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WO1999038524A2 - Administration de ldl-r a des fins therapeutiques contre les infections virales et comme immunoregulateurs - Google Patents

Administration de ldl-r a des fins therapeutiques contre les infections virales et comme immunoregulateurs Download PDF

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Publication number
WO1999038524A2
WO1999038524A2 PCT/IB1999/000149 IB9900149W WO9938524A2 WO 1999038524 A2 WO1999038524 A2 WO 1999038524A2 IB 9900149 W IB9900149 W IB 9900149W WO 9938524 A2 WO9938524 A2 WO 9938524A2
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WIPO (PCT)
Prior art keywords
receptor
immunodeficiency
resultant
ldl
density lipoprotein
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PCT/IB1999/000149
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English (en)
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WO1999038524A3 (fr
Inventor
Patrick Thomas Prendergast
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Patrick Thomas Prendergast
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Priority to AU20697/99A priority Critical patent/AU2069799A/en
Publication of WO1999038524A2 publication Critical patent/WO1999038524A2/fr
Publication of WO1999038524A3 publication Critical patent/WO1999038524A3/fr

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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
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the envelope protein of HIV is a 160-kDa giycoprotein.
  • the protein is cleaved by a protease to give a 120-kDa external protein, gp 120, and a transmembrane giycoprotein, gp 41.
  • the gp 120 protein contains the amino acid sequence that recognizes the receptor on CD4-positive human T-helper cells.
  • human LDL receptor cyste rich domain
  • Anti-serum prepared against synthetic peptides corresponding to amino acid residues 307 -330 and 303 - 321 in gp 120 inhibit HIV-induced syncytium formation.
  • the Figure is a model showing five domains in the structure of the human LDL receptor. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
  • Low density lipoprotein has five structural domains.
  • the deduced amino acid sequence suggests that the human LDL receptor can be divided into five domains which are shown schematically in the Figure.
  • the first domain 1 the cysteine-rich region (ligand binding), consists of the NH 2 -terminal 322 amino acids, which include 47 cysteine residues (15% cysteine). All of these cysteine residues appear to be involved in disulfide bonds, since the intact receptor is labeled only poorly with 3 H-iodoacetamide unless the disulfide bonds are first reduced (W. Schneider, unpublished data).
  • the boundary for the first domain was arbitrarily set at residue 322 on the basis of the computer analysis described below (see Table 1 , page 2). Table 1 shows alignment of repeats TABLE 1
  • sequences are shown in the single letter codex which translates to the tree letter code as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe' G, Gly; H, His; I, lie; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gin; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; Y, Tyr. If the boundary for the first domain were extended to residue 371 , the first domain would include a total of 54 cysteines (15%).
  • the NH 2 - terminal portion of the receptor is composed of a sequence of about 40 residues that is repeated eight times with minor variations (Table 1). With the introduction of a few brief gaps, each of the repeat sequences can be aligned such that the cysteine residues are in register (Table 1 ). At many positions in this sequence, a particular amino acid occurs so frequently that a consensus sequence can be written.
  • arginine and lysine residues are believed to cluster in a small region of the 33 Kilodalton molecule.
  • the three most crucial residues for binding are Arg 142, Arg 145, and Lys 146, all of which are believed to face the same side of a single ⁇ -helix (Innerarity et al., 1984). It seems reasonable to speculate that these arginines and lysines interact with the clustered negatively charged residues in the cysteine-rich region of the LDL receptor (Innerarity and Mahley, 1978; Pitas et al., 1980). Therefore two of the repeats shown in Table 1 may constitute one ligand binding site.
  • the receptor is extremely stable as long as the disulfide bonds are not reduced. It can be boiled in SDS or in guanidine and retain the ability to bind LDL as long as reducing agents are not present (Daniel et al., 1983). Thus the presence of disulfide bonds approximately once every seven residues imparts great stability to the receptor binding site. This stability may protect the molecule against extreme changes in pH when it recycles between coated pits on the cell surface and acidic endosomes within the cell, several times within the cell, several times during its life history (Brown et al., 1983). Second, pre-treatment of the receptor with reducing agents markedly retards its mobility on SDS gets (Daniel et al., 1983). The decreased mobility after reduction of disulphide bonds presumably relates to the unfolding of the extensively cross-linked protein.
  • the second domain 2 (see the Figure) of the human LDL receptor, consisting of approximately 350 residues, begins near the end of the cysteine-rich repeats and extends to amino acid residue 640. Like the cysteine-rich domain, this domain also contains repeat sequences. The optimal alignment for residues 409 to 610 shows that it consists of five repeats of approximately 25 amino acids each; 11 of the amino acids in each repeat are highly conserved.
  • the striking aspect of the second domain is its homology to the amino acid sequence of the polyprotein precursor for mouse EGF. This homology was previously noted when the partial sequence of the bovine LDL receptor was compared with that of the mouse EGF precursor (Russell et al., 1984).
  • the homology between the mouse EGF precursor and the human LDL receptor is even more significant than that previously noted with the bovine receptor.
  • the third domain 3 (see the Figure) of the human LDL receptor, which was previously identified in the bovine sequence (Russell et al., 1984), is a region (residues 700 to 747, see the Figure) rich in serine and threonine that is just outside the membrane spanning region of the receptor (see the Figure).
  • this stretch of 48 amino acids contains 18 serine or threonine residues located immediately outside the membrane-spanning region.
  • this sequence was shown by biochemical studies to contain O-linked carbohydrate chains (Russell et al., 1984). The conservation of this serine and threonine-rich region in the human and bovine LDL receptors suggests that it is also the site to which O-linked carbohydrate chains are added in the human receptor sequence.
  • the external portion of the human LDL receptor contains five potential sites for N-linked glycosylation that exhibit the consensus sequence Asn-X-Ser or Asn-X-Thr (residues sites are in the cysteine-rich domain, and two of the sites are in the second domain that is homologous with the EGF precursor. Quantitative studies have indicated that only two N-linked chains are present in the bovine LDL receptor.
  • the human LDL receptor also contained two N-linked carbohydrate chains (Cummings et al., 1983). It is possible that the three potential N-llnked glycosylatlon sites in the cysteine-rich region are not actually glycosylated because of their occurrence in such an unusual protein environment.
  • the fourth domain 4 is a sequence of 22 hydrophobic amino acids that is believed to be the membrane-spanning region of the receptor (see the Figure).
  • a similarly located hydrophobic sequence in the bovine LDL receptor was shown by proteolysis experiments to span the membrane (Russell et al., 1984).
  • the fifth domain 5 (see the Figure) of the human receptor consists of 50 amino acids at the COOH-terminal end of the protein that are located on the cytoplasmic side of the plasma membrane. This sequence is strongly homologous with the cytoplasmic domain of the bovine LDL receptor (Russell et al., 1984).
  • the 50 residue cytoplasmic domains of the human and bovine LDL receptors are identical with the exception of four residues: serine substituted for threonine at position 825 in the human LDL receptor; asparagine for serine at position 820; histidine for arginine at position 819; and asparagine for serine at position 796.
  • Sequence 'A'-NNTRKSIRIQRGPGRAFVTIGKIG [SEQ. ID NO. 2]
  • the fraction of LDL that binds to the resin-bound Sequence 'A' peptide is useful in preventing syncytium formation in HIV-infected CD4 cells and reduces HIV infectivity.
  • the cysteine-rich domain of the human LDL receptor according to the present invention is useful in the treatment of AIDS and ARC and in the therapy of Hepatitis B and C virus infections.
  • LDL-R-(human) measured by: (i) virus-induced syncytia formation and (ii) levels of P24 virion core antigen in supernatant culture fluid.
  • Virus stock of the GB8 strain prepared from cell-free medium of acutely infected JM cells was diluted in growth medium (RPMI 1640, 10% fetal calf serum) containing different concentrations of test compound. After 15 minutes at room temperature, cells were added and virus adsorption carried out at this temperature for 2 hours to provide a multiplicity of infection (MOI) of 0.001 syncytia-forming units per cell.
  • MOI multiplicity of infection
  • Infected cells were pelleted, washed three times in phosphate buffered saline, resuspended in fresh growth medium containing test peptide (LDL receptor) at appropriate concentrations and distributed into 24 well tissue culture plates. After 3 days incubation at 37° C, numbers of syncytia were scored in quadruple. The supernatant culture fluid was sampled and clarified by centrifugation (2,000 rpm/5 minutes). The level of P24 antigen was determined by the Abbott core antigen Elisa test after treatment with 0.1% Triton X-100.
  • test peptide LDL receptor
  • the anti-HIV activity LDL-R of this invention includes low density lipoprotein receptor as set forth in Table 1 , functionally equivalent salts thereof, functionally equivalent derivatives thereof, and functionally equivalent muteins thereof, as well as any portions of any such materials which exhibit affinity for the resin-bound HIV Sequence 'A'.
  • Such portions may be of at least 7 residues, more commonly at least 30 residues, or at least 100 residues, or at least 300 residues, and preferably comprises a part or all of the cysteine- rich first domain (322 residues).
  • the invention further concerns recombinant DNA molecules comprising the nucleotide sequence coding for said LDL receptor or for its active muteins or fused proteins, expression vehicles comprising them and host cells transformed therewith and to a process for producing the soluble LDL receptor, its active muteins or fused proteins, by culturing said transformant cells in a suitable culture medium.
  • LDL-receptor The isolation of LDL-receptor is well known in the art, and such known methods for isolating LDL-receptor may be used to obtain LDL-receptor for use in accordance with the present invention.
  • mutants refers to analogues of the soluble
  • LDL receptor in which one or more of the amino acid residues of the natural soluble LDL receptor, such as 1-30, preferably 1-10 (e.g., 7) and more preferably 1-5 residues or even only a single residue, are replaced by different amino acid residues or are deleted, or one or more amino acid residues, such as 1-20, preferably 1 - 10 (e.g., 7), more preferably 1-5, or only one residue are added to the natural sequence of the soluble LDL receptor, without changing considerably the antiviral activity of the resulting product.
  • These muteins are prepared by known synthesis and/or site-directed mutagenesis techniques, or any other known technique suitable therefor. The substitutions are preferably conservative. See, e.g., Schulz, G. E.
  • substitutions which may be made in the protein or peptide molecule of the present invention may be based on analysis of the frequencies of amino acid changes between a homologous protein of different species, such as those presented in Table 1-2 of Schulz et al. (supra) and FIGS. 3-9 of Creighton (supra). Based on such an analysis, conservative substitutions may be defined herein as exchanges within one of the following five groups:
  • Conservative amino acid substitutions according to the present invention are known in the art and would be expected to maintain biological and structural properties of the polypeptide after amino acid substitution. Most deletions and insertions, and substitutions according to the present invention are those which do not produce radical changes in the characteristics of the protein or peptide molecule. One skilled in the art will appreciate that the effect of substitutions can be evaluated by routine screening assays, either immunoassays or bioassays.
  • a mutant typically is made by site-specific mutagenesis of the peptide molecule-encoding nucleic acid, expression of the mutant nucleic acid in recombinant cell culture, and, optionally, purification from the cell culture, or a biological sample containing a soluble LDL receptor protein, for example, by immunoaffinity chromatography using a specific antibody on a column (to absorb the mutant by binding to at least one epitope).
  • fused protein refers to a polypeptide comprising the soluble LDL receptor or a mutein thereof fused with another protein which has an extended residence time in body fluids.
  • the soluble LDL receptor may thus be fused to another protein, polypeptide or the like, e.g., an immunoglobulin or a fragment thereof.
  • salts herein refers to both salts of carboxyl groups and to acid addition salts of amino groups of the soluble LDL receptor, muteins and fused proteins thereof.
  • Salts of a carboxyl group may be formed by means known in the art and include inorganic salts, for example, sodium, calcium, ammonium, ferric or zinc salts, and the like, and salts with organic bases as those formed, for example, with amines, such as triethanolamine, arginine or lysine, piperidine, procaine and the like.
  • Acid addition salts include, for example, salts with mineral acids such as, for example, hydrochloric acid or sulfuric acid, and salts with organic acids such as, for example, acetic acid or oxalic acid.
  • “Functional derivatives” as used herein cover derivatives of the soluble LDL receptor and its fused proteins and muteins, which may be prepared from the functional groups which occur as side chains on the residues or the N- or C-terminal groups, by means known in the art, and are included in the invention as long as they remain pharmaceutically acceptable, i.e. they do not destroy the activity of the protein and do not confer toxic properties on compositions containing it.
  • These derivatives may, for example, include polyethylene glycol side-chains which may mask antigenic sites and extend the residence of the soluble LDL receptor in body fluids.
  • derivatives include aliphatic esters of the carboxyl groups, amides of the carboxyl groups by reaction with ammonia or with primary or secondary amines, N-acyl derivatives of free amino groups of the amino acid residues formed with acyl moieties (e.g. alkanoyl or carbocyclic aroyl groups) or acyl derivatives of free hydroxyl groups (for example that of seryl or threonyl residues) formed with acyl moieties.
  • the term "functional derivative” also includes proteins which have an amino acid sequence longer or shorter than the sequence determined, as long as the protein still has the ability to inhibit viral infection.
  • active fractions of the soluble LDL receptor, its fused proteins and its muteins covers any fragment or precursors of the polypeptide chain of the protein molecule alone or together with associated molecules or residues linked thereto, e.g., sugar or phosphate residues, or aggregates of the protein molecule or the sugar residues by themselves, provided said fraction has the ability to inhibit viral infection and/or activity.
  • Such active fractions can be readily determined by testing smaller and smaller portions of the entire soluble LDL receptor or mutein to find the smallest fragment which retains the ability to inhibit viral infections. Any fractions containing the smallest active fraction will also be an active fraction. Undue experimentation would not be involved as the required tests for antiviral activity (as described herein) may be routinely carried out.
  • This invention further concerns DNA molecules comprising the nucleotide sequence encoding the soluble LDL receptor, fused proteins, muteins or active fractions thereof, replicable expression vehicle expression of such transformed hosts.
  • DNA molecules includes genomic DNA, cDNA, synthetic DNA and combinations thereof.
  • the production of the recombinant soluble LDL receptor may be carried out by different techniques.
  • the known cDNA of the entire human LDL receptor is taken from plasmid pLDLR-2 (Yamamoto et al., op cit).
  • the DNA is subjected to site directed mutagenesis with appropriate oligonucleotides so that a termination codon and a polyadenylation site are inserted after codon 292 of the mature LDL receptor.
  • This construct is then inserted into appropriately constructed expression vectors by techniques well known in the art (see Maniatis et al., op cit.).
  • Double-stranded cDNA is linked to plasmid vector by homopolymeric tailing or by restriction linking involving the use of synthetic DNA linkers or blunt-ended ligation techniques.
  • DNA ligases are used to ligate the DNA molecules and undesirable joining is avoided by treatment with alkaline phosphatase.
  • an expression vector In order to be capable of expressing the soluble LDL receptor, its muteins or the fused proteins, an expression vector should comprise also specific nucleotide sequences containing transcriptional and translational regulator information linked to the DNA coding for the desired protein in such a way as to permit gene expression and production of the protein.
  • RNA polymerase a promoter recognizable by RNA polymerase, to which the polymerase binds and thus initiates the transcription process.
  • promoters There are a variety of such promoters in use, which work with different efficiencies (strong and weak promoters). They are different for prokaryotic and eukaryotic cells.
  • Sequence A peptide has been used, other related peptides with up to 5 amino acid substitutions and those related peptides being extended on the amino or carboxy terminal ends or both as well as those related peptides being truncated on the amino or carboxy terminal ends, have been demonstrated to produce substantially similar results and applicants intend that the expression Sequence 'A * peptide and variants thereof when used herein with regard to the preparation of anti-HIV LDL-R will include such related peptides.
  • Table 2 lists certain known variances of the Sequence 'A' region of other HIV isolates. Peptides having these sequences as well as other sequence variations may be substituted for the sequence of sequence 'A' in the process of this invention.
  • other peptide regions in the gp 120/gp 41 that fulfill the criteria for LDL-R binding are included.
  • the at least one Sequence 'A' peptide or variant thereof is covalently bound to a chromatography resin in the usual manner and the resin-bound Table 2 HIV Isolate Sequence 'A' - Variant Sequence
  • SEQ. ID NO.12 sequence 'A' peptide or variant thereof is used to isolate anti-HIV LDL-R by affinity chromatography in the conventional manner.
  • the resin must be insoluble in the solvents and buffers to be employed, it must be mechanically and chemically stable with good flow properties, it must be easily coupled to the sequence A peptide or variant thereof, and it should have a large surface area accessible to the substrate to be absorbed.
  • Anti-HIV LDL-R can be used to prevent syncytium formation in cells infected with HIV-1 virus or other related viruses having gpl20 surface protein.
  • Anti-HIV LDL-R can be used to treat AIDS and ARC and other diseases caused by the retrovirus HIV or other related viruses having gpl20 surface protein.
  • the amount of anti-HIV LDL-R which is needed to prevent syncytium formation in HIV infected cells can be any effective amount.
  • anti-HIV LDL-R or cysteine-rich domain of same
  • concentration of 10 ⁇ g/ml resulted in complete inhibition of syncytium formation as well as reduced the presence of P24 antigen, an indicator of viral replication, to below 2.0 x 10 2 .
  • the amount of anti-HIV LDL-R to be administered in order to treat AIDS or ARC or other disease caused by HIV infection can vary widely according to the particular dosage unit employed, the period of treatment, the age and sex of the patient treated, the nature and extent of the disorder treated, and other factors well-known to those practicing the appropriate arts.
  • anti-HIV LDL-R can be used in conjunction with other agents known to be useful in the treatment of viral diseases and agents known to be useful to treat the symptoms of and complications associated with diseases and conditions caused by retroviruses.
  • the anti-HIV effective amount of anti-HIV LDL-R to be administered will generally range from about 15 mg/kg to 500 mg/kg.
  • a unit dosage may contain from 25 to 500 mg of LDL-R, or the cysteine rich domain of Human LDL receptor, and can be taken one or more times per day.
  • Anti-HIV LDL-R can be administered with a pharmaceutical carrier using conventional dosage unit forms either orally or parentally.
  • the anti-HIV LDL-R can be formulated into solid or liquid preparations such as capsules, pills, tablets, troches, lozenges, melts, powders, solutions, suspensions, or emulsions.
  • the solid unit dosage forms can be a capsule, which can be of the ordinary hard- or soft-shelled gelatine type containing, for example, surfactants, lubricants, and inert fillers such as lactose, sucrose, calcium phosphate, and cornstarch.
  • the compounds of this invention can be tableted with conventional tablet bases such as lactose, sucrose, and cornstarch in combination with binders such as acacia, cornstarch, or gelatin, disintegrating agents intended to assist the break-up and dissolution of the tablet following administration such as potato starch, alginic acid, corn starch, and guar gum, lubricants intended to improve the flow of tablet granulations and to prevent the adhesion of tablet material to the surfaces of the tablet dies and punches, for example, talc, stearic acid, or magnesium, calcium, or zinc stearate, dyes, coloring agents, and flavoring agents intended to enhance the aesthetic qualities of the tablets and make them more acceptable to the patient.
  • conventional tablet bases such as lactose, sucrose, and cornstarch in combination with binders such as acacia, cornstarch, or gelatin
  • disintegrating agents intended to assist the break-up and dissolution of the tablet following administration such as potato starch, alginic acid, corn starch,
  • Suitable excipients for use in oral liquid dosage forms include dilutents such as water and alcohols, for example, ethanol, benzyl alcohols, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptably surfactant, suspending agent, or emulsifying agent.
  • the anti-HIV LDL-R of this invention may also be administered parentally, that is, subcutaneously, intravenously, intramuscularly, or interperitoneally, as injectable dosages of the compound in a physiologically acceptable dilutent with a pharmaceutical carrier which can be a sterile liquid or mixture of liquids such as water, saline, aqueous dextrose and related sugar solutions, an alcohol such as ethanol, isopropanol, or hexadecyl alcohol, glycols such as propylene glycol or polyethylene glycol, glycerol ketols such as 2,2-dimethyl-1,3-dioxolane-4-methanol, others such as poly(ethyleneglycol) 400, an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant such as a soap or a detergent, suspending agent such as pect
  • Suitable fatty acids include oleic acid, steric acid, and isostearic acid.
  • Suitable fatty acid esters are, for example, ethyl oleate and isopropyl myristate.
  • Suitable soaps include fatty alkali metal, ammonium, and triethanolamine salts and suitable detergents include cationic detergents, for example, dimethyl dialkyl ammonium halides, alkyl pyridinium halides, and alkylamines acetates; anionic detergents, for example, alkyl. aryl and olefin sulfonates, alkyl, olefin, ether and monoglyceride sulfates, and sulfosuccinates; nonionic detergents, for example, fatty amine oxides, fatty acid alkanolamides.
  • cationic detergents for example, dimethyl dialkyl ammonium halides, alkyl pyridinium halides, and alkylamines acetates
  • anionic detergents for example, alkyl. aryl and olefin sulfonates, alkyl, olefin, ether and monoglyceride sul
  • compositions of this invention will typically contain from about 0.5 to about 75% by weight of anti-HIV LDL-R in solution. Preservatives and buffers may also be used advantageously. In order to minimize or eliminate irritation at the site of injection, such compositions may contain a non-ionic surfactant having a hydrophile-lipophile balance (HLB) of from about 10 to about 20. The quantity of surfactant in such formulations ranges from about 2 to about 20% by weight.
  • HLB hydrophile-lipophile balance
  • surfactants used in parenteral formulations are the class of polyethylene sorbitan fatty acid esters, for example, sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol, administered in liposomes for oral or parental.
  • the present invention is also directed to the use of human low-density lipoprotein receptor, a functionally equivalent salt thereof, a functionally equivalent derivative thereof, a functionally equivalent mutein thereof, or a part of any such receptor, salt, derivative or mutein in the manufacture of a medicament for use in the treatment of a condition selected from the group consisting of: (a) immunodeficiency resultant from a viral infection;
  • immunodeficiency resultant from the growth of neoplastic tissue (c) immunodeficiency resultant from the growth of neoplastic tissue; (d) immunodeficiency resultant from any cytokine or hormone imbalance or imbalance of any natural product within the patient;
  • the peptide sequence 'A' was synthesized by solid-phase methods using an Applied Biosystems Model 430A synthesizer on 0.5 mmol of a Boc Gly(PAM)-resin (0.8 mmol/g).
  • the N ⁇ -t-Boc-protected amino acids were double coupled as their preformed symmetrical anhydrides, first in N,N-dimethylformamide then in dichloromethane using protocols supplied by the manufacturer.
  • Asn, Arg and Gin were double coupled as their 1-hydroxybenzotriazole esters.
  • An acetic anhydride capping step was included between each successive amino acid.
  • the side chain protection was as follows: Lys(2-CIZ, Arg(Tos), Ser(Bzl), Thr(Bzl).
  • the peptide was deprotected and cleaved from the resin (0.25 mmol) by treatment with liquid hydrofluoric acid (HF) containing 5% anisole at -5° C (salt-ice bath) for 40 minutes. After removal of the HF in vacua the peptide was extracted from the resin with 30% acetic acid followed by 30% acetonitrile. The filtrates were lyophilized and the residue dissolved in 6 M urea. The peptide was purified on a Beckman 2 inch x 150 mm C18 column at 80 mi/min with a 20-25% linear gradient of acetonitrile in 0.1% trifluoroacetic acid over 15 min. The main peak was isolated and lyophilized leaving 137.9 mg of the desired product.
  • HF liquid hydrofluoric acid
  • the coupling reaction is carried out at 40 degrees C. with gentle rocking for 4 hrs. Remaining active esters on the resin are blocked by adding 0.1 ml of 1 M glycine ethyl ester (pH 8) or 0.1 ml of 1 M ethanolamine HCI (pH 8) per ml of gel and then incubated for 1 hour. The resin is then transferred to a column and extensively washed with 0.01 Hepes, 0.05 M NaCI, pH 7 4.
  • CD4 T-cells JM were exposed to a clinical isolate of HIV-1 , GBB.
  • the virus was first incubated with LDL-R for 15 minutes and then the cells were added . After 2 hours adsorption, the virus inoculum was removed and the cells were washed three times to remove traces of input virus.
  • Antiviral activity was determined after 3 days incubation by plotting the mean number of syncytia found in quadruple cultures against logic concentration of LDL-R.
  • the 50% effective dose ED 50 of LDL-R for inhibition of syncytia was estimated as 0.4 ⁇ g/ml.
  • the potency of LDL-R was also measured by assaying viral core antigen (P24 test-Abbott) in the supernatant fluid. ED 50 values of 0.5 ⁇ g/ml were obtained for LDL-R.

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Abstract

L'administration du récepteur des lipoprotéines humaines et animales de faible densité (LDL-R) développe une activité anti-VIH et anti-hépatite significative. Dans la variante préférée, l'invention a trait à l'activité anti virale et régulatrice de l'immunité du domaine riche en cystéine du LDL-R. Lesdits LDL-R, d'origine humaine et animale, qui s'administrent dans le traitement d'infections virales et comme agent régulateur de l'immunité, entrent dans la fabrication de médicaments à cet effet.
PCT/IB1999/000149 1998-01-29 1999-01-28 Administration de ldl-r a des fins therapeutiques contre les infections virales et comme immunoregulateurs WO1999038524A2 (fr)

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AU20697/99A AU2069799A (en) 1998-01-29 1999-01-28 Therapeutic administration of low density lipoprotein receptor for viral infections and immune regulation

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US7298098P 1998-01-29 1998-01-29
US60/072,980 1998-01-29

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WO1999038524A3 WO1999038524A3 (fr) 1999-09-23

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001042429A1 (fr) * 1999-12-10 2001-06-14 Daniel Favre Infection des cellules eucaryotes avec des virus in vitro
EP1245236A4 (fr) * 1999-12-28 2004-09-22 Sumitomo Pharma Produits therapeutiques et prophylactiques pour l'hepatique chronique
EP1592445A4 (fr) * 2000-10-25 2007-01-10 Vincent Agnello Procede d'inhibition de l'infection au virus de l'hepatite c (vhc) et autres virus de la famille des i flaviridae /i , et a tout autre virus formant dans le sang un complexe avec une lipoproteine de basse densite ou tres basse densite, par empechement de l'entree virale dans une cellule
JP2011509254A (ja) * 2008-01-07 2011-03-24 イエダ リサーチ アンド ディベロップメント カンパニー リミテッド ウイルス性肝炎におけるｓＬＤＬＲ

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2628082B2 (ja) * 1988-12-07 1997-07-09 日東電工株式会社 免疫抑制剤
IL102915A (en) * 1992-01-19 2005-12-18 Yeda Res & Dev Soluble ldl receptor and its preparation
DE4222385A1 (de) * 1992-07-08 1994-01-13 Boehringer Ingelheim Int Verfahren zur Isolierung von Inhibitoren des Rhinovirus-Rezeptors

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001042429A1 (fr) * 1999-12-10 2001-06-14 Daniel Favre Infection des cellules eucaryotes avec des virus in vitro
EP1111040A1 (fr) * 1999-12-10 2001-06-27 Daniel Favre L'infection de cellules eukaryotes par des virus in vitro
EP1245236A4 (fr) * 1999-12-28 2004-09-22 Sumitomo Pharma Produits therapeutiques et prophylactiques pour l'hepatique chronique
EP1592445A4 (fr) * 2000-10-25 2007-01-10 Vincent Agnello Procede d'inhibition de l'infection au virus de l'hepatite c (vhc) et autres virus de la famille des i flaviridae /i , et a tout autre virus formant dans le sang un complexe avec une lipoproteine de basse densite ou tres basse densite, par empechement de l'entree virale dans une cellule
JP2011509254A (ja) * 2008-01-07 2011-03-24 イエダ リサーチ アンド ディベロップメント カンパニー リミテッド ウイルス性肝炎におけるｓＬＤＬＲ

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WO1999038524A3 (fr) 1999-09-23
AU2069799A (en) 1999-08-16

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