, succinate, Tris or histidine, typically employed in the range from about 1 mM to 50 mM.
  • the pH of compositions can otherwise be adjusted by using physiologically acceptable acids or bases.
  • Preservatives are generally included in pharmaceutical compositions to retard microbial growth, extending the shelf life of the compositions and allowing multiple use packaging.
  • preservatives include phenol, meta-cresol, benzyl alcohol, para-hydroxybenzoic acid and its esters, methyl paraben, propyl paraben, benzalconium chloride and benzethonium chloride.
  • Preservatives are typically employed in the range of about 0.1 to 1.0 % (w/v) .
  • the pharmaceutically compositions are given to an individual in a "prophylactically effective amount” or a “therapeutically effective amount” (as the case may be, although prophylaxis may be considered therapy) , this being sufficient to show benefit to the individual.
  • compositions are preferably administered to patients in dosages of between about 0.01 and lOOmg of active compound per kg of body weight, and more preferably between about 0.5 and lOmg/kg of body weight .
  • composition may further comprise one or more other pharmaceutically active agents, either further compounds of the invention, inositol phosphoglycans, growth factors such as insulin, NGF or other growth factors listed below, or other drugs, e.g. those in use for the treatment of diabetes or other conditions set out below.
  • other pharmaceutically active agents either further compounds of the invention, inositol phosphoglycans, growth factors such as insulin, NGF or other growth factors listed below, or other drugs, e.g. those in use for the treatment of diabetes or other conditions set out below.
  • the PGM compounds of the invention can be used in the preparation of medicaments for the treatment (either prophylactically or therapeutically) conditions characterised by dyslipidemia or lipodystrophy.
  • the results disclosed herein show that PGMs of the present invention can be used to treat conditions characterised by abnormal or elevated levels of cholesterol, plasma triglycerides, and/or high density lipoproteins (HDL) , especially elevated levels or amounts of these substances, or an abnormal LDL: HDL ratio.
  • HDL high density lipoproteins
  • Lipoproteins are the form in which lipids are transported in mammals and are characterised by their densities as follow: 1.006-1.019, very low density (VLDL) ; 1.019- 1.063, low density (LDL); 1.063-1.21, high density (HDL); and >1.21, very high density (VHDL) .
  • VLDL very low density
  • LDL low density
  • HDL high density
  • VHDL very high density
  • lipidic disorders associated with Type 1 or Type 2 diabetes cardiovascular disorders and their complications such as coronary artery disease (CAD), e.g. other than that associated with diabetes, hyperlipidemia, the treatment of systemic lupus erythematosis (SLE), especially atherosclerotic lesions.
  • CAD coronary artery disease
  • SLE systemic lupus erythematosis
  • NIDDM Non-insulin-dependent diabetes mellitus.
  • TNF Tumour necrosis factor
  • IPGs Inositol phosphoglycans.
  • GPI Glycosylphosphatidyl inositol.
  • PGM Phosphoglycan messenger
  • FBPase Fructose-1, 6-Bisphosphatase.
  • Streptozotocin activated charcoal, ascorbic acid, ammonium molybdate, myo-inositol and bovine serum albumin
  • Collagenase and insulin were obtained from (Boehringer Mannheim GmbH, Germany) .
  • Ion exchange resin AG1-X8 (HO " , 20-50 mesh) was obtained from Bio-Rad Laboratories (Hemel Hempsted, UK) .
  • Cellulose microcrystalline was from (Merck, Germany) .
  • MonoStandardTM was obtained from Dionex Corporation, Sunnyvale, CA, USA. All other materials were of high purity and were obtained from BDH.
  • mice and obese diabetic were bred in our animal colony from parental strains obtained from the National Institute for Medical Research, Mill Hill, London, UK.
  • Wistar rats 120-150 g
  • obese diabetic mice were obtained from Harlan Olac Ltd, Bicester, UK.
  • Male mice 8-12 weeks old were used, when both their blood glucose and insulin levels were markedly raised.
  • Mice were allowed to acclimatize for at least 7 days before being used. All animals had free access to water and were fed ad libitum with normal laboratory chow.
  • mice were made diabetic by 3 daily i.p. injections of lOOmg/kg bw STZ dissolved in 0.01N citrate buffer (pH
  • mice received injections of equivalent volumes of sodium citrate buffer. Blood glucose rose to 15-20 mmol/1 and remained stable for 3 weeks. The syndrome induced in mice or rats by STZ injection closely resembles that observed in patients with Type 1 diabetes.
  • Plasmodium yoelii was maintained by blood passage of 10 4 parasitised red cells in F ⁇ mice. Mice were bled 5-7 days after infection. Parasitized blood (>90% parasitaemia) was washed x 3 with sterile saline by centrifugation at 3000 rpm at 4°C, and the pellet was lysed by incubation in 0.01 % saponin for 3 minutes at room temperature. Parasites were washed x 3 with sterile saline by centrifugation at 3000 rpm at 4°C. The pellet was suspended in 5 ml saline, sonicated for 12 seconds and freeze dried.
  • GPIs were extracted following a procedure described before with some modifications (Gerold et al, 1994) . Briefly, parasites (9 x 10 10 ) from 10 mice (0.1 gram dry weight) were extracted twice with 10 ml of Chloroform: Methanol: Water (10:10:3) (CMW) and centrifuged for 15 minutes at 1800 rpm. The CMW extracted GPIs were pooled, dried in Speed-Vac and suspended in 5 ml of water-saturated n-butanol . An equal volume of water was added, thoroughly mixed, and centrifuged for 10 minutes at 1800 rpm.
  • IPGs inositol phosphoglycans
  • Plasmodium yoelii IPGs were extracted from parasitized erythrocytes as described before (Caro et al, 1996) with some modifications. Briefly, mice infected with the lethal YM line of P. yoelii were bled 5 -7 days after infection into tubes containing heparinised saline. Parasitized erythrocytes were pelleted and washed twice with saline by centrifugation at 3000 rpm at 4°C for 20 min.
  • Parasitized erythrocytes (>90% parasitaemia) containing 20-30 xlO 9 parasites were homogenised and boiled for 5 minutes in 25 ml of a solution of 50 mM formic acid, 1 mM EDTA and ImM 2-mercaptoethanol .
  • the extract was centrifuged at 18,000 x g for 90 minutes at 4°C.
  • the supernatant was stirred for 10 minutes on ice with charcoal (10 mg/ml) and centrifuged at 18,000 x g for 30 minutes at 4°C.
  • the supernatant was then diluted with 10 volumes of distilled water, adjusted to pH 6 with 10% NH 4 OH, and shaken overnight at 4°C with AG1X8 ion exchange resin (formate form) .
  • the resin was poured into a column and washed with 2 bed volumes of water and 2 bed volumes of 1 mM HCl (pH 3) .
  • the resin was sequentially eluted with 5 bed volumes of 10 mM HCl (pH 2) to yield IPG-P, followed by 5 bed volumes of 50 mM HCl (pH 1.3) to yield IPG-A respectively.
  • the two fractions were concentrated and freeze dried twice to remove residual HCl and stored at -20°C. IPGs were injected i.v. in 0.2 ml saline. Control preparations derived from the same number of normal red cell ghosts were made as described above .
  • Plasmodium yoelii IPGs eluted from the anion exchange resin were subjected to cellulose chromatography. IPGs were dissolved in 1 ml of a solvent containing n-butanol, ethanol, and water [4:1:1] (B:E:W) and applied slowly onto a cellulose column (1 ml) pre-equilibrated in B:E:W [4:1:1]. The column was sequentially eluted with 5 ml of B:E:W, 5 ml methanol, 5 ml water, and 5 ml 50 mM HCl (pH 1.3). Different fractions were concentrated and freeze dried.
  • Inositol-free MEM tissue culture medium was used. The medium was supplemented with 0.001% (w/v) Para-amino benzoic acid; L-methionine, 14.92 mg/ml; L-glutamine, 292 mg/ml; L-cystine, 24.02 mg/ml; L-arginine 126.4 mg/ml; L-leucine, 52.46 mg/ml, 5% foetal calf serum and 5.5 mM glucose.
  • Parasites were obtained from 4 infected 12 wk old male (C57/B16 x Balb/c) Fi mice. Parasitaemias were 90-95% comprising mainly trophozoites and schizonts in roughly equal number.
  • PRBCs Parasitized RBCs
  • the pellet was resuspended in 10 ml culture medium (1 xlO 9 parasites/ml) and incubated with 250 ⁇ Ci of myo- [ 3 H] inositol in 25 ml Falcon TC flask for 3 hours at 37°C with 5% C0 2 . Giemsa-stained blood smears showed that the majority of the late trophozoites had transformed into schizonts. Parasites were viable during the course of labelling. Labelled PRBCs were washed and lysed with saponin and GPIs were extracted from labelled PRBCs as described above.
  • GPIs were dissolved in CMW (10:10:3) and applied to silica gel HPTLC plates. These were developed twice in a solvent containing Chloroform: Acetone: Methanol: glacial acetic acid: Water (50:20:10:10:5). GPIs were detected using orcinol and ninhydrin reagents. Biologically active glycolipids that remained at the origin, were scraped and eluted with 2 x 20 ml of methanol, filtered and dried using a rotary evaporator. Control preparations derived from the same number of normal red cell ghosts were made as described earlier. Radioalabelled GPIs were monitored by fluorography after HPTLC sheets were sprayed with En3H enhancer (New England Nuclear) and exposed to BioMax Mj Film (Kodak) at -80°C for 10 days.
  • En3H enhancer New England Nuclear
  • BioMax Mj Film BioMax Mj Film
  • Inorganic phosphate in malaria GPIs and IPGs was determined following the standard procedure with some modifications (Bartlett, 1958) .
  • Disodium hydrogen phosphate was used as standard (0-2 ⁇ M) . Briefly, 10 ⁇ l of samples and standards were dried and hydrolysed with 90 ⁇ l perchloric acid (70%) at 180°C for 45 minutes.
  • the hexosamine content of Plasmodium yoelii of malaria GPIs and IPGs were measured following a procedure described before (Bosworth et al, 1994) . Briefly, 100 ⁇ l samples were deacetylated with 100 ⁇ l 5.5 N HCl at 105°C and adjusted to pH 3 with 100 ⁇ l 6M potassium acetate. 100 ⁇ l of 10 % acetic acid and 50 ⁇ l NaN0 2 were added to the mixture on ice. After 30 minutes 100 ⁇ l of ammonium sulfamate was added. Fluorescence was developed after coupling with 100 ⁇ l of 0.8 % (w/v) 3, 5-diaminobenzoic acid. To the mixture, 20 ⁇ l of 12 N HCl was added and diluted five fold in water. Fluorescence was measured at 514 nm, the excitation wavelength being 422 nm.
  • Plasmodium yoelii GPIs were hydrolysed at 100°C in Teflon-lined screw-capped tubes usinglOO ⁇ l of 4N HCl for 4 hours or 6N HCl for 24 hours. Hydrolysates were centrifuged at 2000 rpm for 20 minutes at 4°C and the supernatants dried in a rotary evaporator. 100 ⁇ l of methanol was added and dried twice and the samples were dissolved in 200-400 ⁇ l water and filtered through a 0.2 mm PTFE filter.
  • Plasmodium yoelii IPGs were hydrolysed at 100°C in Teflon-lined screw-capped tubes using 100 ⁇ l of 4N HCl for 4 hours or 6N HCl for 24 hours. Hydrolysates were centrifuged at 2000 rpm for 20 minutes at 4°C and the supernatants were dried in a Speed Vac. To remove residual acid, 100 ⁇ l of methanol was added and dried, this treatment was repeated twice. Samples were dissolved in 200-300 ⁇ l water and passed through a 0.2 ⁇ m PTFE filtered (Whatman, NJ, USA) and stored at -20°C.
  • Dionex 500 HPLC system carbohydrate analyser from Dionex Corporation, Sunnyvale, CA, USA. It consists of an eluant degassing module, GP40 gradient pump module, AS 50 Autosampler, ED40 amperometric detector with a working gold electrode (Ag/Ag Cl reference electrode) and PeakNet chromatography workstation software was used. Monosaccharides and monosaccharide-alditols of Plasmodium yoelii IPGs hydrolysates were analysed by HPLC using CarbopacTM PA10 and CarbopacTM MAI columns respectively. An AminoTrap guard column was used to eliminate any possible interference of amino acids and peptides.
  • a CarbopakTM PA10 column (4 x 250 mm) with a guard column (4 x 50 mm ) was used for the separation of sugars as recommended by the supplier (Weitzhandler et al, 1996) .
  • columns were regenerated by elution with 200 mM sodium hydroxide for 15 minutes and were equilibrated for 15 minutes with 18 mM sodium hydroxide at a flow rate of 1 ml/minute.
  • the monosaccharides were resolved by isocratic elution with 18 mM sodium hydroxide for 20 minutes.
  • PAD pulsed amperometric detection
  • mice Male Wistar rats (120-150 g) were killed by cervical dislocation and adipocytes were obtained from the epididymal adipose tissue and prepared by digestion with collagenase according to the method of Rodbell (1964) with some modifications. Fat pads from two rats were dissected and placed in Krebs Ringer Hepes (KRH) buffer containing 9.2 mM Hepes, 2.2 mM NaH 2 P0 4 .2H 2 0, 10 mM
  • KRH Krebs Ringer Hepes
  • Lipogenesis was determined as the incorporation of [3- 3 H] glucose into toluene-extractable lipids. Briefly, into a 96-multiwell plate, 100 ⁇ l of adipocyte suspension (3.5 x lOVml) was incubated for 30 minutes at 37°C in a C0 2 incubator with 2 ⁇ l of various concentrations of GPIs or IPGs. Lipogenesis was initiated by the addition of 100 ⁇ l KRH containing 0.2 ⁇ Ci D-[3- 3 H] glucose, and the incubation continued for 2 hours. Adipocytes were harvested onto glass-fibre filter mats using a cell harvester, and rinsed with 5 mM glucose in 0.154M NaCl . 3 ml of a toluene-based scintillation cocktail were added to each filter disc for counting the radioactivity incorporated into lipids.
  • HPTLC-purified GPI was sonicated in 100 ⁇ l of water and tested for its effect on bovine heart PDH-phosphatase. GPIs were sonicated in water before assay. Different fractions of IPGs eluted from cellulose columns were tested for their effects on bovine heart PDH-phosphatase. 2-16 ⁇ l of IPGs (containing 1-2 nmol phosphate/ml) were used to stimulate PDH phosphatase. Activated PDH was determined spectrophotometrically following the procedure described earlier (Caro et al, 1997) by measuring the rate of production of NADH at 340nm (Jasco V560 spectrophotometer, Jasco corporation, Tokyo, Japan) .
  • GPIs were sonicated in dilution buffer. The ability of
  • GPIs and IPGs to inhibit PKA activity was determined using a colourimetric assay kit and a standard PKA preparation (Pierce, Rockford, IL, USA) .
  • Kemptide labelled with a fluorescent probe was used as PKA substrate.
  • Phosphorylated Kemptide was detected by measuring absorbance at 570nm in OPTI maxTM microplate reader (Molecular Device Corporation, Sunnyvale, CA, USA) .
  • Plasma Blood was collected from the trunk after decapitation into heparinised tubes. Plasma was separated by centrifugation and frozen -20°C. Plasma was assayed for total cholesterol and total triglycerides using kits from Sigma diagnostic. HDL cholesterol was assayed after precipitation of LDL cholesterol using phosphotungistic acid supplied by Sigma Diagnostics'. Results, in mmol/1, are expressed as means + SEM.
  • Plasmodium yoelii GPI was found to be labelled with H 3 -_nyo-inositol.
  • PKA cAMP-dependent protein kinase activity
  • Plasmodium yoelii IPGs to inhibit PKA activity was determined after fractionation of IPGs on cellulose chromatography columns ( Figure 3) . Different concentrations (0.001-7 ⁇ M) of acid and water eluates were tested. Both IPG-P and IPG-A induced a dose- related inhibition of cAMP-dependent protein kinase activity, with IPG-P being more inhibitory. Acid eluates of IPG-A and IPG-P inhibited PKA by more than 95%. 50% inhibitory dose concentrations (IC 50 ) for the acid eluates of IPG-A and IPG-P were 0.9 and 0.09 ⁇ M respectively.
  • IPG-P and IPG-A eluted from the anion exchange resin were subjected to cellulose chromatography column (1 ml) and eluted with different solvents (refer to the method section) and 1 ⁇ M of IPGs (equivalent of phosphate) was used for the lipogenesis assay.
  • IPG-P from mammalian sources normally has no significant lipogenic activities.
  • Plasmodium IPG-P increased lipogenesis in adipocytes by 20-30% from basal (Table 2, with methanol fraction stimulation was statistically significant, p ⁇ 0.01).
  • IPG-P In the presence of a maximal dose of insulin (10 ⁇ 8 M) , IPG-P increased lipogenesis by 20-30% (Table 1, with acid fraction stimulation was statistically significant, p ⁇ 0.01). However, different fractions of IPG-A had no significant lipogenic activity (Table 2) .
  • Plasmodium yoelii IPGs to stimulate PDH was determined after fractionation of IPGs on cellulose chromatography columns. Only IPG-P (acid fraction) stimulated PDH-phosphatase. Different concentrations of IPG-P (0.006, 0.0125 and 0.02 ⁇ M) stimulated PDH-phosphatase by 60, 100 and 130% above the base line value and at 0.05 ⁇ M by 101% ( Figure 4). 50% effective dose concentration of IPG-P (ED 50 ) was 0.005 ⁇ M. IPG-A fractions had no effect on PDH phosphatase (data not shown) .
  • Plasmodium yoelii IPGs from 15 infected mice contained 0.568 ⁇ mole and 0.265 ⁇ mole inorganic phosphate in IPG-A and IPG-P respectively.
  • Hexosamine concentrations in IPG-A and IPG-P were 0.117 ⁇ mole and 0.106 ⁇ mole respectively.
  • Keppler & Decker in Methods of Enzymatic Analysis (Bergmeyer, H.-U., ed) , pp.1127- 1131, Academic Press,

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Abstract

L'invention concerne des messagers phosphoglycane (PGM) qui sont des phosphatidyl cyclitols dérivés de glucides, ainsi que la découverte selon laquelle ces matières sont biologiquement actives, notamment dans la modulation du cholestérol plasmatique, de triglycérides plasmatiques et/ou de taux de lipoprotéines de haute densité, et/ou dans la modulation du rapport LDL/HDL, et pour traiter la lipodystrophie ou la dyslipidémie. Ces composés se distinguent d'ancres GPI divulguées précédemment en ce que lesdites ancres GPI se lient aux protéines et ne sont pas biologiquement actives.
PCT/GB2001/002775 2000-06-26 2001-06-21 Messagers phosphoglycane et leurs applications medicales WO2002000674A1 (fr)

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Publication number Priority date Publication date Assignee Title
CN112114063A (zh) * 2020-08-06 2020-12-22 浙江省农业科学院 大豆中d-松醇的检测方法

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WO1998011116A1 (fr) * 1996-09-11 1998-03-19 Rademacher Group Limited Hydrates de carbone contenant cyclitol, qui proviennent de tissus humains et regulent l'activite lipogene
WO1999006421A1 (fr) * 1997-07-29 1999-02-11 The University Of Virginia Patent Foundation Substances insulinomimetiques de synthese
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WO1998011116A1 (fr) * 1996-09-11 1998-03-19 Rademacher Group Limited Hydrates de carbone contenant cyclitol, qui proviennent de tissus humains et regulent l'activite lipogene
WO1999006421A1 (fr) * 1997-07-29 1999-02-11 The University Of Virginia Patent Foundation Substances insulinomimetiques de synthese
WO2000024406A1 (fr) * 1998-10-27 2000-05-04 The Walter And Eliza Hall Institute Of Medical Research Procede d'activation des lymphocytes t et agents utiles a cet effet

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GIGG R ET AL: "SYNTHESIS OF GLYCOSYLPHOSPHATIDYLINOSITOL ANCHORS", GLYCOPEPTIDES AND RELATED COMPOUNDS, DEKKER, NEW YORK,, US, 1997, pages 327 - 392, XP000897779 *
MARTIN-LOMAS M ET AL: "The solution conformation of glycosyl inositols related to inositolphosphoglycan (IPG) mediators", TETRAHEDRON: ASYMMETRY, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 11, no. 1, January 2000 (2000-01-01), pages 37 - 51, XP004202370, ISSN: 0957-4166 *
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112114063A (zh) * 2020-08-06 2020-12-22 浙江省农业科学院 大豆中d-松醇的检测方法

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