WO1992005791A1 - Inhibition de replication virale - Google Patents

Inhibition de replication virale Download PDF

Info

Publication number
WO1992005791A1
WO1992005791A1 PCT/US1991/007232 US9107232W WO9205791A1 WO 1992005791 A1 WO1992005791 A1 WO 1992005791A1 US 9107232 W US9107232 W US 9107232W WO 9205791 A1 WO9205791 A1 WO 9205791A1
Authority
WO
WIPO (PCT)
Prior art keywords
polysaccharide
ncmcs
subunits
therapeutic
virus
Prior art date
Application number
PCT/US1991/007232
Other languages
English (en)
Inventor
Sharon V. Vercellotti
Ruth M. Ruprecht
Miguel A. Gama Sosa
Original Assignee
Dana-Farber Cancer Institute, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Dana-Farber Cancer Institute, Inc. filed Critical Dana-Farber Cancer Institute, Inc.
Publication of WO1992005791A1 publication Critical patent/WO1992005791A1/fr

Links

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
    • A61K31/737—Sulfated polysaccharides, e.g. chondroitin sulfate, dermatan sulfate
    • 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
    • A61K31/716—Glucans
    • A61K31/722—Chitin, chitosan
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

Definitions

  • the invention relates to methods of inhibition of viral replication, especially by therapeutic agents including polysaccharides.
  • AIDS Acquired Immune Deficiency Syndrome
  • HAV-1 Human Immunodeficiency Virus Type 1
  • ABT nucleoside analog 3'-azido-3'-deoxythymidine
  • ddC dideoxcytidine
  • ddl dideoxyinosine
  • Sulfated polysaccharides have been shown to act as potent inhibitors of HIV-1 replication.
  • Sulfated polysaccharides with anti-HIV-l activity include: pentosan polysulfate, dextran sulfate, xylofuranan sulfate, ribofuranan sulfate, the bacterial sulfated glycosaminoglycan termed Org 31581 as well as chemically degraded heparin termed Org 31733, fucoidan sulfate, prunellin, lentinan sulfate, glycyrrhizin, heparin, carrageenans and mannan sulfate.
  • dextran sulfate is currently undergoing clinical trials.
  • the invention features a method for prevention of infection of a mammal, including humans by, or treatment of a mammal infected with, a virus, said method comprising the steps of providing an active agent in the form of a therapeutic polysaccharide comprising an ampholytic (l ⁇ 4)-2-amino-2-deoxy-0-D-glucan of polydispersed molecular weight comprising, at physiological pH, subunits of the structure
  • R ⁇ CH 2 OS0 3 " , CH 2 OH, CH 2 OCH 2 COO " , CH 2 OCH 2 CHOHCH 2 OH,
  • R 2 S0 3 " , H, CH 2 COO " , CH 2 CHOHCH 2 OH, or CEL j O j OH;
  • R 3 S0 3 ⁇ or H;
  • R 4 ⁇ E, S0 3 " , COCH 3 , CH 2 COO " , CHCH 3 CH 2 CH 2 COO " , CH 2 CHOHCH 2 OH,
  • R 4 for between 10-85% of the subunits R 4 is C0CH 3 , for between 5-85% of the subunits R 4 is CH 2 COO ⁇ , and for between 0-85% of the subunits R 4 is
  • R 4 is H
  • R 3 is S0 3 ⁇ .
  • the virus is preferably a retrovirus, especially HIV-1.
  • the method of administration is preferably oral, parenteral, or topical.
  • polydispersed molecular weight is meant that the ratio of the weight average molecular weight and the number average molecular weight for a sample preparation is greater than 1 and preferably greater than 2.
  • ampholytic is meant that the polysaccharide is an amphoteric electrolyte and can, at one time, behave as both an acid and a base.
  • substituent amino groups having in general a pK a of above 8.5, will usually be positively charged
  • substituent carboxyl, or sulfate groups having in general a pK a of below 5.5, will usually be negatively charged.
  • therapeutic polysaccharide is meant a polysaccharide useful for the treatment of a disease or disorder.
  • a therapeutically effective amount is that quantity which produces a significant physiological effect in the patient and is recognized by those of ordinary skill in the art to depend upon the size and weight of the mammal as well as other well known factors.
  • the polysaccharide as described above can be prepared from chitosan, itself a deacetylated derivative of chitin. The latter is an unbranched (l ⁇ 4)-2- acetamido-2-deoxy-3-D-glucan that occurs in the exoskeleton of crustaceans and insects and in the cell wall of some bacteria and fungi.
  • Ampholytic sulfated or carboxylated derivatives of chitosan are active inhibitors of HIV-1 replication with no significant toxic effects on cultured human or murine cells. They also have a wide spectrum of anti-retroviral activity, including inhibition of replication of oncogenic retroviruses such as Rauscher Murine Leukemia Virus (RLV) .
  • RLV Rauscher Murine Leukemia Virus
  • the compounds are highly soluble in aqueous solutions and are easily prepared according to conventional methods.
  • Other features and advantages of the invention will be apparent from the following description of the preferred embodiment thereof and from the claims. Brief Description of the drawing The Figure shows a typical preparation of N- carboxymethylchitosan-N,0-sulfate (NCMCS) , representative of the class.
  • N-carboxymethyl- chitosan-N,0-sulfate N-carboxymethyl- chitosan-N,0-sulfate
  • amphoteric electrolyte or ampholyte an active inhibitor of the replication of
  • NCMCS can be used alone or in combination therapy with other anti-retroviral agents in the treatment of patients with AIDS.
  • the zwitterionic effect occurring between the positive and negative centers of the molecule can enhance binding to the target virus or to virus-infected cells expressing viral antigens.
  • the amphoteric nature of NCMCS is maintained even with a variation of the charge distribution on the polymer with different substitutients and varying degrees of substitution. Structure of a typical preparation of NCMCS.
  • NCMCS is a polydispersed molecular weight polysaccharide composed of (l ⁇ 4)-2-amino-2-deoxy-)0-D- glucan subunits.
  • 66% of the subunits 12 contained an amino group 18 at the 2-position of the pyranose ring
  • 16% of the units 14 contained an acetamido group 20 at the 2- position
  • 18% of the units 16 contained an N- substituted carboxymethyl group 22 at the 2-position.
  • An undetermined fraction of units 12 and 16 also contained an N-substituted sulfate group 24 at the 2-position, and an undetermined fraction of all three units 12, 14, and 16, in addition, was substituted at the 3-position 26 or at the 6-position 28 with an O-substituted sulfate group.
  • the individual subunits were arranged in random order in the polysaccharide.
  • N-Carboxymethylchitosan (NCMC) was prepared by a Schiff reaction of chitosan with glyoxylic acid (Muzzarelli et al.. Carbohydrate Res. 107:199-204, 1982; Muzzarelli, Carbohydrate Polymers 8.:1-21, 1988).
  • Chitosan 80 gm was added to 8.1 liters of 1.25% acetic acid with overhead stirring, and the chitosan gradually dissolved. After thirty minutes of stirring, the pH was 4 and the solution was clear.
  • N-(carboxymethylidene) chitosan was effected by gradual addition of sodium borohydride (46 gm, 1.4 mole, in 250 ml water) with stirring over a 1 hour period.
  • MW nominal molecular weight
  • NCMC gel Both the gel and the soluble form were separately precipitated by gradually pouring the NCMC into dry ethanol with stirring. The white precipitate formed was collected by centrifugation and dried to a white powder. Yield: 80 gm: NCMC gel - 85%, NCMC soluble - 15%.
  • the starting material for sulfation was an N- carboxymethylchitosan, as a dry powder from NCMC gel (the precipitate from NCMC soluble could have been used as well) , with degree of substitution 16% N-acetyl; 18% N- carboxymethyl; and 66% free NH 2 as determined by 13 C nuclear magnetic resonance.
  • NCMC gel (5 gm) was suspended in 50 ml of dry pyridine (Horton et al., Carbohydrate Res.
  • Chlorosulfonic acid (26 ml) was added carefully with a dropping funnel to dry pyridine (105 ml) in a closed flask fitted with a stirring motor, condenser, and drying tube. Ten ml of the complex were removed and stored in a dry flask for the second addition.
  • the NCMC suspension was added to the pyridine-chlorosulfonic acid complex, and the mixture was heated in a water bath at 90-93°C for 4 hours. Stirring was then continued at room temperature. After 18 hours the remaining 10 ml of pyridine- chlorosulfonic acid mixture were added to the NCMC mixture. The mixture was heated for 8 hours at 90°C in a water bath and then stirred for 14 hours.
  • PWXL series 2500, 3000, 4000, (Supelco, Bellefonte, PA) with 10% acetonitrile in 0.2M sodium chloride as solvent.
  • the calibration equation having a correlation coefficient of 0.98, was developed from ten runs of the molecular weight standards.
  • the molecular weight of the N- carboxymethylchitosan-N,O-sulfate (NCMCS) preparation was determined using the above system. Eighty-one and five- tenths per cent (81.5%) of the sample was in the inclusion volume.
  • the weight average molecular weight (M w ) is 32,800 and the number average molecular weight ( jj ) is 7,400.
  • the ratio M ⁇ /M j . is equal to 4.4, indicating a high degree of polydispersity.
  • NCMCS gives a positive metachromatic reaction with toluidine blue, indicative of an O-sulfate ester or an N- sulfoamino group of a polysaccharide in a regular substitution pattern.
  • NCMCS With cetyl pyridinium chloride, NCMCS forms a precipitate that dissolves in 1M sodium chloride, indicative of an O-sulfate ester or an N- sulfoamino group on a polysaccharide. NCMCS is precipitable in 50% ethanol, and it is burned in the presence of sulfuric acid to an ash that is typical of sodium sulfate, indicating that a sodium salt of a sulfate ester was present in the original preparation. NCMCS does not give a positive ninhydrin reaction, indicating essentially complete N-substitution with N- acetyl, N-carboxymethyl, and/or N-sulfoamino. Preparation of N-carboxymethylchitosan-N.O-sulfate for antiretroviral studies.
  • N-Carboxymethylchitosan-N,O-sulfate prepared as described above, was obtained from V-LABS, INC. (Covington, LA) .
  • the material was dissolved in RPMI medium 1640 (Gibco, Gaithersburg, MD) at concentrations between 0.1-1.0 g/ml and sterilized through a 45 ⁇ m Millipore filter. Stock solutions were stored at -20°C until use. Dilutions of NCMCS were made in complete RPMI medium 1640 or Dulbecco modified Eagle's medium supplemented with 10% fetal calf serum, penicillin/streptomycin and L-glutamine. For enzyme kinetics experiments NCMCS stocks were prepared in sterile RNase-free water. Inhibition of HIV-1 replication bv NCMCS.
  • HIV-1 2000 reverse transcriptase cpm units/well
  • production of HIV-1 was determined by reverse transcriptase assays using poly(A) n * oligo(dT) as template primer and
  • NCMCS 3 H-TTP as substrate as previously described (Roy-Burman et al., J. Virol. 19:1107-1110, 1976). Simultaneously, the cytotoxicity of NCMCS was determined by trypan blue exclusion.
  • NCMCS inhibited HIV-1 with an inhibitory concentration 50 IC 5 o) of 7 g/ml* No significant cytotoxicity was noted over a wide concentration range, including a concentration of 0.17 mg/ml. Thus, the therapeutic index of NCMCS remains undetermined because of a lack of cytotoxicity.
  • NCMCS was very active in this assay; RLV-induced plaques were inhibited at an IC 50 of 0.4 ⁇ g/ml. Again, at the concentrations that were tested, no cytotoxicity was detected in the SC-1 fibroblasts. This lack of toxicity prevented the estimation of a therapeutic window. Inhibition of HIV Reverse Transcriptase by NCMCS.
  • NCMCS The kind of inhibition of HIV-l reverse transcriptase by NCMCS was determined by standard enzyme kinetics. Reactions were performed in 50 ⁇ l of 50 mM Tris-HCl, pH 8.0, 1 mM MgCl 2 , 80 mM KC1, 1 mM dithiothreitol, 1 ⁇ M TTP, 250 ⁇ Ci/ml 3 H-TTP (New England Nuclear, specific activity 86.3 Ci/mmol) , poly(A) n * oligo(dT) , 1 unit of human placental RNase inhibitor (RNasin, Promega Biotech) and NCMCS at different concentrations.
  • NCMCS When the concentration of poly(A) n * oligo(dT) and the concentration of the inhibitor were varied in the standard enzyme assay, NCMCS was observed to competitively inhibit the reverse transcriptase of HIV-l. While not being bound to any theory, it appears that in addition to inhibiting retroviral reverse transcriptase, NCMCS may inhibit retroviral adsorption to the cellular receptor due to its ampholytic nature. Presence of HIV-l Proteins in NCMCS-treated Cells To confirm the data obtained in Jurkat cells by measuring reverse transcriptase activity in tissue culture supernatants, the presence of HIV-l proteins in NCMCS-treated cells was examined by immunoprecipitation. Jurkat cells pretreated with NCMCS were incubated with fully infectious HIV-l as described above.
  • NCMCS is an active inhibitor of HIV-l replication with no significant toxic effects on cultured human cells
  • NCMCS can be used therapeutically in the prevention of or treatment of Acquired Immune Deficiency Syndrome, or AIDS.
  • NCMCS may be administered orally, parenterally, or topically by routine methods in pharmaceutically acceptable inert carrier substances. Optimal dosages and modes of administration can readily be determined by conventional protocols.
  • the compounds may be ad instered prophylactically or after infection to treat infection by any virus, especially any retrovirus, including oncogenic retroviruses such as RLV.
  • the compounds may be administered as a vaccine adjuvant to enhance the production of antibodies.
  • the compounds may also be coadministered with other therapeutic agents to aid in the delivery of the therapeutic agent and to enhance the effectiveness of the agent.
  • the compounds may be used to coat or microencapsulate the vaccine or therapeutic agent or may be coadministered orally, parenterally, or topically.
  • NCMCS may also be used in combination therapy with antiviral agents with different mechanisms of inhibition and non-overlapping toxicity. Other embodiments are within the following claims.
  • any sulfated and/or carboxylated derivative of chitosan having the water solubility and amphoteric properties of NCMCS is suitable as a therapeutic polysaccharide in the method of the invention.
  • some subunits may contain an O-substituted carboxymethyl group at position 6 or position 3 of the pyranose ring and no sulfate substituents, or the carbon atom itself at position 6 may be substituted by a carboxyl group.
  • Other charged substituents at position 2 may include N-substituted carboxyalkyl and N-ortho-carboxybenzyl residues, and aromatic rings having at least one carboxyl group.
  • Non- charged substituents including hydroxyalky1, aldose or ketose residues attached via the aldehyde or keto carbon atom, and aromatic rings having at least one hydroxyl group, may be N-substituted at position 2, and hydroxyalkyl residues may be O-substituted at position 6, to enhance the solubility of the polysaccharide.

Landscapes

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

Abstract

Procédé destiné à prévenir ou à traiter l'infection d'un mammifère, en particulier un humain, par un virus, en particulier le VIH-1, consistant à administrer une quantité thérapeutiquement efficace d'un dérivé de chitosan sulfaté et/ou carboxylé au patient.
PCT/US1991/007232 1990-10-01 1991-10-01 Inhibition de replication virale WO1992005791A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US59150990A 1990-10-01 1990-10-01
US591,509 1990-10-01

Publications (1)

Publication Number Publication Date
WO1992005791A1 true WO1992005791A1 (fr) 1992-04-16

Family

ID=24366764

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1991/007232 WO1992005791A1 (fr) 1990-10-01 1991-10-01 Inhibition de replication virale

Country Status (1)

Country Link
WO (1) WO1992005791A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1853213A1 (fr) * 2005-02-17 2007-11-14 Chitogenics, Inc. Methode et composition pour traiter un trouble de tissu mucosal

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4840941A (en) * 1986-04-04 1989-06-20 Kabushiki Kaisha Ueno Seiyaku Oyo Kenkyujo Method for inhibiting infection of human T-cells

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4840941A (en) * 1986-04-04 1989-06-20 Kabushiki Kaisha Ueno Seiyaku Oyo Kenkyujo Method for inhibiting infection of human T-cells

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1853213A1 (fr) * 2005-02-17 2007-11-14 Chitogenics, Inc. Methode et composition pour traiter un trouble de tissu mucosal
JP2008530224A (ja) * 2005-02-17 2008-08-07 キトジェニックス インコーポレイテッド 粘膜組織の異常の治療のための方法及び組成物
EP1853213A4 (fr) * 2005-02-17 2009-08-12 Chitogenics Inc Methode et composition pour traiter un trouble de tissu mucosal

Similar Documents

Publication Publication Date Title
Sosa et al. N-Carboxymethylchitosan-N, O-sulfate as an anti-HIV-1 agent
Baba et al. Pentosan polysulfate, a sulfated oligosaccharide, is a potent and selective anti-HIV agent in vitro
CA1277239C (fr) Traitement des maladies causees par un retrovirus
Yoshida et al. Synthesis of curdlan sulfates having inhibitory effects in vitro against AIDS viruses HIV-1 and HIV-2
Jayakumar et al. Sulfated chitin and chitosan as novel biomaterials
EP0270317A2 (fr) Médicament pour le traitement des maladies virales
EP1274446B1 (fr) Esters polysaccharidiques de derives n- de l'acide glutamique
CN102329397A (zh) 一种岩藻糖化糖胺聚糖衍生物及其制备方法
US20110306567A1 (en) Polysaccharides with antithrombotic activity, including a covalent bond and an amino chain
CN102558389A (zh) 低分子量岩藻糖化糖胺聚糖的羧基还原衍生物及其制备方法与用途
US5053398A (en) Sulfated homopolysaccharides as anti-aids virus agents
CN101039963B (zh) 生物素化十六糖，它们的制备方法及其用途
Barzu et al. Preparation and anti-HIV activity of O-acylated heparin and dermatan sulfate derivatives with low anticoagulant effect
WO1992005791A1 (fr) Inhibition de replication virale
US7268122B2 (en) Use of oversulfated polysaccharides as inhibitors of HIV
JP2010518251A (ja) ビオチンまたはビオチン誘導体との少なくとも１つの共有結合を含むヘパリン、これらの調製方法およびこれらの使用
EP0066379A2 (fr) Composition pour le traitement du virus herpès
EP3060223A1 (fr) Polysaccharides sulfatés destinés à traiter un cancer
WO2018129647A1 (fr) Composé oligosaccharidiquee pour inhiber un complexe enzymatique du facteur x de coagulation endogène, son procédé de préparation et ses utilisations
Yoshida et al. Anti-HIV activity of sulfonated arabinofuranan and xylofuranan
EP0588855B1 (fr) Heparines acylees utilisees comme inhibiteurs de la replication de retrovirus
Ito et al. Potent and selective activity of dextrin sulphate against human immunodeficiency virus type 1 in vitro
JPH04308531A (ja) 抗エイズウイルス活性を有する硫酸化βグルカンのレトロウイルス感染症治療用薬剤
US20110212907A1 (en) Hexadecasaccharides with antithrombotic activity, including a covalent bond and an amino chain
JPH03218317A (ja) 抗レトロウイルス剤

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IT LU NL SE

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA