EP2007400A1 - Neue medikamente für behandlungen gegen retroviren - Google Patents

Neue medikamente für behandlungen gegen retroviren

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Publication number
EP2007400A1
EP2007400A1 EP07731297A EP07731297A EP2007400A1 EP 2007400 A1 EP2007400 A1 EP 2007400A1 EP 07731297 A EP07731297 A EP 07731297A EP 07731297 A EP07731297 A EP 07731297A EP 2007400 A1 EP2007400 A1 EP 2007400A1
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EP
European Patent Office
Prior art keywords
group
agents
degree
polysaccharide
glucose
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EP07731297A
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English (en)
French (fr)
Inventor
Jean-Claude Yvin
Jean-Claude Chermann
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ASE AND BIO
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Laboratoire de la Mer SAS
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Publication of EP2007400A1 publication Critical patent/EP2007400A1/de
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    • 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
    • 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
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the subject of the invention is new drugs for retrovirus treatments, by acting on their replication cycle by inhibition of RT.
  • Retroviruses belong to a family of viruses whose genome is made of RNA.
  • the peculiarity of retroviruses is to replicate in a host cell via DNA steps, which is made possible by the Reverse Transcriptase, or Reverse Transcriptase, designated by RT in what follows, and which is an enzyme allowing the transcription of I 1 viral RNA into complementary DNA molecule called provirus.
  • the provirus is able to circularize and integrate into the genome of the host cell.
  • the retroviruses thus integrated into the genome of the host cell can either use the cellular machinery to multiply, or remain latent in the host cell.
  • their genes are transmitted to the descending cells at each mitosis and are temporarily silent; the organism carrying the retrovirus does not present any pathological sign.
  • the retrovirus family has three subfamilies: oncoviruses, lentiviruses and spumaviruses.
  • Oncoviruses are responsible for cancers and, in particular, certain leukemias.
  • Cancer-causing oncoviruses include Roux sarcoma virus or VSR.
  • HTLV Roux sarcoma virus
  • HTLV Human T cell Leukemia Virus
  • felines leukemia is caused by the FTLV virus.
  • Lentiviruses are responsible for slow-growing viral infections such as acquired immunodeficiency syndrome or AIDS.
  • lentiviruses responsible for AIDS are part of HIV or "Human Immunodeficiency Virus” (type I, HIV-I or type II, HIV-II), or French HIV (Human Immunodeficiency Virus) ; in monkeys, these are SIV ("Simian Immunodeficiency Virus") and in FIV cat (Feline Immunodeficiency Virus).
  • Spumaviruses are poorly known, both in terms of their structure and the exact mode of their integration into the cellular genome. In the current state of knowledge, no disease seems to be associated with spumaviruses. However, their responsibility in triggering certain autoimmune diseases seems likely.
  • oncoviruses transform the T cells they infect and cause uncontrolled proliferation of these cells; lentiviruses destroy the cells they infect.
  • helper T4 cells that express on their surface the CD4 molecule (as a membrane glycoprotein molecule), which is a receptor that allows HIV to penetrate inside these cells. .
  • gp120 a surface glycoprotein of the retrovirus
  • the retrovirus here an HIV
  • the host cell here the T4 lymphocyte
  • the RT which intervenes in the manner indicated below.
  • the nucleus or core of the virus once it has penetrated inside the host cell, releases two copies of single-stranded RNA.
  • These two copies of single-stranded RNA are associated with RT as well as other proteins such as protease and integrase. This synthesizes a strand of DNA complementary to the viral RNA.
  • RT-associated RNase activity degrades the RNA strand, while a second strand of DNA is synthesized.
  • the double-stranded DNA thus obtained is then circularized and integrated, thanks to an integrase enzyme, into the cellular genome to give a provirus.
  • This provirus contains 3 structural genes, namely the gag (antigen group), pol (polymerase) and env (envelope) genes; gag genes code for p24 viral proteins, pol genes for RT and its associated activities which are polymerase, RNase, integrase and protease activities, and env genes for envelope glycoproteins such as gp120.
  • the provirus is transcribed into messenger RNA using the machinery of the nucleus of the host cell, which allows the production of the constitutive proteins of the retrovirus on the one hand and the replication of the viral genomic material on the other hand. go.
  • the whole constitutes a new virus produced by budding of the membrane of the host cell.
  • fusion phenomena occur between the infected cells, which have on their surface, in particular, the gp120 protein, and the uninfected T4 lymphocytes, which comprise on their surface the CD4 molecule.
  • an infected cell can bind healthy uninfected T4 cells and form what is called a syncytium, i.e. infected and uninfected lymphocytes, which is unable to survive.
  • a single infected cell can cause the death of many healthy T4 cells. This results in a gradual decrease in cellular immunity which results in the development of opportunistic infections, which may be accompanied by certain types of tumors.
  • T4 cells other cells also have the CD4 molecule on their surface, and are therefore sensitive to HIV viruses; these include macrophage monocytes, certain cells found in the ganglia, skin and other organs, and some B cells.
  • HIV viruses can also attack certain CNS central nervous system cells: in this case, they cause neurological syndromes.
  • chemokines Other molecules located on the surface of the cell that normally function as receptors for chemokines have also been identified as co-receptors that allow the entry of HIV viruses into the host cell.
  • the retroviruses responsible for AIDS that is to say lentiviruses and especially HIV are characterized by extreme genetic and antigenic variability.
  • HIV-I HIV-I
  • HIV-II HIV-II
  • HIV-1 strains belong to the group M (Major), which comprises at least ten subtypes or clades (A, B, C, D, ...); we meet these clades in various geographical areas.
  • M Major
  • clades A, B, C, D, ...
  • the HIV-1 strains of the O and N groups have so far been isolated only in African populations.
  • the high genetic variability of these HIV strains is mainly due to the high error rate of RT. Indeed, during successive replication cycles, genetic variants appear. Mutations occur in particular on the env gene and more precisely on the coding part for gp120.
  • RT is still essential for their replication. And when this viral enzyme is inhibited, the oncovirus can no longer replicate in the host cell.
  • RTs reverse transcriptase inhibitors
  • RT reverse transcriptase
  • nucleoside RT inhibitors include nucleoside RT inhibitors, non-nucleoside RT inhibitors and nucleotide analogues.
  • nucleoside inhibitors of RT mention may be made of AZT
  • nucleoside inhibitors compete with natural nucleosides and prevent elongation of the DNA chain; they were the first to be used as RT inhibitors, first alone and then in combination with other RT inhibitors or viral enzymes such as protease and integrase.
  • RT inhibitors first alone and then in combination with other RT inhibitors or viral enzymes such as protease and integrase.
  • side effects are known and numerous.
  • mutations of the reverse transcriptase confer resistance to INRTs that can be crossed between several INRTs.
  • Non-nucleoside RT inhibitors act as noncompetitive antagonists by binding to a hydrophobic region adjacent to the catalytic site of RT, thereby inhibiting RT; among them, ritonavir, saquinavir, efavirenz, rescriptor, sustiva and viramune may be mentioned.
  • the second therapeutic group is constituted by protease inhibitors (PIs).
  • PIs protease inhibitors
  • They are powerful anti-retroviral agents that inhibit the proteolytic activity of the viral protease; for example, amprenavir, tipranavir, indinavir, saquinavir, lopinavir, posanprenavir, ritonavir, atazanavir and nelfinavir.
  • the third therapeutic group corresponds to the fusion and entry inhibitors, of which several molecules are under study. Only enfuvirtide is currently on the market. It acts at the first stage of virus replication by preventing fusion between virus / cell by competitive inhibition.
  • AIDS is mainly due to viral resistance phenomena, although other factors such as the toxicity of the agents used as well as some side effects also affect the effectiveness of treatments, although to a lesser extent. It is to cope with this situation that much research has been and is being conducted to discover novel antiretroviral agents capable of inhibiting RT, particularly among polysaccharides.
  • EP 0240 098 recommends the use as antiretroviral agents of synthetic or natural sulfated polysaccharides via connector groups.
  • EP 0240 098 particularly described sulphates of chondroitin, dermatan, keratan, hyaluronic acid, carrageenan, fucoidan, heparin and dextran.
  • the patent application EP 0 464 759 also describes sulfated polysaccharides via a specific group intended for the long-term prophylaxis of diseases caused by viruses. These two patent applications, EP 0 240 098 and EP 0 464 759, have the major disadvantage of providing complex oligosaccharides to be synthesized due to the presence of an intermediate group necessary for sulfation.
  • Japanese Patent Application Publication 01-103601 describes the antiviral activity, especially with regard to HIV, lentinan sulfate, certain ⁇ -1, 3 glucans such as curdlan, pachymane, and those of the cell walls of yeast and those of cellulose.
  • the published Japanese patent application 03-145 425 describes the antiviral activity, with respect to HIV, of certain sulphated laminarioligosaccharides and in particular of sulphated laminaripentaose.
  • the problem to be addressed by the present invention is therefore the availability to the medical profession of new antiretroviral drugs with a high therapeutic index, particularly active against lentiviruses and oncoviruses, especially against HIV, as well as against strains resistant to certain antiretroviral agents already known, and having a low anticoagulant activity in vivo.
  • the subject of the present invention is therefore the use, for the manufacture of a medicament for the treatment of retroviral diseases, of a polysaccharide of formula (I)
  • R 1 represents either a hydrogen atom, a sulfate group or a phosphate group, or a sulfated or phosphate glucose preferably linked by a ⁇ -type bond (1 ⁇ 6) to the saccharide structure,
  • R 2 represents a hydrogen atom, a sulfate group or a phosphate group
  • R 1 and R 2 can not simultaneously represent a hydrogen atom
  • X and Y represent, each independently, an OH group, a glucose, a sulfated or phosphated glucose, a mannitol, or a sulfated or phosphated mannitol,
  • n represents an integer from 11 to 30, preferably from 20 to 30, more preferably from 25 to 30, said polysaccharide having a degree of sulfation greater than 2, preferably from 2.2 to 2.4, or a degree of phosphatation greater than 1, preferably from 1.5 to 2.5.
  • the polysaccharide used is a polysaccharide of formula (I) in which R 1 and R 2 may be identical and then represent a sulfate or phosphate group, or different from each other, Ri representing then a sulfated or phosphated glucose unit preferably linked by a ⁇ -1 ⁇ -bond to the saccharide structure, X and / or Y representing a mannitol group and n an integer of 11 to 30, more particularly of 25 to 30.
  • R 1 and R 2 may be identical and then represent a sulfate or phosphate group, or different from each other
  • Ri representing then a sulfated or phosphated glucose unit preferably linked by a ⁇ -1 ⁇ -bond to the saccharide structure
  • X and / or Y representing a mannitol group
  • n an integer of 11 to 30, more particularly of 25 to 30.
  • the drug manufactured using a polysaccharide of formula (I) acts on the retrovirus replication cycle by inhibiting the RT of the latter.
  • sulfation degree means the average number, per saccharide unit, of sulphated OH groups.
  • a degree of sulfation greater than 2 means that, on average, over the entire polysaccharide, more than 2 OH groups per saccharide unit are sulfated.
  • degree of phosphatation means the average number, per saccharide unit, of phosphate-containing OH groups. A degree of phosphatation greater than 1 means that, on average, over the entire polysaccharide, more than 1 OH group per saccharide unit is phosphated.
  • sulfate group means a group of the type (-SO 3 H).
  • phosphate group means a group of the type (-PO3H2).
  • Another subject of the invention is the use of one of the polysaccharides defined above for the implementation of a method for treating retroviral diseases.
  • the retroviral diseases are preferably chosen from those caused by lentiviruses and oncoviruses, more particularly by HIV, and by the strains of these retroviruses resistant to anti-retroviral RT inhibitors agents already known.
  • the drug obtained by use according to the invention of a polysaccharide of formula (I) makes it possible to treat the acquired immunodeficiency syndrome in humans.
  • a retroviral disease within the meaning of the invention is the acquired immunodeficiency syndrome or AIDS in humans.
  • polysaccharides of formula (I) as used according to the invention are also particularly active against oncoviruses, in particular against HTLV type I and II viruses.
  • the use according to the invention of a polysaccharide of formula (I) makes it possible to treat the cancers associated with these retroviruses.
  • the polysaccharide of formula (I) is a sulfated laminarine having a degree of polymerization of 11 to 28.
  • the polysaccharide of formula (I) is a laminarine of degree of sulfation equal to about 2.3, called "laminarin PS3".
  • the term "degree of polymerization” is intended to mean the number of monosaccharide units bonded to each other by ⁇ (1-> 3) -type bonds constituting the main linear chain.
  • a degree of polymerization of 11 to 28 means a polysaccharide composed of 11 to 28 saccharide units, in particular glucose, bound together by ⁇ (1-> 3) type bonds. This degree of polymerization does not take into account glucose units bound in ⁇ (1 ⁇ 6) to the main chain of the polysaccharide.
  • the degree of polymerization is equal to n + 2 when X and Y simultaneously represent OH, to n + 3 if only one of X or Y represents OH, and at n + 4 neither X nor Y represents OH.
  • sulphated laminarine having a degree of sulphation greater than 2, preferably of 2.2 to 2.4, and a degree of polymerization of 11 to 28, was particularly effective for the treatment of retroviral diseases, preferably selected from those caused by lentivirus and oncovirus, more particularly by HIV, and by strains of these retroviruses resistant to anti-retroviral agents RT inhibitors already known, particularly I 1 AZT .
  • This sulphated laminarine also had a low anticoagulant activity, thus confirming its great interest in the manufacture of a medicament intended for human or animal administration.
  • the invention relates to the use of a polysaccharide, obtained from sulphated laminarine with a degree of sulphation greater than 2 and preferably from 2.2 to 2.4, of degree of polymerization of 11 to 28, for the preparation of a medicament for the treatment of retroviral diseases, preferably chosen from those caused by lentiviruses and oncoviruses, more particularly by HIV, and by the strains of these retroviruses resistant to antiretroviral agents already known RT inhibitors.
  • the polysaccharide of formula (I) is a phosphatized laminarine having a degree of polymerization of 11 to 28.
  • the laminarin phosphate according to the invention has a degree of phosphatation greater than 1 and preferably from 1.5 to 2.5 and is particularly suitable for the treatment of retroviral diseases, preferably chosen from those caused by lentiviruses and oncoviruses, more particularly by HIV, and by strains of these retroviruses resistant to anti-retroviral RT inhibitors already known.
  • the invention relates to the use of a polysaccharide obtained from laminarine phosphate with a degree of phosphatation greater than 1 and preferably from 1.5 to 2.5, with a degree of polymerization of 11. at 28, for the preparation of a medicament for the treatment of retroviral diseases, preferably chosen from those caused by lentiviruses and oncoviruses, more particularly by HIV, and by the strains of these retroviruses resistant to inhibitory antiretroviral agents of the RT already known.
  • a particular embodiment of the invention relates to the use of a sulfated laminarine, characterized in that it has a degree of sulfation greater than 2, preferably from 2.2 to 2.4, and a degree of polymerization. from 11 to 28 for the manufacture of a medicament for the treatment of retroviral diseases.
  • Another particular embodiment of the invention relates to the use of a sulfated laminarine, characterized in that it has a degree of sulfation greater than 2, preferably from 2.2 to 2.4, and a degree of polymerization from 11 to 28, for the implementation of a method for treating retroviral diseases.
  • Another particular embodiment of the invention relates to the use of a phosphatized laminarine, characterized in that it has a degree of phosphatation greater than 1, preferably from 1.5 to 2.5, and a degree of polymerization from 11 to 28, for the manufacture of a medicament for the treatment of retroviral diseases.
  • Yet another particular embodiment of the invention relates to the use of a phosphatized laminarin, characterized in that it has a degree of phosphatation greater than 1, preferably from 1.5 to 2.5, and a degree of polymerization from 11 to 28, for the implementation of a method for treating retroviral diseases.
  • a phosphatized laminarin characterized in that it has a degree of phosphatation greater than 1, preferably from 1.5 to 2.5, and a degree of polymerization from 11 to 28, for the implementation of a method for treating retroviral diseases.
  • multi-therapies not only two or even three antiretroviral agents, but also other pharmacological agents intended to fight against the associated pathogenesis.
  • the use of these numerous drugs represents a considerable bond for patients.
  • the subject of the invention is also a combination product comprising an effective quantity
  • R 1 represents either a hydrogen atom, a sulphate group or a phosphate group, or a sulphated or phosphatic glucose bonded, preferably, by a ⁇ -type bond (1- 6) to the saccharide structure
  • R 2 represents a hydrogen atom, a sulfate group or a phosphate group
  • X and Y represent, each independently, an OH group, a glucose, a sulfated or phosphated glucose, a mannitol, or a sulfated or phosphated mannitol
  • n represents an integer from 11 to 30, preferably from 20 to 30, more preferably from 25 to 30, said polysaccharide having a degree of sulfation greater than 2, preferably from 2.2 to 2.4, or a degree of phosphatation greater than 1, preferably from 1.5 to 2.5
  • R 1 represents either a hydrogen atom, a sulphate group or a phosphate group, or a sulphated or phosphat
  • At least one antiretroviral agent chosen from the group comprising:
  • nucleoside reverse transcriptase inhibitors including I 1 AZT, ddI, ddC, d4T, 3TC and ABC,
  • NRTIs non-nucleoside reverse transcriptase inhibitors
  • protease inhibitors including Agenerase and Kaletra
  • fusion inhibitors including enfuvirtide (Fuzeon)
  • Input inhibitors including, in particular, AMD-3100 and, optionally, at least one pharmacological agent chosen from the group comprising anti-nausea agents, anti-diarrheal agents, anti-hyperbilirubinemia agents, agents anti-pain agents for dermatological treatments, anti-nephrotoxic agents, for simultaneous, separate or spread over time.
  • pharmacological agent chosen from the group comprising anti-nausea agents, anti-diarrheal agents, anti-hyperbilirubinemia agents, agents anti-pain agents for dermatological treatments, anti-nephrotoxic agents, for simultaneous, separate or spread over time.
  • the skilled person is able to define the best adapted administration to obtain the best therapeutic index for the patient.
  • Each active substance in HAART can be administered sequentially, on different routes, or at the same time.
  • the term "effective amount" means an amount of active substance that is sufficient to obtain a therapeutic effect on a patient.
  • the subject of the invention is also the use of an effective amount of a polysaccharide of formula (I) as described above,
  • At least one antiretroviral agent chosen from the group comprising:
  • nucleoside reverse transcriptase inhibitors including I 1 AZT, ddI, ddC, d4T, 3TC and ABC,
  • NRTIs non-nucleoside reverse transcriptase inhibitors
  • protease inhibitors including Agenerase and Kaletra
  • fusion inhibitors including enfuvirtide (Fuzeon)
  • Input inhibitors including, in particular, AMD-3100 and, optionally, at least one pharmacological agent chosen from the group comprising anti-nausea agents, anti-diarrheal agents, anti-hyperbilirubinemia agents, agents anti-pain agents for dermatological treatments, anti-nephrotoxic agents, for the manufacture of a medicament for the treatment of retroviral diseases, preferably caused by lentiviruses and oncoviruses, more preferably by HIV, in particular by the strains of these retroviruses resistant to antiretroviral agents already known.
  • pharmacological agent chosen from the group comprising anti-nausea agents, anti-diarrheal agents, anti-hyperbilirubinemia agents, agents anti-pain agents for dermatological treatments, anti-nephrotoxic agents, for the manufacture of a medicament for the treatment of retroviral diseases, preferably caused by lentiviruses and oncoviruses, more preferably by HIV, in particular by the strains of these retroviruses resistant
  • At least one antiretroviral agent chosen from the group comprising:
  • nucleoside reverse transcriptase inhibitors including AZT, ddl, ddC, d4T,
  • NRTIs non-nucleoside reverse transcriptase inhibitors
  • protease inhibitors including Agenerase and Kaletra
  • fusion inhibitors including enfuvirtide (Fuzeon),
  • input inhibitors including the AMD-3100 and possibly at least one pharmacological agent chosen from the group comprising anti-nausea agents, anti-diarrheal agents, anti-hyperbilirubinemia agents, anti-pain agents, agents for dermatological treatments, anti-nephrotoxic agents, the implementation of a method for treating retroviral diseases, preferably caused by lentiviruses and oncoviruses, more preferably by HIV, in particular by the strains of these retroviruses resistant to antiretroviral agents already known.
  • pharmacological agent chosen from the group comprising anti-nausea agents, anti-diarrheal agents, anti-hyperbilirubinemia agents, anti-pain agents, agents for dermatological treatments, anti-nephrotoxic agents, the implementation of a method for treating retroviral diseases, preferably caused by lentiviruses and oncoviruses, more preferably by HIV, in particular by the strains of these retroviruses resistant to antiretroviral agents already known.
  • the present invention also relates to a method for treating a retroviral disease, preferably caused by lentiviruses and oncoviruses, more preferably by HIV, in particular by the strains of these retroviruses resistant to antiretroviral agents already known, comprising administering, in a patient affected by said retroviral disease, an effective amount of a medicament comprising as active agent at least one polysaccharide of formula (I) as previously described; or a combination product as described above.
  • the term "patient” means any warm-blooded animal, particularly mammals and especially humans.
  • the present invention also relates to a treatment method as defined above, in which the polysaccharide of formula (I) is a sulfated laminarine, characterized in that it has a degree of sulfation greater than 2, preferably 2, 2 to 2.4, and a degree of polymerization of 11 to 28.
  • the subject of the present invention is also a treatment method as defined above, in which the polysaccharide of formula (I) is a phosphatized laminarine, characterized in that it has a degree of phosphatation greater than 1, preferably of 1, 5 to 2.5, and a degree of polymerization of 11 to 28.
  • a sulfation step is carried out preferably following the protocol described by Alban S, Kraus J, and Franz G in "Synthesis of laminarin sulfates with anticoagulant activity" , Artzneim.Forsch./drug Res (1992) 42; 1005-1008.
  • the sulfation reaction is advantageously carried out under conditions corresponding to an absolute absence of water.
  • the polysaccharide is therefore preferably dried, for example on phosphorus pentoxide (P 2 O 5 ) and then dissolved in dimethylformamide or DMF.
  • P 2 O 5 phosphorus pentoxide
  • DMF dimethylformamide
  • the SO 3 -pyridine complex can advantageously be used.
  • the S ⁇ 3 -pyridine complex has, compared to other complexes, the advantage of being neither too reactive nor too stable, that is to say, too slowly from the reaction point of view. Due to the fact that the degree of sulfation obtained is proportional to the molar excess of sulfation reagent and since it is sought to obtain a degree of substitution greater than 2, it is advantageously implemented a concentration of 6 moles of SO 3 - pyridine per mole of glucose.
  • pyridine is added to the sulfation reagent in an equimolar amount, in order to directly capture the sulfuric acid that may be formed by reaction of the complex S ⁇ 3 -pyridine with water .
  • concentration of the polysaccharide as that of the sulfation reagent should preferably be as high as possible, the solubility of the polysaccharide and sulfation reagent limiting the degree of final sulfation.
  • the solution of the S ⁇ 3-pyridine complex in DMF could not be added all at once but in a manner continue for a period of 4 hours.
  • the sulfation reaction can be carried out at a temperature of 20 to 60 ° C., preferably of approximately 40 ° C. Higher temperatures result in a more efficient substitution but also a degradation of the chains.
  • the mixture is preferably stirred for several hours around 60 ° C. At this temperature, an additional substitution occurs without degradation of the chains.
  • the supernatant of the mixture is then advantageously separated by decantation.
  • the residue is dissolved, preferably in NaOH, and then mixed with 10 times its volume of ethanol.
  • the precipitate which occurs at a temperature of 4-8 ° C overnight is isolated and then preferentially dissolved in dilute sodium hydroxide (pH solution of about 9).
  • the solution is dialysed to remove salts and low weight molecules molecularly then advantageously brought to a pH of 7.0 by addition of NaOH and then lyophilized.
  • the resulting sulfated polysaccharide is in the form of the sodium salt.
  • the degree of sulphation is preferably determined by conductimetric titration of the free acid of the sulphated polysaccharide, or alternatively by ion chromatography after hydrolysis using an HPLC type system.
  • the first method has the advantage of being also suitable for stability research (the consumption of soda increases when sulphate groups are eliminated) whereas the HPLC method requires less substance and can be automated. As a control, it is possible to determine the sulfur content by elemental analysis.
  • the degree of sulfation obtained by proceeding as indicated above is greater than 2, more precisely from 2 to 2.5 and especially from 2.2 to 2.4.
  • the polysaccharide of formula (I), and preferably sulphated laminarine are used for the preparation of a medicament for retrovirus treatments intended for general administration and preferably for oral, rectal, pulmonary topical (including transdermal, oral and sublingual) and parenteral (including subcutaneous intramuscular, intravenous, intradermal and intravitreous).
  • the daily dose is generally from 0.01 to 250 mg per kilogram of weight of the patient and preferably from 0.10 to 100 mg, more preferentially still from 0.5 to 30 mg, and more particularly from 1.0 to 20 mg.
  • the daily dose can be administered per unit dose in one, two, three, four, five or six times or more at different times of the day.
  • the unit doses can comprise from 10 to 1000 mg, from 50 to 400 mg, and preferably from 50 to 100 mg of active substance.
  • the medicaments obtained according to the invention, using at least one of the polysaccharides of formula (I), comprise the conventional formulation ingredients and optionally one or more other therapeutic agents.
  • the polysaccharide of the invention can be advantageously combined with other active substances.
  • Their mode of administration can be simultaneous or sequential. They can also be administered by different routes as previously described.
  • the laminarine was first dried over phosphorus pentoxide (P2O5) and then dissolved in dimethylformamide or DMF.
  • the supernatant was then decanted from the mixture, the residue dissolved in 2.5 M NaOH, and then mixed with 10 times its volume of 99% ethanol.
  • the resulting solution was then placed at a temperature of 4-8 ° C overnight and a precipitate was obtained which was isolated and then dissolved in dilute sodium hydroxide (pH solution of about 9).
  • the solution was then dialyzed using a 1000 D cutoff Spectrapor membrane and then brought to pH 7.0 by the addition of NaOH. Finally, the solution thus dialysed was lyophilized. A laminarin sulphate in the form of a sodium salt was obtained.
  • the degree of sulfation by conductimetric titration of the free acid of the sulfated polysaccharide was then determined using 0.1 N sodium hydroxide.
  • the degree of sulfation of the laminarine obtained was 2.3.
  • the degree of polymerization of the laminarin sulphate thus obtained was 23 to 28.
  • Example 2A or by observation, depending on the amount of sulfated polysaccharides used and the timing of this implementation that can take place before, during or after infection or permanently, the decrease in the number of syncitia , appearing in a culture of MT 4 cells following infection with an HIV retrovirus, (Example 2A) - either by evaluation as a function of the amount of sulfated polysaccharides used and the moment of this implementation that can take place before, during or after infection, or permanently throughout the infection, inhibition of RT, which results in a measurable decrease in RT activity, and which reflects the slowing down of the replication of the infecting virus in a culture of CEM cells infected with an HIV retrovirus, (Example 2B) it being understood that for comparison purposes the same experiments were carried out using as comparison products on the one hand the dextran ulfate and on the other hand 3'-azidothymidine or AZT.
  • the above-mentioned MT 4 and CEM cells are human
  • the retroviruses that have been implemented are constituted by
  • RTMC virus (clade B) known to be resistant to the AZT antiviral agent
  • PIC CH an isolate derived from a patient resistant to several antiviral agents known as d4T and Zerit.
  • PTA 1 at the concentrations indicated below was also diluted with AZT (Sigma origin A2169), widely known to be an inhibitor of RT.
  • Example 2A Measuring the number of syncitia
  • the cells were then infected by adding to each well, except those corresponding to the cell controls, 50 .mu.l of viral dilution previously titrated. After a further hour of incubation, the plate was centrifuged and the supernatants containing the residual virus removed.
  • the sulfated laminarin PS3 and the comparison products were brought into contact with the cells for one hour, then the cells were washed twice to eliminate the products used and finally the infection was carried out. of MT 4 cell culture.
  • the sulfated laminarin PS3 was contacted with the comparison products at the time when the cells were infected.
  • the culture thus treated was kept as it was for one hour, followed by two washes, which resulted in the removal of the sulfated laminarin PS3 and comparison products as well as viruses that did not penetrated into the cells.
  • the sulfated laminar PS3 and the comparison products were first contacted with the cells for one hour at 37 ° C, then the culture was infected for one hour at 37 ° C. . Then two washes were made, which led to the elimination of viruses that did not penetrate the cells. The cell pellets were then transferred to 24-well plates at 3.10 5 cells / ml per well in the presence of the sulfated laminarin PS3 and comparison products at the selected concentrations.
  • Washes and rinses were performed with RPMI culture medium without fetal calf serum.
  • Ci uninfected cell culture
  • FIG. 2 shows an infected cell culture in which certain Ci cells agglutinate in syncytia identified by S, and
  • FIG. 3 shows, at a larger magnification, a culture of infected Ci cells, some of the Ci cells being agglutinated in syncytia identified by S.
  • AZT was used as a comparison product at a concentration of 0.01 ⁇ m
  • sulphated laminarin PS3 was used at two concentrations of 5 and 10 ⁇ g / ml, respectively.
  • the BRU (clade B) virus was used for the infection of MT 4 cells at a viral dilution of 10 5 which corresponds to the amount of virus particles that can infect 80% of the MT4 cultures (Tissue Culture Infections Dose 80% TCID 8 %).
  • the results observed from day D3 to day D4 in the four types of experiments previously defined are shown in Table A.
  • MT 4 cell cultures do not form syncitia when treated continuously, as soon as they are cultured (before, during and after infection), with concentrations of 10 ⁇ g / ml of PS3.
  • MT 4 cell cultures do not form syncitia when treated with concentrations of 10 and 5 ⁇ g / ml PS3 after infection with the BRU viral strain. The effect is observed from the 3rd day after the viral infection.
  • ⁇ ZT and dextran sulphate were used as comparison products respectively at concentrations of 0.4 ⁇ M and 10 ⁇ g / ml, the latter concentration also being that of PS3.
  • the NDK virus (clade D) used was used at the viral dilution of 2.5 ⁇ 10 -5, which corresponds to the amount of viral particles that can infect 80% of the MT4 cultures (Tissue Culture Infections Dose 80% TCID 80 % The results observed from D3 to D6 are collated in Table B. TABLE B
  • MT 4 cell cultures do not form syncitia when treated continuously, during and after infection, with a concentration of 10. ⁇ g / ml of PS3.
  • Dextran sulphate on the other hand, has an optimal effect when the cells are treated before viral infection (2 out of 2 wells inhibited).
  • Example 2B Measurement of inhibition of RT activity
  • the number of viruses present in the culture was demonstrated by measuring RT activity in the culture supernatant, the detected amount of RT activity being proportional to the number of viruses produced.
  • Example 2A The same experimental protocol as that used in Example 2A was used on MT 4 cells, except that the CEM cells were incubated with the sulphated laminarin PS3 or the comparison product and then cultured at the same time. concentration of 0.5 ⁇ 10 6 cells / ml. Every three days, the cells were counted and the cultures were diluted for culture at 0.5. 10 6 cells / ml; the RT activity was assayed according to a protocol comprising:
  • the culture samples were made in a protected P3 laboratory.
  • each well (1 ml) were centrifuged for 5 min at 1500 rpm (Jouan GR 422 centrifuge), and then ultracentrifuged the culture supernatant thus obtained at 40 ° C., 95000 rpm (Beckman TL100 ultracentrifuge) to obtain the viral pellet. .
  • the viral pellet was then taken up in a test tube containing 10 ⁇ l of NTE buffer supplemented with 0.1% triton, which releases the viral enzymes and in particular the RT; the tube was then vortexed, capped with parafilm and kept for 10 minutes at 4 ° C. and then frozen at -20 ° C.
  • reaction mixture previously discussed in the biochemistry laboratory was prepared and this mixture comprises for a 5 mL test tube.
  • composition of the aforesaid 5X base buffer is as follows
  • the tubes in question were held for one hour in a water bath at 37 ° C with stirring every 15 minutes. The reaction was then stopped by introducing into each tube 1 ml of 0.1 M NaP (sodium pyrophosphate) prepared in trichloroacetic acid or 5% TCA.
  • NaP sodium pyrophosphate
  • the synthesized DNA contained in the mixture was precipitated at 4 ° C. by addition of 3.5 ml / tube of 20% trichloroacetic acid and then filtered through 0.45 ⁇ Millipore nitrocellulose filters. To do this, the contents of the tubes were poured into the corresponding wells of a sample collector (Millipore), and the tubes and wells were rinsed three times with 5% TCA.
  • the filters were then dehydrated with 70% alcohol before being dried in the oven at 80 ° C. for 20 minutes. After cooling, the filters were individually placed in flasks containing a scintillation or scintillating agent sold under the tradename Emulsifier Safe cat. N 0 6013389 by Perkin Elmer Company. The count was then carried out using a liquid scintillation analyzer marketed under the trademark "PACKARD 2100T", the results of which are expressed in dpm / ml (disintegration per minute and per ml of supernatant).
  • the amount of radioactivity measured is proportional to the RT activity present and, consequently, to the number of viruses produced by replication.
  • the sulphated laminarine PS3 was tested at concentrations of 5 and 10 ⁇ g / ml and the comparison product of AZT at the concentration of 0.1 ⁇ M.
  • the RTMC virus (clade B) was used for the infection of the CEM cells, which has the particularity of being resistant to AZT and which has been used at a dilution of 5.10 -4 .
  • results recorded on days 3, 7 and 10 are collated in Table C. These results include, for each experiment, the RT activity expressed in dpm / ml and the number of cells expressed in 10 6 cells / ml.
  • the viral dilution being 5.10 -4 .
  • PS3 was tested at concentrations of 5 and 10 ⁇ g / ml and I 1 AZT at a concentration of 0.1 ⁇ M, the virus used being, this time, the NKD virus (clade D) at dilution. 2.5.10 "5 .
  • FIG. 5 which illustrates Table D
  • the RT activity expressed in dpm / ml measured at day 7 in the case of I 1 AZT and the two concentrations of PS3, as well as in the case of the cells was plotted on a graph. control not having been treated with an antiretroviral agent, for the four experiments corresponding to the administration of the antiretroviral agent continuously, before, during and after the infection with the NDK virus at a dilution of 2.5.10 "5 .
  • a viral concentration corresponding to a RT activity of 220,000 dpm was used; the results obtained appear on the graph of FIG. 7, the examination of which shows that PS3 at the concentration of 2.5 ⁇ g / ml causes an inhibition of RT of 35%, whereas dextran sulfate does not inhibit RT than 6% at the same concentration.
  • NDK virus For the NDK virus, a viral concentration corresponding to a RT activity of 81,000 dpm was used; the results obtained appear on the graph of FIG. 9, the examination of which shows that at the concentration of 0.6 ⁇ g / ml, the PS3 causes an inhibition of the RT of 43% compared to only 11% in the case of the sulphate of dextran; to the concentration of 0.8 ⁇ g / ml is obtained 57% inhibition respectively in the case of PS3 against 18% in the case of dextran sulfate.
  • PS3 is particularly effective on the RT of the CH CH virus (clade B) which is resistant to several antiviral agents, as well as on the NDK virus (clade D).
  • the anticoagulant activity of the laminar sulfate PS3 obtained in Example 1 was determined as a function of its concentration in comparison with that of heparin in conventional APTT coagulation tests or "Partial Activator Thromboplastin-Zeit", the duration of prothrombin, the test called “HEPTEST” and the duration of thrombin.
  • the APTT reflects an interaction with the intrinsic coagulation system while the prothrombin time reflects an interaction with extrinsic coagulation;
  • the so-called "HEPTEST” test is the standard test for the measurement of heparin inhibitory activity with respect to factor Xa and the duration of thrombin corresponds to the last stage of coagulation, namely the formation of fibrins induced by thrombin. It has been found that unlike heparin, the activity of laminar sulfate PS3 in the test called “HEPTEST" is more than 20 times lower. Similarly, in relation to the duration of prothrombin sulfate laminarine PS3 showed no pronounced anticoagulant effect, as in the case of heparin.
  • the specific activity (ILJ / mg) in the APTT represents 30% of the activity of heparin and in the case of the thrombin time 60%.
  • the concentration of APTT was 4 times higher and, in the case of thrombin time, 20 times higher.
  • laminar sulfate PS3 unlike heparin, does not exhibit either a significant anti-thrombin-dependent anti-factor Xa activity. an anti-thrombin activity.
  • the effect in the case of thrombin time may be considered to be due to heparin cofactor II-dependent thrombin inhibition.
  • the inhibitory properties of RT of laminarin sulfate PS3 can be advantageously exploited without fear of undesirable side effects on coagulation.
  • Example 1 The in vitro cytotoxicity of the sulfated laminar PS3 obtained in Example 1 was determined simultaneously with that of a comparison product.
  • 24-well plate CEM cells were cultured in 1 ml of RPMI supplemented with 10% fetal serum and calf, 1% Penicillin-Streptomycin, 2 mM glutamine, 2 ⁇ g / ml polybrene and different concentrations of sulphated laminarine PS3 and the comparison product consisting of dextran sulphate.
  • the cells were counted daily and the increase in number was compared to that of a control culture consisting of cultured CEM cells in the absence of sulphated PO3 laminarin or dextran sulfate (O ⁇ g / ml).
  • the white rabbits were tested on the one hand for ocular irritation and on the other hand for the primary skin irritation test. The outcome of the first of these tests led to the conclusion that the action was slightly irritating and in the second to a non-irritating action.
  • the rats were then subjected to an acute dermal toxicity test and to an acute oral toxicity study.
  • the dermal lethal dose 50 is greater than 2 g / kg of body weight, which makes it possible to affirm that the product is not toxic.
  • the acute oral toxicity may be considered greater than 2 g / kg of body weight which again allows the product to be classified as nontoxic.
  • Example 9 Composition of an Aerosol Solution Based on Sulphated Laminarine PS3
  • EXAMPLE 10 Composition of a suppository based on a sulfate of 3 1-3 qlucan
  • a suppository based on sulfate of a ⁇ 1-3 glucan having the following composition:
  • Example 11 Composition of a Laminaritol Sulphate Injectable Solution An injectable solution based on laminaritol sulphate having the following composition was prepared:
  • Example 12 Composition of a Vaginal Solution Based on a ⁇ 1-3 Glucan Sulfate
  • vaginal solution based on ⁇ 1-3 glucan sulfate having the following composition was produced:
  • the time of implementation at which sulfate laminar PS3 is effective demonstrates a specific action on the early events of the replication cycle of the virus.
  • the anti-retroviral activity of the polysaccharides of formula (I), in particular that of sulphated laminarine, is not only better than that of the products previously used, but, moreover, it is exerted even on viruses resistant to known inhibitors. of the RT, which is resistant to d4T and Zerit and CHT virus that is resistant to AZT.
  • the polysaccharides of formula (I), and especially sulfated laminarine inhibit the RT of isolated viruses, which seems to rule out the hypothesis of a mechanism of action related to the simple anionic nature of the sulfated polysaccharides of the invention.

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