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WO1990003791A1 - Procedes d'interruption de la multiplication du vih et composition pour le traitement du sida - Google Patents

Procedes d'interruption de la multiplication du vih et composition pour le traitement du sida Download PDF

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Publication number
WO1990003791A1
WO1990003791A1 PCT/US1989/004249 US8904249W WO9003791A1 WO 1990003791 A1 WO1990003791 A1 WO 1990003791A1 US 8904249 W US8904249 W US 8904249W WO 9003791 A1 WO9003791 A1 WO 9003791A1
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Prior art keywords
heparin
hiv
molecular weight
cells
tion
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PCT/US1989/004249
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English (en)
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Nicola Diferrante
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Publication of WO1990003791A1 publication Critical patent/WO1990003791A1/fr
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Classifications

    • 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
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • 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

Definitions

  • This invention relates to antiviral agents which inter ⁇ fere with viral intracellular multiplication.
  • a mutated virus may not be recognized by antibodies elicited by a slightly different antigen derived from a previous mutation.
  • T-4 viral envelope glycoproteins which engage with the cell receptors prior to internalization of the whole virus
  • CD4 antigen soluble fragments of T-4 cell viral receptors
  • interference should be a permanent and complete one if it were to prevent healthy cells from becom ⁇ ing infected either from endogenous virus released by dying cells or exogenous virus acquired through the usual means of transmission.
  • An inexpensive, easily available, low molecu ⁇ lar weight, non-protein compound, capable of diffusing throughout the organism and capable of binding the virus or the viral receptors would be more suitable to this purpose than bulky peptides or proteins of finite half-life, costly preparation and potential immunogenic properties.
  • the present invention provides low molecular wight degra ⁇ dation products of naturally occurring linear polyanion heparin (glycosaminoglycan, acid mucopolysaccharide) lacking antithrombic and anticoagulant activities (i.e., less than 50 units antithro bin activity) , yet retaining sufficient nega ⁇ tive charges, as effective agents which interfere with the replication of the human immunodeficiency virus (HIV) .
  • the methods of invention provide for inhibiting T cell death and replication of human immunodeficiency virus (HIV) comprising exposing the human T cells to a pharmaceutically effective amount of a low molecular weight heparin degradation products lacking anticoagulant activity.
  • the present invention fur ⁇ ther provides a method of treating HIV infection by adminis ⁇ tering to the infected host a therapeutically effective amount of a low molecular weight heparin degradation product lacking anticoagulant activity.
  • the administration can be accomplished through any of many routes including intrave ⁇ nously, intraperitoneally, orally, subcutaneously, intramus ⁇ cularly, rectally, topically or by inhalation.
  • heparin is prepared from beef lung or, prefer ⁇ entially from bovine or porcine intestinal mucosa by extrac ⁇ tion procedures based on the high negative charge of mole ⁇ cule. After digestion of the crude material with proteases, the clear supernatant obtained by centrifugation is treated with quaternary ammonium salts. This precipitation may be achieved step-wise, at decreasing salt concentrations, the most highly charged molecules being obtained as a precipitate at the high salt concentration.
  • This higher charged heparin may be precipitated with quaternary ammonium salts above 0.35 M NaCl, while the other contaminating glycosaminglycans (chondroitin sulfates, heparan sulfate) remain in solution, to be precipitated upon further dilution of the solution.
  • the heparin-quaternary ammonium salt precipitate is repeatedly washed in the centrifuge with 95% ethanol/10% potassium acetate in order to remove the quaternary ammonium salt.
  • the potassium salt of heparin is dissolved in water, cleared by centrifugation and further purified by column chromatography on anion or cation exchange resins.
  • hepa ⁇ rin fractions of different molecular weight Several methods are available for the isolation of hepa ⁇ rin fractions of different molecular weight: a) anion ex ⁇ change chromatography in which the heparin is retained on the column at low ionic strength and fractions of increasing molecular weight may be eluted by step-wise increase in the ionic strengths of the eluting solution (from 0.2 M to 3.0 M NaCl) ; or b) gel filtration, in which larger fractions emerge first from the column, to be followed by gradually smaller ones. In this technique, the influence of sulfate content may be minimized by using as eluant a solution of 0.2 M NaCl.
  • Heparin and heparan sulfate are glycosminoglycans which uniquely contain N-sulfate groups. Older methods of de-N- sulfation with controlled hydrolysis in dilute acids (0.04N HC1 at 56° for 4 hours) caused hydrolysis of N-sulfated groups accompanied by some cleavage of glycosidic linkages (depolymerization) and of 0-sulfate groups (de-O-sulfation) .
  • the pyridinium salt of N-sulfated glucosamine residues (present in heparin and heparan sulfate) is desulfated much more rapidly and selec ⁇ tively in dimethyl sulfoxide containing small amounts of water or methanol.
  • the sodium salt or heparin (molecular weight 12,00 to 15,000) is dissolved in 25 ml of water and the solution is passed through a column of Dowex 50x8 at 4° C. The effluent and washing are combined, neutralized with pyridine and lyophilized to give the pyridinium salt of heparin.
  • This salt is dissolved in 100 ml of dimethyl sulfoxide containing 5% water and the solution is kept for 90 minutes at 50 * C, then diluted with equal volume of water.
  • the pH of the solution is adjusted to 9.0-9.5 with 0.1 M NaOH, dialyzed over night against tap water and then against distilled water for 20 hours. Filtration and lyophilization of the retentate gives the sodium salt of completely de-N-sulfated heparin.
  • This large molecular weight compound (12,000 to 15,000 daltons) has no anticoagulant activity.
  • This compound may be acetylated by the method of Danishefsky, et al. Arch. Biochem. , Biophys. 90:114 (1960).
  • Antithro bin (commercially available) bound to an insoluble matrix (Sepharose) may be used to separate large MW heparin into two fractions: one with high and one with low affinity for antithrombin (HA and LA respectively) .
  • the first has approximately 300 U anticoagulant activity/mg; the second is essentially inactive. This technique has allowed to identify the structure of the pentasaccharide of heparin responsible for binding to antithrombin.
  • the internal glucosamine unit carries a 0-sulfate group in position C-3, previously consid ⁇ ered essential for interaction with antithrombin and absent in LA heparin. More recently, however, the consensus is that the 3-O-sulfate is an artifact produced during deamination of the 2-N-sulfate group, which migrates to position 3. In fact, the 3-O-sulfate group is never found in products of bacterial degradation of heparin. However, three additional sulfate groups, are required for high affinity to antithrom ⁇ bin. One of those, 2-N sulfate on the internal glucosamine, may be removed by solvolytic cleavage with methyl sulfoxide, leaving a highly sulfated, nonanticoagulant product.
  • Both these fractions may be depolymerized with nitrous acid, to obtain fragments of various length which may be separated by gel chromatography. It is well documented that anticoagulant activity of heparin is highly dependent on molecular weight. Thus, oligosaccharides of less than 18 monosaccharides (less than 5,000 MW) length have lost their anticoagulant activity but retained their anti ⁇ thrombic activity (anti-Xa activity) .
  • N-sulfated glucosamine is transformed into 2,5- anhydromannose.
  • This residue located at the reducing end of the oligosaccharide products, may be conveniently reduced with sodium borohydride labeled with tritium (NaB 3 H 4 ) , to yield tritium-labeled mannose at the reducing end.
  • This labeling technique allows one to follow the fate of these oligosaccharides 1 , once added to a cell culture.
  • heparinase II ob ⁇ tained originally from Flavobacterium heparinum and now commercially available.
  • 200 mg of heparin in 0.005 M phos ⁇ phate buffer pH 7.0, containing 0.02% bovine serum albumin is incubated at 25° C for up to 125 hours with heparinase II (10 units in 10 ml buffer) .
  • heparinase II 10 units in 10 ml buffer
  • a series of disaccharides is obtained, which may be separated by high pressure liquid chromatography. Shorter periods of incubation, with the enzyme, may yield trisaccharides or oligosaccharides which may be separated by gel chromatography. All these products
  • Sample B used in these experiments is a degradation product of heparin (MW 4.000-5,000 daltons) prepared by mild oxidation of heparin with copper, H 2 02 and ascorbate.
  • the desired sample was then desalted, by gel filtration and precipitated with methanol and dried with ether as de ⁇ scribed in 3.
  • the virus a source of reverse transcriptase
  • concentrations 1.0; 0.3; 0.1.
  • Concentra ⁇ tion 1 is arbitrary and it is defined, for the purpose of this assay, as that amount of virus which provides sufficient enzyme to cause incorporation of tritiated thymidine triphos- phate (3 H TTP) into the template to the extent of 40,000 cpm.
  • the two heparin preparations A and B have been used at sever ⁇ al, decreasing concentrations: 1.0; 0.3; 0.1; 0.03 mg/ml.
  • HIV virus suspension HAV pp213, stock of UTMB Galveston
  • the concentration of the virus (1:10 dilution from the stock solution) was adopted so as not to kill the cells immediately but nevertheless to provide total infection of the cells within 4-5 days.
  • cells were exposed to the heparin preparations A or B by adding the heparin sample to the cells 1 hour before or 1 hour after challenging with virus.
  • CPE cytopathic effects
  • Control cells + Heparin B * for each of # 1.0; 0.3; 01.; and 0.03mg/ml
  • Heparin B protects the cells from being killed by the virus even though they had been infected for 48 hours. Control cells, infected but not treated show morphological changes and fluorescence by day 2 and 3, with cell death by day 5.
  • Control cells plus Heparin B do not show changes of toxicity. However, cells infected at time 0 and treated 48 hours later do not show cytopathic effects, even though they display the presence of viral core protein (P-24) .
  • Heparin B administered 48 hours after viral infection does not prevent the synthesis of the core P-24 antigen of the virus. The latter apparently must have had time to shed the glycoprotein coat, to free its RNA from the core protein, to have the RNA transcripted into DNA and this incorporated into host DNA.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'héparine et des dérivés d'héparine de faible masse moléculaire ne présentant pas d'activité anticoagulante sont efficaces pour interrompre le cycle de vie du VIH. Des cellules exposées à l'héparine ou à ses dérivés de faible masse moléculaire, une heure avant ou après la confrontation avec le VIH, n'ont montré aucune trace d'infection virale lorsqu'on les a examinées de quatre à six jours après la confrontation avec le VIH. Des cellules témoin confrontées de manière similaire avec le VIH ont présenté une infection par le VIH de 100 % six jours après la confrontation avec le VIH. Des cellules confrontées avec le VIH jusqu'à 48 heures avant l'exposition à l'héparine ou à ses dérivés de faible masse moléculaire, ont montré peu de traces de multiplication du VIH et ont manifesté des caractéristiques morphologiques saines.
PCT/US1989/004249 1988-10-05 1989-09-27 Procedes d'interruption de la multiplication du vih et composition pour le traitement du sida Ceased WO1990003791A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US25368688A 1988-10-05 1988-10-05
US253,686 1988-10-05

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WO1990003791A1 true WO1990003791A1 (fr) 1990-04-19

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992022588A1 (fr) * 1991-06-14 1992-12-23 Italfarmaco S.P.A. Heparines acylees utilisees comme inhibiteurs de la replication de retrovirus
EP0583360A4 (fr) * 1991-05-02 1994-04-06 Yeda Research And Development Co. Ltd.
US5861382A (en) * 1992-05-01 1999-01-19 Yeda Research And Development Co. Ltd. Methods for regulation of active TNF-α
US6399078B1 (en) 1998-06-01 2002-06-04 University Of Maryland Biotechnology Institute Chemokine—glycosaminoglycan complexes and their use in treating or preventing receptor mediated diseases
US6750207B1 (en) 1992-05-01 2004-06-15 Yeda Research And Development Co. Ltd. Compositions for the regulation of cytokine activity
WO2003078960A3 (fr) * 2002-03-11 2005-01-27 Momenta Pharmaceuticals Inc Analyse de polysaccharides sulfates
US7285536B2 (en) 2001-12-05 2007-10-23 Yeda Research And Development Co., Ltd. Anti-cancer therapeutic compounds
US7790466B1 (en) 2007-01-26 2010-09-07 Momenta Pharmaceuticals, Inc. Evaluating mixtures of low molecular weight heparins by chain profiles or chain mapping
CN101942039A (zh) * 2010-09-16 2011-01-12 山东海科化工集团有限公司 一种帕肝素生产方法
US7968082B1 (en) 2007-01-26 2011-06-28 Momenta Pharmaceuticals, Inc. Evaluating mixtures of low molecular weight heparins by NMR
US8435795B2 (en) 2010-01-19 2013-05-07 Momenta Pharmaceuticals, Inc. Evaluating heparin preparations
US9068957B2 (en) 2011-02-21 2015-06-30 Momenta Pharmaceuticals, Inc. Evaluating heparin preparations
US9139876B1 (en) 2007-05-03 2015-09-22 Momenta Pharmacueticals, Inc. Method of analyzing a preparation of a low molecular weight heparin

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2053953T3 (es) * 1988-08-24 1994-08-01 Akzo Nv Uso de fracciones de heparina o fragmentos de heparina.

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1986006729A1 (fr) * 1985-05-17 1986-11-20 Opocrin S.P.A. Laboratorio Farmacobiologico Sulfates d'hexosaminoglucane depolymerises dotes d'une activite antithrombotique fibrinolytique et anti-inflammatoire, leur procede de preparation et compositions pharmaceutiques associees

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1986006729A1 (fr) * 1985-05-17 1986-11-20 Opocrin S.P.A. Laboratorio Farmacobiologico Sulfates d'hexosaminoglucane depolymerises dotes d'une activite antithrombotique fibrinolytique et anti-inflammatoire, leur procede de preparation et compositions pharmaceutiques associees

Cited By (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0583360A4 (fr) * 1991-05-02 1994-04-06 Yeda Research And Development Co. Ltd.
US5474987A (en) * 1991-05-02 1995-12-12 Yeda Research And Development Co. Ltd. Methods of using low molecular weight heparins treatment of pathological processes
US5686431A (en) * 1991-05-02 1997-11-11 Yeda Research And Development Co., Ltd. Methods of using low molecular weight heparins for treatment of pathological processes
US5908837A (en) * 1991-05-02 1999-06-01 Yeda Research And Development Co. Ltd. Methods of using low molecular weight heparins for prevention or treatment of pathological processes
WO1992022588A1 (fr) * 1991-06-14 1992-12-23 Italfarmaco S.P.A. Heparines acylees utilisees comme inhibiteurs de la replication de retrovirus
US7332480B2 (en) 1992-05-01 2008-02-19 Yeda Research And Development Co. Ltd. Compositions for the regulation of cytokine activity
US5861382A (en) * 1992-05-01 1999-01-19 Yeda Research And Development Co. Ltd. Methods for regulation of active TNF-α
US6020323A (en) * 1992-05-01 2000-02-01 Yeda Research And Development Co. Ltd. Compositions and methods for regulation of active TNF-α
US6750207B1 (en) 1992-05-01 2004-06-15 Yeda Research And Development Co. Ltd. Compositions for the regulation of cytokine activity
US6399078B1 (en) 1998-06-01 2002-06-04 University Of Maryland Biotechnology Institute Chemokine—glycosaminoglycan complexes and their use in treating or preventing receptor mediated diseases
US7285536B2 (en) 2001-12-05 2007-10-23 Yeda Research And Development Co., Ltd. Anti-cancer therapeutic compounds
US7575886B2 (en) 2002-03-11 2009-08-18 Momenta Pharmaceuticals, Inc. Analysis of sulfated polysaccharides
WO2003078960A3 (fr) * 2002-03-11 2005-01-27 Momenta Pharmaceuticals Inc Analyse de polysaccharides sulfates
US7947507B2 (en) 2002-03-11 2011-05-24 Momenta Pharmaceuticals, Inc. Analysis of sulfated polysaccharides
US8715953B2 (en) 2002-03-11 2014-05-06 Momenta Pharmaceuticals, Inc. Analysis of sulfated polysaccharides
US7790466B1 (en) 2007-01-26 2010-09-07 Momenta Pharmaceuticals, Inc. Evaluating mixtures of low molecular weight heparins by chain profiles or chain mapping
US7968082B1 (en) 2007-01-26 2011-06-28 Momenta Pharmaceuticals, Inc. Evaluating mixtures of low molecular weight heparins by NMR
US8076149B1 (en) 2007-01-26 2011-12-13 Momenta Pharmaceuticals, Inc. Evaluating mixtures of low molecular weight heparins by chain profiles or chain mapping
US8252597B1 (en) 2007-01-26 2012-08-28 Momenta Pharmaceuticals, Inc. Evaluating mixtures of low molecular weight heparins by chain profiles or chain mapping
US8617896B1 (en) 2007-01-26 2013-12-31 Zachary Shriver Evaluating mixtures of low molecular weight heparins by chain profiles or chain mapping
US9139876B1 (en) 2007-05-03 2015-09-22 Momenta Pharmacueticals, Inc. Method of analyzing a preparation of a low molecular weight heparin
US8435795B2 (en) 2010-01-19 2013-05-07 Momenta Pharmaceuticals, Inc. Evaluating heparin preparations
CN101942039A (zh) * 2010-09-16 2011-01-12 山东海科化工集团有限公司 一种帕肝素生产方法
US9068957B2 (en) 2011-02-21 2015-06-30 Momenta Pharmaceuticals, Inc. Evaluating heparin preparations

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Publication number Publication date
AU4426289A (en) 1990-05-01

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