+

WO2006033012A2 - Procede - Google Patents

Procede Download PDF

Info

Publication number
WO2006033012A2
WO2006033012A2 PCT/IB2005/003070 IB2005003070W WO2006033012A2 WO 2006033012 A2 WO2006033012 A2 WO 2006033012A2 IB 2005003070 W IB2005003070 W IB 2005003070W WO 2006033012 A2 WO2006033012 A2 WO 2006033012A2
Authority
WO
WIPO (PCT)
Prior art keywords
cell
seq
fusion
assay
cells
Prior art date
Application number
PCT/IB2005/003070
Other languages
English (en)
Other versions
WO2006033012A3 (fr
Inventor
Romuald Gaston Corbau
Derek John Falconer
Helen Bostock Lavender
Manoussos Perros
Original Assignee
Pfizer Limited
Pfizer 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
Priority claimed from GB0421360A external-priority patent/GB0421360D0/en
Application filed by Pfizer Limited, Pfizer Inc. filed Critical Pfizer Limited
Publication of WO2006033012A2 publication Critical patent/WO2006033012A2/fr
Publication of WO2006033012A3 publication Critical patent/WO2006033012A3/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • G01N33/5767—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis non-A, non-B hepatitis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/18—Togaviridae; Flaviviridae
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the present invention relates to an assay method and compounds suitable for use in the treatment of hepatitis C virus infection.
  • Hepatitis C virus is one of the most important causes of chronic liver disease. In the United States it accounts for about 15 percent of acute viral hepatitis, 60 to 70 percent of chronic hepatitis and up to 50 percent of cirrhosis, end-stage liver disease and liver cancer. Hepatitis C is the most common blood-borne infections in the United States, infecting more than 1.8 percent of the population and causing an estimated 8,000 to 10,000 deaths annually (National Institute of Health Consensus Development Conference Statement: Management of Hepatitis C: June 10-12 (2002); http://www.consensus.nih.gov).
  • HCV is a small (50 to 60 nm in diameter), enveloped, positive, single-stranded RNA virus in the Flaviviridae family.
  • the genome is approximately 10,000 nucleotides and encodes a single polyprotein of about 3,000 amino acids.
  • the polyprotein is processed by host cell and viral proteases into three major structural proteins and several non ⁇ structural protein necessary for viral replication (Bartenschlager and Lohmann, J. Gen. Virol. (2002) 81, 1631-1648). Because the virus mutates rapidly, changes in the envelope protein are thought to contribute to evasion of the immune system.
  • Genotypes 1a and 1b are the most common in the United States while genotypes 2 and 3 are present in only 10 to 20 percent of patients. There is little difference in the severity of disease or outcome of patients infected with different genotypes. However, this genetic diversity impacts negatively on both treatment options and effectiveness.
  • genotype 1 which accounts for >70 percent of all HCV infections in the US and has infected millions of people around the world via contaminated blood transfusions, is associated with the poorest response to treatment. Patients with genotypes 2 and 3 are more likely to respond to alpha interferon treatment
  • Alpha interferon is a host protein that is made in response to viral infections and has natural antiviral activity. Recombinant forms of alpha interferon have been produced, and several formulations are available as therapy of hepatitis C. However, these standard forms of interferon are now being replaced by pegylated interferons (peginterferons). Peginterferon is alpha interferon that has been modified chemically by the addition of a large inert molecule of polyethylene glycol. Pegylation changes the uptake, distribution and excretion of interferon, prolonging its half-life, and therefore is more effective than standard interferon in inhibiting HCV, yielding higher sustained response rates with similar side effects.
  • peginterferons pegylated interferons
  • Alpha interferon has multiple neuropsychiatry effects and strict abstinence from alcohol is also recommended during therapy with interferon. Prolonged therapy can cause marked irritability, anxiety, personality changes, depression and even suicide or acute psychosis.
  • Alpha interferon therapy can induce auto-antibodies and a 6- to 12-month course triggers an autoimmune condition in about 2 percent of patients, particularly if they have an underlying susceptibility to autoimmunity. Exacerbation of a known autoimmune disease (such as rheumatoid arthritis or psoriasis) occurs commonly during interferon therapy. Alpha interferon also has bone marrow suppressive effects.
  • Ribavirin causes red cell haemolysis to a variable degree in almost all patients. Therefore, patients with a pre-existing haemolysis or anaemia should not receive ribavirin. Similarly, patients who have significant coronary or cerebral vascular disease should not receive ribavirin, as the anaemia caused by treatment can trigger significant ischaemia. Fatal myocardial infarctions and strokes have been reported during combination therapy with alpha interferon and ribavirin. Ribavirin is excreted largely by the kidneys and patients with renal disease can develop haemolysis that is severe and even life-threatening. Ribavirin also causes birth defects in animal studies, while alpha interferon has direct antigrowth and antiproliferative effects.
  • HCV replicons such as those described in EP 1043399, provide a useful alternative for the study of processes involved in HCV genome replication, they do not involve processes linked to HCV maturation or entry.
  • vaccinia based fusion assays in which the envelope protein of the virion of interest is only transiently expressed in the virion host cell, an example of which is described by Takikawa et al (J. Virology (2000) 74(11 ), 5066-5074). While such vaccinia based cell fusion assays have been useful, they suffer from certain disadvantages. In particular, they are unsuitable for adaptation to high throughput screening.
  • Successful high throughput screens preferably require stable cell lines. This is in contrast to the vaccinia- or transient transfection-based methods of the prior art in which the generation of a high quantity of virus or plasmids is not compatible with a safe and robust high throughput assay.
  • a limitation with a transfection-based method is the difficulty in obtaining highly reproducible transfections.
  • the use of a stable cell line method, as opposed to a vaccinia-based method allows the use of a reporter system such as the Tat and HIV LTR reporter system.
  • Tat may be expressed in one cell and, on fusion with a second cell, the Tat protein is able to reach the nucleus of the second cell where it will function by enabling phosphorylation of the carboxy terminal domain of RNA-pol II.
  • a vaccinia-based method is not suitable for use with such a reporter system since the reporter system will be expressed in the cytoplasm.
  • the reporter protein that drives expression from the reporter system will be in a different cellular compartment and so a signal will never be produced.
  • HCV has been proposed to contain a class Il fusion protein (E2).
  • Enveloped viruses penetrate the host cells by a process of fusion between the viral and cell membranes that is catalyzed by a fusogenic activity harboured by viral surface glycoproteins (E1 and E2 for HCV).
  • E1 and E2 for HCV viral surface glycoproteins
  • activation of the fusion proteins occurs in an acid pH-dependent manner, via acidified endosomal vesicles into which the virions are routed following receptor binding.
  • HCV pseudo-particles HCV pseudo-particles
  • E1 E2 High level cell surface expression of E1 E2 is possible, typically by modifying E1 E2 in such a way that the protein is directed to the cell surface. To date, only two possibilities have been described:
  • Chimeric E1 E2 Takikawa et al (ibid.) designed a chimeric E1 E2 in which the native transmembrane domains of E1 E2 were replaced with those of Vesicular Stomatitis Virus G protein.
  • the Takikawa assay uses HCV envelope protein that is not in its natural conformation.
  • E1 E2 mutations in the transmembrane domain of E1 and E2 have been shown to allow cell-surface expression of the glycoproteins (Cocquerel et al, J. Virol. (2000) 74(8), 3623-33). However, most mutants do not retain the ability for E1 E2 to form functional proteins when expressed at the cell surface.
  • the present invention encompasses a cell-based assay for HCV-induced membrane fusion.
  • the present invention relates to an assay method for determining whether an agent is capable of modulating HCV infection, the method comprising (a) contacting the agent with (i) a first cell line expressing E1 and E2 proteins on the cell surface, and (ii) a permissive second cell line, and (b) determining whether the agent inhibits fusion between the first and second cell lines, wherein the E1 and E2 proteins are full length.
  • HCV Hepatitis C virus
  • HCV is a generic term that embraces individual species and/or genotypes of HCV. "HCV” should thus be understood to include all HCV genome types, particularly 1b and/or 1a as appropriate, unless the context and/or specific statements indicate otherwise.
  • An example of a suitable HCV is a 1 b subtype whose sequence may be found under the accession number AJ238799 (EMBL).
  • the native envelope proteins E1 and E2 of the present assay are native forms rather than chimeric constructs such as those incorporating the VSV-G transmembrane domain.
  • the native forms are full-length sequences from approximately amino acids 192 to 383 and 384 to 748 respectively within the HCV polyprotein (for example from 192 ⁇ 2 to 383 ⁇ 3 for E1 and from 384 ⁇ 2 to 748 ⁇ 7 for E2).
  • Expression of high-level full-length native E1 and E2 proteins at the surface of the cells unexpectedly leads to cell-cell fusion that occurs at the cell surface, rather than in the low-pH intracellular components. It is to be understood that expression of the E1 and E2 proteins includes the glycosylated forms of the E1 and E2 proteins.
  • the E1 and E2 sequences may also include some of the HCV core protein (protein C), for example the last 60 amino acids of the core protein.
  • protein C HCV core protein
  • An example of a suitable nucleotide and corresponding amino acid sequence is given below:
  • PVVVGTTDRFGVPTYSWGENETD VLLLNNTRPPQGNWFGCTWMNSTGFTKTCG GPPCNIGGIGNKTLTCPTDCFRKHPEATYTKCGSGPWLTPRCLVHYPYRLWHYPC TVNF ⁇ IFKVRMYVGGVEHRI ⁇ AACNWTRGERCNLEDRDRSELSPLLLSTTEWQVL PCSFTTLPALSTGLIHLHQNWDVQYLYGIGSAVVSFAIKWEYVLLLFLLLADARV CACLWMMLLIAQAEA
  • a permissive cell line is one that permits or is susceptible to HCV entry and/or infection.
  • Examples of such cell lines include Huh7, HepG2-CD81 and MOLT-4, and preferably is a hepatocyte cell line such as Huh7 and its derivatives.
  • the first cell line may be selected from HEK293T, HeLa or any other cell line that will allow high expression of
  • E1 E2 on the cell surface and that will not form syncytae with permissive cells, and is preferably a HEK293T cell line.
  • the inhibition of fusion is understood to be an indication that an agent is capable of modulating HCV infection.
  • the E1 and E2 proteins are stably expressed on the cell surface.
  • the first cell line comprises a coding sequence for the proteins that is stably incorporated into the cell's genome such that the proteins are expressed within the cell and directed to the cell surface. Stable expression ensures long-term expression of the proteins and is in contrast to the vaccinia based systems of the prior art.
  • the envelope proteins are transiently expressed on the cell surface.
  • Cell-cell fusion may be measured and quantified by any suitable system.
  • E1 and E2 are capable of supporting syncytium formation
  • inhibition of fusion may be determined by assaying for syncytium formation, for example using microscopy, such as low power light or phase contrast microscopy, or fluorescence activated cell analysers or sorters (FACS).
  • microscopy such as low power light or phase contrast microscopy, or fluorescence activated cell analysers or sorters (FACS).
  • a preferred method for determining inhibition of fusion is a reporter system, in particular one in which the first cell line has one component of a stably-expressed, two- component, signal-producing system and the second cell line has the second component.
  • the system provides a detectable signal upon fusion between the first and second cells.
  • the system includes a transactivating protein, such as the HIV Tat protein or any homologous protein, and a responsive expression construct, such as a Tat responsive expression construct, that encodes for a reporter.
  • a transactivating protein such as the HIV Tat protein or any homologous protein
  • a responsive expression construct such as a Tat responsive expression construct, that encodes for a reporter.
  • the Tat protein is HIV-1 Tat and the responsive expression construct is HIV-1 LTR.
  • the detectable signal may be by way of fluorescent proteins or a reporter enzyme such as beta-lactamase, galactosidase or luciferase.
  • the detectable signal is the reporter enzyme, luciferase. It is desirable that the detectable signal or reporter enzyme is proportional to the amount of fusion that occurs between the two cell lines. By this it is meant that, for example, a smaller amount signal detected is proportional to the amount of antiviral activity of the agent being tested, such that the amount of signal has an inverse correlation with the amount of antiviral activity of the agent.
  • the first cell line stably expresses Tat and the second cell line expresses luciferase driven by an HIVLTR promoter.
  • the first cell line stably expresses luciferase driven by an HIV LTR promoter and the second cell line expresses Tat.
  • An alternative method for determining whether the agent inhibits fusion may be by monitoring, measuring or detecting the mixing of cell contents or cell membranes. Such methods may involve the mixing of dyes loaded into one or both cell lines of the assay
  • the dyes being visually detectable or detectable by fluorescence (including detection of Fluorescent Resonance Energy Transfer or FRET, or detection by a decrease in fluorescence by quenching). Detection may also be achieved by expression of beta-lactamase enzyme in the first cell line while the second cell line is loaded with a dye such as CCF-4 that, on cleavage by beta-lactamase, yields a detectable fluorescent signal. Alternatively, the first cell line may be loaded with dye and the beta-lactamase expressed in the second cell line.
  • Alternative configurations suitable for use in the assay of the present invention will be apparent to a person skilled in the art.
  • Agents may be any chemical entity, including small molecules such as synthetic or natural compounds, polymeric molecules such as oligonucleotides, peptides, polypeptide or protein molecule, macromolecules such as one or more antibodies and fragments thereof.
  • Such agents may be screened using the assay of the present invention to determine whether they are capable of modulating HCV infection. Modulation may be to enhance, accelerate, increase, stimulate or otherwise promote fusion, or the effect may be to retard, prevent, restrict, reduce or otherwise inhibit fusion.
  • An agent may affect the rate of fusion, or may affect its equilibrium, or may affect the final ration of fused to unfused cells, or may affect any combination of such factors.
  • Preferred agents according to the invention have the effect of inhibiting and/or reducing fusion, whether by prevention, restriction, down-regulation or any other mechanism.
  • the Applicant has identified, and from another aspect the present invention relates to, an isolated peptide which is a subsequence of HCV E1 or E2, or a fragment, variant or homologue of such a subsequence, and which inhibits fusion in the assay of the present invention.
  • the isolated peptide comprises a motif corresponding to the E1 fusion peptide region, the fusion peptide region which has the amino acid sequence CSAMYVGDLC (SEQ ID NO:6).
  • the motif is flanked by a sequence or sequences corresponding to the sequence or sequences flanking the native E1 fusion peptide region.
  • a particular motif corresponds to the fusion peptide region if the motif is functionally equivalent to that region such that the motif acts as a mimic of the fusion peptide region in the natural fusion mechanism of HCV infection. Therefore, a motif that is identical to the full-length region, or to a truncated fusion peptide region, or to a variant or homologue of the full length of truncated region is encompassed, provided it has that functionality.
  • the isolated peptide contains a sequence that is at least 70% identical to all or part of amino acids 81 to 91 of the E1 amino acid sequence.
  • the isolated peptide has 20 to 30 amino acids.
  • An example of a suitable peptide is one having the sequence LVGAAALCSAMYVGDLCGSVFLVAQ (SEQ ID NO:3).
  • the isolated peptide comprises a motif corresponding to the E2 hydrophobic heptad repeat region, the heptad repeat region having the sequence
  • the motif is flanked by a sequence or sequences corresponding to the sequence or sequences flanking the native hydrophobic heptad repeat region.
  • a particular motif corresponds to the hydrophobic heptad repeat region if the motif is functionally equivalent to that region such that the motif acts as a mimic of the hydrophobic heptad region in the natural fusion mechanism of HCV infection. Therefore, a motif that is identical to the full length region, or to a truncated fusion peptide region, or to a variant or homologue of the full length of truncated region is encompassed, provided it has that functionality.
  • HLHQNVVDVQYLYGIGSAVVSFAIK (SEQ ID NO: 5)
  • the present invention encompasses an isolated peptide having an amino acid sequence selected from the group comprising: LVGAAALCSAMYVGDLCGSVFLVAQ (SEQ ID NO:3);
  • HLHQNVVDVQYLYGIGSAVVSFAIK (SEQ ID NO: 5) and variants, fragments and homologues thereof.
  • fragment include any substitution, variation, modification, replacement, deletion or addition of one or more amino acid (for example, up to 30%, up to 20%, up to 10%, up to 5% or up to 2 or 3% of the amino acids in the parent sequence) from or to the sequence, providing the resultant peptide has activity in the assay of the invention.
  • a homologue has close identity with respect to the parent sequence structure and/or function.
  • sequence homology there may be at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95% homology to the sequences shown in SEQ ID NOS: 3 to 5. Most preferably there may be at least 98% homology to the sequences shown in SEQ ID NOS: 3 to 5.
  • Identity refers to sequence identity between two peptides or between two nucleic acid molecules. Identity between sequences can be determined by comparing a position in each of the sequences which may be aligned for purposes of comparison. When a position in the compared sequences is occupied by the same base or amino acid, then the sequences are identical at that position. A degree of identity between nucleic acid sequences is a function of the number of identical nucleotides at positions shared by these sequences. A degree of identity between amino acid sequences is a function of the number of identical amino acids at positions shared by these sequences. Since two polynucleotides may each (1) comprise a sequence (i.e.
  • sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity.
  • a “comparison window” refers to a conceptual segment of at least twenty contiguous nucleotide positions wherein a polynucleotide sequence may be compared to a reference sequence of at least twenty contiguous nucleotides and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e. gaps) of twenty percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • Optimal alignment of sequences for determining a comparison window may be conducted by the local homology algorithm of Smith and Waterman (1981), by the homology alignment algorithm of Needleman and Wunsch (1972), by the search for similarity method of Pearson and Lipman (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Solftware Package Release 7.0, Genetics Computer Group, 575, Science Dr. Madison, W1), or by inspection.
  • the best alignment i.e., resulting in the highest percentage of identity over the comparison window
  • sequence identity means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison.
  • the types of amino acid substitutions that may be made may be of a conserved or non-conserved nature and should maintain the hydrophobicity/hydrophilicity of the amino acid sequence.
  • conserved amino acid substitutions consist of replacing one or more amino acids with amino acids of similar charge, size and/or hydrophobicity characteristics, such as a glutamic acid (E) to aspartic acid (D) amino acid substitution.
  • Non-conserved substitutions consist of replacing one or more amino acids possessing dissimilar charge, size and/or hydrophobicity characteristics, such as a glutamic acid (E) to valine (V) substitution.
  • Amino acid substitutions may be made, for example from 1 , 2 or 3 to 10 or 20 substitutions provided that the modified sequence retains its antiviral activity in accordance with the present invention. Amino acid substitutions may include the use of non-naturally occurring analogues.
  • Amino acid insertions may consist of single amino acid residues or stretches of residues.
  • the insertions may be made at the carboxy or amino terminal end of the peptides, as well as at a position internal to the peptide. Such insertions will generally range from 2 to 10 amino acids in length.
  • One or more such insertions may be introduced into the peptide sequences of the present invention provided such insertions result in peptides that still exhibit antiviral activity.
  • deletions may involve a single or contiguous or greater than one discrete portion of the peptide sequences.
  • One or more deletions may be introduced, provided the resulting peptide exhibits antiviral activity.
  • Derivatives of the peptides of the invention are also contemplated. Examples of such derivatives include peptides that have been modified, for example to enhance solubility or formulation. Examples of modifications are the attachment of fusion sequences or pegylation.
  • the peptides of the invention may also be used as inhibitors of viral membrane fusion- associated events, in particular HCV.
  • the peptides may also be used to inhibit or reduce the level of membrane fusion between two or more cells relative to the level of membrane fusion that occurs between the cells in the absence of the peptide.
  • the present invention also contemplates a pharmaceutical composition including a peptide of the invention, or variants, fragments and homologues thereof, together with one or more pharmaceutically acceptable excipients, diluents or carriers.
  • the pharmaceutical composition may include one or more additional therapeutic agents.
  • treating means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition.
  • treatment refers to the act of treating as “treating” is defined immediately above.
  • therapeutically effective amount and “effective amount” are intended to mean the amount of an inventive agent that, when administered to a mammal in need of treatment, is sufficient to effect treatment for injury or disease conditions alleviated by the inhibition of HCV infection.
  • amount of a given HCV-inhibiting agent used in the method of the invention that will be therapeutically effective will vary depending upon factors such as the particular HCV-inhibiting agent, the disease condition and the severity thereof, the identity and characteristics of the mammal in need thereof, which amount may be routinely determined by artisans.
  • Administration of the peptides of the invention and pharmaceutical compositions thereof may be performed according to any of the accepted modes of administration available to those skilled in the art.
  • suitable modes of administration include oral, nasal, parenteral, topical, transdermal, rectal, inhalation, injection and implantation.
  • compositions of the present invention may be administered in any suitable pharmaceutical form, including solid, semisolid, liquid, or lyopholized formulations, such as tablets, powders, capsules, suppositories, suspensions, liposomes, and aerosols.
  • compositions are known or may be routinely determined by those skilled in the art.
  • pharmaceutical preparations may be prepared following conventional techniques of the pharmaceutical chemist involving steps such as mixing, granulating, and compressing when necessary for tablet forms, or mixing, filling, and dissolving the ingredients as appropriate, to give the desired products for oral, parenteral, topical, intravaginal, intranasal, intrabronchial, intraocular, intraaural, and/or rectal administration.
  • compositions of the invention may also include suitable excipients, diluents, vehicles, and carriers, as well as other pharmaceutically active agents, depending upon the intended use.
  • Solid or liquid pharmaceutically acceptable carriers, diluents, vehicles, or excipients may be employed in the pharmaceutical compositions.
  • Illustrative solid carriers include starch, lactose, calcium sulfate dihydrate, terra alba, sucrose, talc, gelatin, pectin, acacia, magnesium stearate, and stearic acid.
  • Illustrative liquid carriers include syrup, peanut oil, olive oil, saline solution, and water.
  • the carrier or diluent may include a suitable prolonged-release material, such as glyceryl monostearate or glyceryl distearate, alone or with a wax.
  • a suitable prolonged-release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax.
  • the preparation may be in the form of a syrup, elixir, emulsion, soft gelatin capsule, sterile injectable liquid (e.g., solution), or a nonaqueous or aqueous liquid suspension.
  • peptides may be formulated with excipients such as cyclic lactones and cyclic ketones which enhance permeability of the skin or mucosa, while iontophoretic and piezoelectric devices may be used for transdermal delivery.
  • excipients such as cyclic lactones and cyclic ketones which enhance permeability of the skin or mucosa
  • iontophoretic and piezoelectric devices may be used for transdermal delivery.
  • Dispersal of peptides in liposomes, microvesicles, or micelles, or encapsulation in nanoparticles enables administration by a variety of routes, including transdermal, transmucosal, intranasal and intrabuccal.
  • Bioadhesive polymers formulated with peptides, or applied as coatings to standard encapsulation, may be used to retain a therapeutically effect dose within the mouth, throat and/or oesophagus where the peptide may be absorbed.
  • starch, gel-based lozenges that dissolve in the mouth may be used.
  • Coating of peptides with an acid-resistant vehicle, carrier or coating also facilitates oral absorption.
  • Lyophilisation, or spray drying, or dispersal with nano-particulate additives or GRAS Generally Recognised As Safe
  • excipients such as magnesium stearate, lecithin, hyaluronic acid, octyl phenoxypolyethoxyethanol, glycolic acid, lactic acid, citric acid, organic acids, amorphous glass-forming materials especially polyhydroxy compounds, or leucine enable intra-oral delivery via inhalation.
  • polyhydroxy compounds are sugars, such as sucrose, trehalose and lactose, or carbohydrate polymers, such as dextran, inulin and polyhydric alcohols (e.g. mannitol).
  • glass-forming materials include proteins such as albumin and hydrolysed gelatine, as well as polymers such as polyvinyl pyrrolidine) (PVP).
  • PVP polyvinyl pyrrolidine
  • Encapsulation of peptides within biodegradable, non-silicone-based, hydrophilic polymer matrices, foams and microspheres, or printing onto microchip devices enables implantation, or administration by injection, and slow release over time. Such administration may be site-directed. For example, the therapeutic dose may be administered directly to the liver.
  • suitable polymers include poly(lactic acid- co-glycolic acid), polyactide co-glycolide, poly(DL-lactide-co-glycolide, poly(DL-lactide), poly(DL-lactide-co-caprolactone), or polyvinyl pyrrolidine).
  • An example of a suitable hydrogel is the thermo-sensitive ReGel® injectable system. Below body temperature, ReGel® is an injectable liquid while at body temperature it forms a gel reservoir that slowly erodes and dissolves into known, safe, biodegradable polymers. The active drug is delivered over time as the biopolymers dissolve.
  • PEG polyethylene glycol
  • pegylation an alternative to PEGylation is polysialic acid.
  • a similar method of formulation is fusion of peptides to the Fc or CH region of human antibodies, such as IgGI and lgG4.
  • a dose of the pharmaceutical composition may contain at least a therapeutically effective amount of one or more peptides of the present invention and preferably is made up of one or more pharmaceutical dosage units.
  • the selected dose may be administered to a mammal, for example, a human, in need of treatment mediated by inhibition of HCV activity, by any known or suitable method of administering the dose, including topically, for example, as an ointment, cream or transdermal patches or devices; orally; rectally, for example, as a suppository; parenterally by injection or implant; intravenously; subcutaneously; intramuscularly; or continuously by intravaginal, intranasal, intrabronchial, intraaural, or intraocular infusion.
  • a dose that may be employed is from about 0.001 to about 1000 mg/kg body weight, or from about 0.1 to about 100 mg/kg body weight, or from about 1 to about 50 mg/kg body weight, or from about 0.1 to about 1 mg/kg body weight, with courses of treatment repeated at appropriate intervals.
  • the subject invention also includes isotopically-labelled peptides, which are identical to those recited in the peptides of the present invention, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature.
  • isotopes that can be incorporated into peptides of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine, such as 2 H, 3 H, 13 C, 14 C, 15 N, 18 O, 17 O, 31 P, 32 P, 35 S, 18 F, and 36 CI, respectively.
  • Peptides of the present invention and prodrugs thereof which contain the aforementioned isotopes and/or other isotopes of other atoms are within the scope of this invention.
  • Certain isotopically-labelled peptides of the present invention, for example those into which radioactive isotopes such as 3 H and 14 C are incorporated, are useful in drug and/or substrate tissue distribution assays. Tritiated, i.e., 3 H, and carbon-14, i.e., 14 C, isotopes are particularly preferred for their ease of preparation and detectability.
  • lsotopically labelled peptides of the present invention and prodrugs thereof can generally be prepared by substituting a readily available isotopically labelled reagent for a non-isotopically labelled reagent.
  • the active peptides may be applied as a sole therapy or may involve one or more other antiviral substances, for example those selected from, for example, HCV inhibitors such as interferon alphacon-1 , natural interferon, interferon beta-la, interferon omega, interferon gamma-1b, interleukin-10, BILN 2061 (serine protease), amantadine
  • HCV inhibitors such as interferon alphacon-1 , natural interferon, interferon beta-la, interferon omega, interferon gamma-1b, interleukin-10, BILN 2061 (serine protease), amantadine
  • viramidine thymozine alpha-1
  • Other antiviral substances include HIV inhibitors such as nelfinavir, delavirdine, indinavir, nevirapine, saquinavir, and tenofovir.
  • HIV inhibitors such as nelfinavir, delavirdine, indinavir, nevirapine, saquinavir, and tenofovir.
  • Such conjoint treatment may be achieved by way of the simultaneous, sequential or separate dosing of the individual components of the treatment.
  • Figure 1 E1 and E2 expression at the cell surface; cell surface levels of E1 and E2 were measured by FACS scan using either HCM-081-5 antibody (recognises E1) or AP33 antibody (recognises E2) on a HEK239T parental cell line that does not express E1 E2 ( Figure 1A) or on a clone, HEK293TcE1 E2TAT6 known to express E1 E2 at the cell surface ( Figure 1 B), under the following condition: Shaded histogram: cells were incubated with no antibody,
  • FIG. 1 Schematic principle of HCV cell-cell fusion assay;
  • the HCV fusion assay is based on either the transient or stable expression of E1 E2 and Tat in HEK293T cell line (or any other cell line easy to transfect). In this assay, E1 E2 envelope proteins need to be expressed at the cell surface. The corresponding cells are then mixed together with a permissive cell line (e.g. Huh7) expressing a reporter
  • HIV-1 LTR under the control of HIV-1 LTR.
  • an HIV- LTR-reporter stable cell line would have been selected for a minimal expression of the reporter in absence of the transactivator Tat and for an up-regulation in the presence of Tat. If the two cell lines fuse, Tat (expressed in HEK293T cell background) can reach and transactivate HIV LTR (expressed in the Huh7 cell background), resulting in the expression of the reporter;
  • FIG. 3 Correlation between E2 cell surface expression and fusion activity; HEK293T cells were transfected with E1 E2 (strain 1a). Thirty-six hours post- transfection, selection medium (zeocin) was added to the cells. Approximately six weeks later, clones grown from individual cells were selected and subjected to
  • E1E2 or a vector expressing chimeric E1 E2 envelop protein (pBudVSVGtmd- E1 E2, E1 E2 proteins with the authentic transmembrane domain of E1 E2 replaced with that of VSV-G protein).
  • the resulting cells were mixed respectively with Huh7 cells expressing HIV-1 LTR-luciferase.
  • the resulting cells were mixed with Huh7-LTR-luciferase and, two days later, luciferase expression was measured.
  • the data is plotted in a histogram showing that fusion activity is enhanced when the cells expressing E1 E2 are grown at low pH;
  • Figure 6 screening for cells permissive for fusing with HEK293TE1 E2 cell line;
  • HEK293T parental cell line expressing Tat or HEK293T cells expressing E1 E2 and Tat were mixed with one of the following cell line: Huh7, HepG2, Vero, HeLa, HEK293T, CHOK1 or MOLT-4 respectively.
  • Hek293T cell expressing E1 E2 and Tat were mixed with Huh7-LTR-luciferase cells together with two concentrations of AP33, a blocking antibody directed against E2 (Clayton et al, J. Gen. Virol. (2001) 82, 1877-1883). Forty-eight hours later, the level of luciferase was monitored in the first plate, while cytotoxicity was monitored in the second plate using a wst-1 kit;
  • Figure 9 Inhibition of HCV cell-cell fusion by peptide derived from E1 region; Overlapping peptides (25mers) covering the entire E1 region were tested in the cell-cell fusion assay of the invention.
  • Figure 9A shows a consensus sequence for E1. This consensus sequence was made using the following strains (HCV-BK, accession number M8335; H-1, accession number AB107944; H-77 accession number AF009606; HC-J2, accession number D10074; HC-J5, accession number D10075; HC-J7, accession number D10077; HC-J8, accession number D10988;
  • FIG. 9B is a histogram representing the % inhibition each peptide achieves in the cell-cell fusion assay using two different peptide concentrations
  • Figure 10 Inhibition of HCV cell-cell fusion by peptide derived from E2 region; Overlapping peptides (25mers) covering the entire E2 region were tested in the cell-cell fusion assay of the invention.
  • Figure 10A shows a consensus sequence for E2.
  • E1 and E2 include a carboxy-terminal signal sequence that retains the proteins in the endoplasmic reticulum. Previous attempts at developing an HCV fusion assay have deleted the signal sequence, thus enabling translocation of truncated E1 and E2 to the cell surface.
  • the present invention provides full-length E1 and E2 which have been shown to be expressed in their natural conformation on the surface of HEK293T cells.
  • HEK293T a HEK293Tcell line was maintained in Dulbecco Modified Eagles Medium
  • DMEM fetal calf serum
  • FCS foetal calf serum
  • FCS 1 mM sodium pyruvate
  • G418 G418
  • Cells were split 1 :5 - 1 :20 twice a week using Accutase (ITC, AT104) to avoid clumping of the cells.
  • HEK293TcE1 E2TAT6 HEK293T cells were stably transfected to express HCV E1 and
  • E2 glycoproteins and the HIV Tat transactivator by Zeocin selection (0.2mg/ml) (selection agent for E1E2 expression; Invitrogen 45-0430) and 10 ⁇ g/ml blasticidin (selection agent for Tat expression; Invitrogen 46-1120).
  • the E1E2 domain expressed comprises the last 60 amino acids of the core protein and the complete E1 and E2 sequences. The sequence was derived from a 1 B consensus strain and the expression vector used was pBud.
  • HEK293TcE1E2 cells were stably transfected to express the HIV Tat peptide by Blasticidin selection (10 ⁇ g/ml) using the pTAT plasmid.
  • Cells were maintained in DMEM and glutamax-l containing 10% FCS, 1mM sodium pyruvate, 0.4mg/ml G418, 0.2mg/ml zeocin and 10 ⁇ g/ml blasticidin. Cells were split 1 :5 - 1 :20 twice a week using Accutase to avoid clumping of the cells.
  • AP33 was provided by Arvind Patel and is a monoclonal antibody that specifically recognise the amino acid region 412 to 423 in E2 (Clayton et al, J. Virol. (2002) 76(15). 7672-7682).
  • HCM-081-5 monoclonal antibody that recognise HCV E1 (Austral biologicals, # HCM- 081-5). Secondary antibody: R-phycoerythrine labelled goat anti mouse monoclonal antibody (Sigma, P9287).
  • HEK293T and HEK293TcE1 E2 TAT6 cells were washed once with 5ml/flask PBS, then detached using trypsin and diluted to a final volume of 10ml with ice cold sterile PBS containing 0.1% BSA.
  • the cells were dispersed by repeated vigorous pipetting using a 10ml pipette and bulb to minimise clumping of the cells. 1ml aliquots of each suspension were counted using an automated cell counter (CEDEXTM) to determine cell density and viability.
  • the cells were re- suspended at 2.2 x 10 7 cells/ml in ice-cold sterile PBS containing 0.1% bovine serum albumen (BSA). Aliquots of 45 ⁇ l were transferred to 1.5ml eppendorf tubes and 5 ⁇ l of either AP33 or HCM-081-5 or PBS were added to the tubes and mixed. The tubes were left to incubate for 1hour on ice. The cells were then spun down (1 min, 5000rpm), washed twice in PBS containing 0.1% BSA and the pellet was re-suspended in 45 ⁇ l of PBS containing 0.1% BSA.
  • BSA bovine serum albumen
  • Figure 1 shows the result of the FACS analysis of E1 and E2 expression on the parental HEK293T cells and on HEK293TcE1 E2TAT6 clone cells respectively. Looking at Figure 1
  • the parental HEK293T cells show no difference in fluorescence between the cells incubated with no antibody (shaded histogram), with the secondary antibody only (pale grey line) or with the cells incubating with the primary and the secondary antibody (dark and pale grey lines, respectively), indicating that there is no expression of either E1 or E2 on surface of these cells.
  • El antibody or anti-E2 antibody show a shift in fluorescence when compared to incubation in the absence of any antibody or in the presence of the secondary antibody ( Figure 1b).
  • the cell-cell fusion assay of the present invention requires the combination of a cell expressing viral envelope protein on its cell surfaces with a second cell expressing at least one of the human receptor that mediate viral infection.
  • the assay mimics viral infection in a non-infectious environment.
  • a reporter assay for fusion is achieved by expressing a transactivator protein (HIV-1 Tat) in one cell line whilst the other cell line contains a reporter system (HIV-1 LTR) under the control of HIV-1 Tat.
  • HEK293TcE1 E2 TAT6 HEK293T cells were stably transfected to express HCV E1 and E2 proteins and the HIV Tat transactivator by Zeocin selection (0.2mg/ml) as described in Example 1.
  • Huh7tl_TR-luc Huh7 cells were stably transfected to contain a luciferase reporter under the control of a truncated version of the HIV LTR using G418 selection (0.8mg/ml). Cells were maintained in Modified Eagles Medium (MEM; Gibco 21090-022) containing 10% FCS, 2mM L-glutamine (Sigma G7531), 1mM sodium pyruvate, 1x Non Essential Amino
  • NEAA Human 7tLTR-luc growth medium without G418, and 1% penicillin-streptomycin (Gibco 15140-122).
  • HEK293TcE1 E2 TAT6 cells and Huh7tLTR-luc cells were washed once with 5ml/flask PBS, then detached using trypsin and diluted to a final volume of 10ml with fusion medium. Cells were then dispersed by repeated vigorous pipetting using a 10ml pipette and bulb to minimise clumping of the Huh7tLTR-luc cells. 1ml aliquots of each suspension were counted using an automated cell counter (CEDEXTM) to determine cell density and viability.
  • CEDEXTM automated cell counter
  • Huh7tLTR-luc cells and HEK293TcE1 E2 TAT6 cells were mixed in fusion medium to give a suspension containing 3 x 10 5 Huh7tLTR-luc and 3 x 10 5 HEK293TcE1 E2 TAT6 per ml of medium, i.e. a total cell density of 6 x 10 5 viable cells/ml.
  • Cell suspensions were added to 96-well flat-bottom tissue culture plates (Corning Costar 3585) to give 100 ⁇ l/well containing 6 x 10 4 total cells/well (3 x 10 4 Huh7tLTR-luc + 3 x 10 4 HEK293TcE1 E2 TAT6). This cell density provided a near-confluent layer of cells. The plates were then incubated for 24 to 48 hours at 37°C, 5% CO 2 .
  • the fusion medium removed was from the wells (by careful inversion of plate and draining on gauze squares) and the cells washed once with 100 ⁇ l/well PBS.
  • the PBS was drained and 25 ⁇ l/well Promega passive lysis buffer (Cat # E194A) added.
  • the plate was incubated for ⁇ 10mins at 37 0 C before being placed on a plate shaker (e.g. Heidolph Titramax 100, speed setting 4) for ⁇ 30sec to detach cell debris from the plate. 20 ⁇ l aliquots were then transferred from each well to wells on a white 96-well plate (Packard picoplate-96 Cat # 6005162). 20 ⁇ l passive lysis buffer was then added to empty wells for determination of "reagent background" (20 ⁇ l/well lysis buffer +
  • luciferase substrate 100 ⁇ l/well luciferase substrate. 100 ⁇ l Promega luciferase substrate (Cat # E1501) was added to each well and the plate read immediately on a Victor 2TM plate reader using the manufacturer's luminescence protocol. The reagent background reading was subtracted from all other readings.
  • the assay was also carried out using transient transfection of HEK293T cells.
  • the protocol was as set out above with the exception that preparation of the cells was begun two days before they were required for experiments.
  • day -2 a T75 flask was inoculated with -3x10 6 HEK293T cells in 15ml medium without antibiotics (no G418, zeocin or penicillin/streptomycin).
  • day -1 the cells were transfected by adding 24 ⁇ g plasmid EV279 (pcDNA3.1 containing the HIV tat exon 1 ; provided by Eric Verdin) DNA to 1.5ml Gibco Optimem-1 reduced serum medium (Cat # 11058-021).
  • TAT cells that were allowed to grow in over-confluence gave a higher signal than usual (data not show). These results were investigated further by incubating these cells in either their normal media (at ph 7.0) or at pH 5.5.
  • HCV envelope proteins are believed to belong to the class Il of fusion envelope protein which need to be internalised in an acidic environment before they can drive the fusion of the viral and the cellular membrane (Jardetzky and Lamb, Nature (2004) 427, 307-308). Cultivating the cells for 2 days at pH 5.5 is believed to mimic this acidification step needed for HCV envelope to fuse and therefore gives a higher fusion activity compared to cells grown at pH 7.0.
  • Huh7 cells have been shown to be permissive for HCV infection and to express the putative receptors CD81, LDL-R and SR-BI. Although the HCV cell-cell fusion of the present invention uses Huh7 cell line, alternative cell lines have been investigated ( Figure 6).
  • Huh7 Cells were maintained in MEM containing 10% FCS, 2mM L-glutamine, 1mM sodium pyruvate and 1x NEAA. Cells were split 1 :3 — 1 :10 twice a week using Accutase
  • HepG2 Cells were maintained in MEM containing 10% FCS, 2mM L-glutamine, 1mM sodium pyruvate and 1x NEAA. Cells were split 1 :3 - 1 :10 twice a week using Accutase
  • Vero Cells were maintained in MEM containing 10% FCS, 2mM L-glutamine, 1mM sodium pyruvate and 1x NEAA. Cells were split 1 :5 - 1 :20 twice a week using Accutase
  • HeLa Cells were maintained in DMEM and glutamax containing 10% FCS. Cells were split 1 :5- 1 :20 twice a week using Accutase Il to avoid clumping of the cells.
  • HEK293T Cells were maintained in DMEM and glutamax-l containing 10% FCS, 1mM sodium pyruvate. Cells were split 1 :5 - 1 :20 twice a week using Accutase to avoid clumping of the cells.
  • CHOK1 Cells were maintained in DMEM and glutamax containing 10% FCS. Cells were split 1 :5 - 1 :20 twice a week using Accutase Il to avoid clumping of the cells.
  • MOLT-4 Cells were maintained in RPMI 1640 (Gibco 31870-025) containing 10% FCS,
  • HEK293TcE1 E2 TAT6 HEK293T cells were stably transfected to express HCV E1 and
  • E2 proteins and the HIV Tat transactivator by Zeocin selection (0.2mg/ml) as described in Example 1.
  • Each of the cell lines to be tested (i.e. Huh7, HepG2, Vera, HeLa, HEK293T, CHOK1 and MOLT-4) was mixed respectively with HEK293TcE1 E2 TAT6 cells in fusion medium to give a suspension containing 3 x 10 5 of cells to be tested and 3 x 10 5 HEK293TcE1 E2 TAT6 per ml of medium, i.e. a total cell density of 6 x 10 5 viable cells/ml.
  • Cell suspensions were added to 96-well flat-bottom tissue culture plates (Corning Costar 3585) to give 100 ⁇ l/well containing 6 x 10 4 total cells/well (3 x 10 4 cells to be tested + 3 x 10 4 HEK293TcE1 E2 TAT6). This cell density provided a near-confluent layer of cells. The plates were then incubated for 24 to 48 hours at 37°C, 5% CO 2 .
  • the fusion medium removed was from the wells (by careful inversion of plate and draining on gauze squares) and the cells washed once with 100 ⁇ l/well PBS.
  • the PBS was drained and 25 ⁇ l/well Promega passive lysis buffer (Cat # E194A) added.
  • the plate was incubated for ⁇ 10mins at 37 0 C before being placed on a plate shaker (e.g. Heidolph Titramax 100, speed setting 4) for ⁇ 30sec to detach cell debris from the plate. 20 ⁇ l aliquots were then transferred from each well to wells on a white 96-well plate (Packard picoplate-96 Cat # 6005162).
  • the present experiment confirms that the other well- characterised human hepatocellular carcinoma cell line, HepG2, is not permissive for HCV entry as it lacks CD81 receptor (Bartosch et al, J. Biol. Chem. (2003) 278(43). 41624-30).
  • the human lymphoblastic leukaemia Molt-4 cell has also been shown to be permissive for HCV entry, consistent with observations made by Laggings et al. (J. Virol. (1998) 72(5), 3539-3546) but different from those made by Bartosch et al (ibid).
  • the present experiment also shows
  • Vero, HeLa, Hek293T and CHOK1 cell lines to be non-permissive to HCV entry in the assay of the present invention.
  • Example 4 Development of the cell-cell fusion assay has enabled high-throughput screening for inhibitors of HCV virus entry.
  • the assay has been developed in a format suitable for use with 384-well plates or more.
  • Assay plates were prepared with compound by adding 10 ⁇ l/well compound to white opaque tissue culture treated plates (Greiner 781080). On receipt of cells at 2x10 6 cells/ml, the same volume of 2% FCS HCV fusion media (see example 2) was added to each cell line to obtain solutions at 1x10 6 cells/ml. 10//I of each cell line was added per well. Plates were lidded and incubated at 37°C and 5% CO2 in a humidified incubator for 48 hours. Bright-Glo Luciferase reagent (Promega E2620) was prepared as described by the manufacturer and, after 48 hours, 10//I was added to each well. The plates were shaken for 3 minutes before being read on a LeadseekerTM plate reader. The data were exported into Microsoft ® Excel 97 SR-2 (k) and the %inhibition calculated and plotted against the compound concentration. These percent inhibition values were used to obtain IC50 values.
  • SR-BI scavenger receptor class B type I
  • BLT-1 inhibited HCV cell-cell fusion in a dose-dependent manner and displayed an IC50 of 6.9 ⁇ M. No significant cell toxicity was observed at the IC 50 concentration.
  • the development of the cell-cell fusion assay has enabled the screening of peptides libraries to identify domains in E1 and E2 that are essential for virus entry and potentially useful as therapeutics.
  • overlapping (25mers) covering the E1 E2 sequence were tested in the cell-cell fusion assay of the present invention at concentrations of 10 ⁇ g/ml and 100 ⁇ g/ml.
  • SEQ ID NO:16 Peptide derived from SEQ ID NO:8 residues 158 to 182
  • SEQ ID NO:30 Peptide derived from SEQ ID NO:19 residues 183 to 207
  • SEQ ID NO:36 Peptide derived from SEQ ID NO:19 residues 291 to 315

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Cell Biology (AREA)
  • Communicable Diseases (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un procédé de dosage et des composés destinés au traitement d'une infection par le virus de l'hépatite C. Le procédé de dosage selon l'invention est notamment destiné à déterminer si un agent est capable de moduler une infection par le virus de l'hépatite C (HCV). Ledit procédé consiste (a) à amener l'agent en contact avec (i) une première lignée cellulaire exprimant des protéines E1 et E2 sur la surface de la cellule et (ii) une deuxième lignée cellulaire permissive ; et (b) à déterminer si l'agent inhibe la fusion entre la première et la deuxième lignée cellulaire, les protéines E1 et E2 étant des protéines de longueur totale.
PCT/IB2005/003070 2004-09-24 2005-09-19 Procede WO2006033012A2 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GB0421360.9 2004-09-24
GB0421360A GB0421360D0 (en) 2004-09-24 2004-09-24 Method
US62237704P 2004-10-26 2004-10-26
US60/622,377 2004-10-26

Publications (2)

Publication Number Publication Date
WO2006033012A2 true WO2006033012A2 (fr) 2006-03-30
WO2006033012A3 WO2006033012A3 (fr) 2006-08-24

Family

ID=36090386

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2005/003070 WO2006033012A2 (fr) 2004-09-24 2005-09-19 Procede

Country Status (1)

Country Link
WO (1) WO2006033012A2 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009030872A1 (fr) * 2007-09-07 2009-03-12 Mats Axel Atterdag Persson Matériaux et procédés pour le traitement de l'hépatite c
US20130129671A1 (en) * 2011-05-27 2013-05-23 Bristol-Myers Squibb Company Tripeptides Incorporating Deuterium as Inhibitors of Hepatitis C Virus
CN105330730A (zh) * 2014-07-29 2016-02-17 中国科学院上海巴斯德研究所 一种丙型肝炎病毒重组蛋白的制备及其应用
CN114062070A (zh) * 2021-09-27 2022-02-18 苏州德运康瑞生物科技有限公司 用于制备循环肿瘤细胞质控品的试剂盒及循环肿瘤细胞质控品的制备方法

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0947525A1 (fr) * 1998-03-27 1999-10-06 Innogenetics N.V. Epitopes de protéines d'enveloppe virale et anticorps spécifiques pour cet epitope: usage pour le diagnostic de l'antigène de virus HCV dans le tissu hÔte
AU765940B2 (en) * 1998-06-24 2003-10-02 Innogenetics N.V. Particles of HCV envelope proteins: use for vaccination
US20020160936A1 (en) * 1999-09-29 2002-10-31 Howard J. Worman Hcv e2 protein binding agents for treatment of hepatitis c virus infection
EP1355947A2 (fr) * 2000-12-01 2003-10-29 The Government of the United States of America, as Represented by the Secretary, Department of Health and Human Services Anticorps monoclonaux specifiques a la glycoproteine e2 du virus de l'hepatite c et leurs utilisations dans le diagnostic, le traitement et la prevention de l'hepatite c
GB0114629D0 (en) * 2001-06-15 2001-08-08 Pfizer Ltd Method

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009030872A1 (fr) * 2007-09-07 2009-03-12 Mats Axel Atterdag Persson Matériaux et procédés pour le traitement de l'hépatite c
US20130129671A1 (en) * 2011-05-27 2013-05-23 Bristol-Myers Squibb Company Tripeptides Incorporating Deuterium as Inhibitors of Hepatitis C Virus
US20140079664A1 (en) * 2011-05-27 2014-03-20 Bristol-Myers Squibb Company Tripeptides Incorporating Deuterium as Inhibitors of Hepatitis C Virus
CN105330730A (zh) * 2014-07-29 2016-02-17 中国科学院上海巴斯德研究所 一种丙型肝炎病毒重组蛋白的制备及其应用
CN114062070A (zh) * 2021-09-27 2022-02-18 苏州德运康瑞生物科技有限公司 用于制备循环肿瘤细胞质控品的试剂盒及循环肿瘤细胞质控品的制备方法

Also Published As

Publication number Publication date
WO2006033012A3 (fr) 2006-08-24

Similar Documents

Publication Publication Date Title
Yu et al. A peptide-based viral inactivator inhibits Zika virus infection in pregnant mice and fetuses
Berger et al. Daclatasvir-like inhibitors of NS5A block early biogenesis of hepatitis C virus–induced membranous replication factories, independent of RNA replication
Lee et al. Apolipoprotein E likely contributes to a maturation step of infectious hepatitis C virus particles and interacts with viral envelope glycoproteins
Lavillette et al. Characterization of fusion determinants points to the involvement of three discrete regions of both E1 and E2 glycoproteins in the membrane fusion process of hepatitis C virus
Chevaliez et al. HCV genome and life cycle
Lundin et al. Topology of the membrane-associated hepatitis C virus protein NS4B
Meunier et al. Apolipoprotein c1 association with hepatitis C virus
Lindenbach et al. The ins and outs of hepatitis C virus entry and assembly
Friesland et al. Sigma-1 receptor regulates early steps of viral RNA replication at the onset of hepatitis C virus infection
US20090081636A1 (en) Pharmaceutical compositions for and methods of inhibiting HCV replication
Zimmerman et al. Entry receptors—the gateway to alphavirus infection
Khaliq et al. Hepatitis C virus p7: molecular function and importance in hepatitis C virus life cycle and potential antiviral target
Li et al. Mutagenesis of the fusion peptide-like domain of hepatitis C virus E1 glycoprotein: involvement in cell fusion and virus entry
Pan et al. New therapeutic opportunities for hepatitis C based on small RNA
von Hahn et al. Hepatocytes that express variants of cyclophilin A are resistant to HCV infection and replication
Tong et al. Functional analysis of hepatitis C virus (HCV) envelope protein E1 using a trans-complementation system reveals a dual role of a putative fusion peptide of E1 in both HCV entry and morphogenesis
Ndjomou et al. Fluorescent primuline derivatives inhibit hepatitis C virus NS3-catalyzed RNA unwinding, peptide hydrolysis and viral replicase formation
Guo et al. Identification of a new region of SARS-CoV S protein critical for viral entry
Ma-Lauer et al. Oxysterole-binding protein targeted by SARS-CoV-2 viral proteins regulates coronavirus replication
Sornjai et al. The interaction of GRP78 and Zika virus E and NS1 proteins occurs in a chaperone-client manner
JP2012504647A (ja) 新規なｈｃｖ侵入因子、オクルディン
WO2006033012A2 (fr) Procede
Lee Interaction of hepatitis C virus core protein with Janus kinase is required for efficient production of infectious viruses
Castelain et al. Hepatitis C Virus p7 membrane protein quasispecies variability in chronically infected patients treated with interferon and ribavirin, with or without amantadine
Izumida et al. The roles of HCV core protein and its binding host factor in virus assembly and release

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KM KP KR KZ LC LK LR LS LT LU LV LY MA MD MG MK MN MW MX MZ NA NG NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SM SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU LV MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

DPEN Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed from 20040101)
NENP Non-entry into the national phase

Ref country code: DE

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载