Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
  • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/20—Antivirals for DNA viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/20—Antivirals for DNA viruses
  • A61P31/22—Antivirals for DNA viruses for herpes viruses
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/10—Tetrapeptides
  • C07K5/1002—Tetrapeptides with the first amino acid being neutral
  • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
  • C07K5/1008—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/10—Tetrapeptides
  • C07K5/1019—Tetrapeptides with the first amino acid being basic
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/16011—Herpesviridae
  • C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
  • C12N2710/16722—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2730/00—Reverse transcribing DNA viruses
  • C12N2730/00011—Details
  • C12N2730/10011—Hepadnaviridae
  • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
  • C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
  • C12N2760/00011—Details
  • C12N2760/16011—Orthomyxoviridae
  • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
  • C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• • 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
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
  • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
  • G01N2333/4701—Details
  • G01N2333/4718—Lipocortins

Definitions

• the present invention relates to the finding that 1,4-benzodiazepines and 1,4- benzothiazepines derivatized with a peptide chain inhibit binding of HBsAg to annexin V.
• the present invention concerns such molecules and their use to treat infections with hepatitis B virus and hepatitis delta virus, or any other disease in which a protein interaction with annexin family members is involved.
• Hepatitis B virus belongs to the Hepadnaviridae, which are characterised by a significant hepatotropism and species specificity.
• Hepatitis delta virus represents a naturally occurring subviral satellite of HBV (Rizetto et al, 1986). HBV causes major medical problems, such as chronic liver disease and hepatocellular carcinoma (Schroder and Zentgraf, 1990). HDV superinfection is usually more severe compared to HBV infection solely. It is estimated that there are 300 million human carriers of the virus worldwide, while in the US only 70,000 carriers are coinfected with HDV.
• Genotypes A to F of HBV are designated based on this sequence divergence (for review, see Magnius and Norder, 1995).
• the HBV envelope consists of three related glycoproteins, termed hepatitis B surface antigens (HBsAg), which are the product of the S gene: 1) the "small" transmembrane protein, also termed major protein or small S-protein, composed of 226 amino acids (aa), 2) the "middle” protein which comprises the small S-protein and 55 additional amino acids at the N-terminus corresponding to the pre-S2 region of the S gene, and 3) the "large” protein composed of 389 or 400 amino acids corresponding to the following regions: S + pre-S2 + pre-Sl (108-119 N- terminal aa) (Heerman et al., 1984; Robinson et al., 1987).
• the envelope of HDV is also entirely derived from HBV and consists predominantly of small HBsAg, 5-10% of middle HBsAg and no or less then 1% of large HBsAg (Bonino et al., 1986).
• Influenza viruses cause recurrent epidemics and even global pandemics. During these outbreaks absenteeism from work leads to high economic losses and is accompanied with increases of hospitalisation and even death due to respiratory diseases (for review Cox and Fukuda, 1998).
• Cytomegalovirus is a member of the herpes virus family and is an important pathogen in immunocompromised individuals such as AIDS patients.
• a protein referred to as glycoprotein B is present within the envelope of herpes viruses . This protein has been shown to be essential for infectivity including host cell entry and cell-to-cell spread (Cai et al., 1988).
• Annexin V (also termed endonexin II, placental anticoagulant protein, PP4 or lipocortin V) is a member of the family of structurally related Ca 2+ -dependent phospholipid-binding proteins, known as annexins, which have molecular weights between 32 and 67 kDa (Klee, 1988; Zaks and Creutz, 1990). Annexin V is found in various tissues such as liver, spleen, lung, intestine and placenta (Walker et al., 1990).
• the protein has been described to bind, in a Ca 2+ -dependent manner, to placental membranes (Haigler et al., 1987) and to inhibit blood coagulation (Grundmann et al., 1988) and phospholipase A2 activity in vitro (Pepinsky et al., 1988).
• Other investigators have demonstrated that annexin V behaves like an integral membrane protein and forms calcium- selective cation channels (Rojas et al., 1990; Bianchi et al., 1992).
• 1,4-benzodiazepines Hofmann et al., 1998) and 1,4- benzothiazepines (Kaneko et al., 1997a, b) can bind to annexin V.
• Annexin II is related to annexin V and also binds 1,4-benzodiazepines (Hofmann et al., 1998).
• Annexins also display a large variety of binding to non-viral proteins such as collagen, tenascin, actin, synapsin, calspectin, insulin receptor, plasminogen,... (for review see Sheldon and Chen, 1996).
• annexin V present on human liver plasma membranes, specifically binds to "small" HBsAg in a Ca 2+ -dependent manner (Hertogs et al., 1993; WO 94/01554).
• the receptor-ligand relationship between HBsAg and annexin V is further supported by the observation that rabbits, immunised with native human liver annexin V or recombinant annexin V, or chickens, immunised with F(ab') 2 -fragments of rabbit anti-annexin V IgG, spontaneously develop anti-idiotypic antibodies (Ab2) which specifically recognise HBsAg (Hertogs et al., 1994).
• HDV particles are binding to annexin V via the HBsAg containing envelope of HBV (de Bruin et al., 1994).
• the mapping of the sites on HBsAg which are involved in annexin V binding are described in WO 97/07268. These sites reside within the region composed of amino acids 100 to 160 of "small" HBsAg, which has been predicted to be located on the outer surface of the virus (for review, see Berting et al., 1995).
• HBV and/or HDV infection as well as other viral infections such as influenza and cytomegalovirus infections, are still a major health problem.
• the development of new drugs interfering with the life cycle of HBV and/or HDV or of these other viruses, is therefore still a major need.
• Compounds that bind annexin in such a way that the binding of HBV and/or HDV or of these other viruses to annexin is inhibited could be good candidates for interfering with the life cycle of these viruses and consequently, for the use as a drug to treat viral infections. Therefore, there is an urgent need to provide molecules that specifically interact with annexin, simultaneously inhibiting the interaction of HBV and/or HDV or of these other viruses with annexin.
• the present invention relates to 1,4-benzodiazepines and 1,4-benzothiazepines derivatized with one or more peptides.
• the present invention relates to 1,4-benzodiazepines and 1,4- benzothiazepines derivatized with one or more peptides such that the derivative inhibits the interaction between annexin and certain annexin binding proteins.
• 1,4-Benzodiazepines and 1,4-benzothiazepines which do bind annexin V, a molecule involved in the viral entry of HBV, do not inhibit the binding of HBV to annexin V.
• the present invention is based on the surprising finding that upon derivation of these compounds with a peptide ligand, the interaction between annexin V and the HBsAg of HBV can be inhibited.
• These new derivatives of 1,4-benzodiazepines and 1,4-benzothiazepines are therefore a new lead in the discovery of drugs interfering with the life cycle of HBV and/or HDV and other viruses binding to annexins in general, or more specifically, to annexin V. In general these molecules may interfere with any interaction of an annexin with another protein.
• any of these interactions may be targeted in a specific way.
• annexin refers to any member of the family of structurally related Ca 2+ -dependent phospholipid-binding proteins, known as annexins, which have molecular weights between 32 and 67 kDa (Klee, 1988; Zaks and Creutz, 1990). More specifically, the annexin can be annexin V shown to bind to the HBsAg of HBV and to the influenza virus, or annexin II known to bind to glycoprotein B of cytomegalovirus. But the annexins referred to in the present invention are not restricted to these annexins, but can also be any other annexin that binds with an annexin binding protein.
• the 1,4-benzodiazepine or 1,4-benzothiazepine may be any modification known in the art such as the ones mentioned in WO 94/11360 and WO 92/12148.
• the 1,4- benzodiazepine or 1,4-benzothiazepine has a structure as shown in formula I (figure 1), wherein:
• - benzothiazepines have a sulphur atom (X represents S); X may also be SO n with
• - benzodiazepines have a nitrogen atom (X represents N) which allows additional modifications with a peptide as defined in the claims as "a peptide containing an annexin binding epitope of an annexin binding protein" or part thereof, linked via the C-terminus of the peptide or linked via the N-terminus of the peptide after additional modification of position 1 with carboxymethyl (- CH 2 COOH).
• modification can be H, alkyl, phenyl, -COZ in which Z stands for H, alkyl, phenyl or substituted phenyl;
• positions 1, 2, 3, 4, 5 may form double bonds with adjacent positions; if this is the case the side chains R 2 , R ⁇ , R5 or R 7 are non-existing;
• Ri and/or R 2 is amine, this may be further substituted with a peptide defined in the claims as "a peptide containing an annexin binding epitope of an annexin binding protein" or part thereof, either directly linked via the C-terminus of the peptide or linked to the N-terminus of the peptide via a linker such as glutaraldehyde or succinicanhydride;
• R 3 and/or R 4 may represent at position 3 and/or 3' H, amine or alkyl.
• R 3 and/or R» is amine, this may be further substituted with a peptide defined in the claims as "a peptide containing an annexin binding epitope of an annexin binding protein" or part thereof, either directly linked via the C-terminus of the peptide or linked to the N-terminus of the peptide via a linker such as glutaraldehyde or succinicanhydride;
• R 5 may represent at position 4 a side chain defined in the claims as "a peptide containing an annexin binding epitope of an annexin binding protein", or part thereof, either directly linked via the C-terminus of the peptide or linked via the N-terminus of the peptide after additional modification of position 4 with carboxymethyl (-CH 2 COOH).
• R 5 may represent H, alkyl, CO-R ⁇ 2 or 3-(l-(4-benzyl)piperidinyl)propionyl);
• R 7 represent H or alkyl or phenyl, possibly further substituted with alkyl, cyano, halo, nitro, alkylalkoxy, alkanoyl, carboxy, alkanoylalkoxy, carbamoyl;
• R 8 , R9, Rio and/or R u represent H, alkyl, cyano, halo, nitro, alkylalkoxy, alkanoyl, carboxy, alkanoylalkoxy, carbamoyl;
• R ⁇ 2 represents H or alkyl or phenyl possibly further substituted with alkyl, cyano, halo, nitro, alkylalkoxy, alkanoyl, carboxy, alkanoylalkoxy, carbamoyl.
• the 1,4-benzodiazepine or 1,4-benzothiazepine used for the preparation of a molecule of the present invention can also be any pharmaceutically acceptable salt of this formula.
• the 1,4 benzodiazepine used for the preparation of a molecule of the present invention may be any derivative known in the art such as but not limited to alprazolam, bromazepam, chloordiazepoxide, clobazam, clonazepam, clorazepate, diazepam, fludiazepam, flunitrazepam, flurazepam, lorazepam, nitrazepam, oxazepam, temazepam, triazolam, BDA 250, BDA 452, BDA 753 (see also figure 6 and Hoftman et al., 1998).
• the compound of the present invention is not BDA753, a compound according to formula II (FR2479818; figure 2) or a compound according to formula III (Nachman et al., 1998; figure 3).
• the 1,4 benzothiazepine may be any known derivative known in the art such as but not limited to K-201 (Kaneko et al., 1997a).
• these 1,4-benzodiazepine or 1,4-benzothiazepine are derivatized with a polypeptide that can be linked at positions 2, 2', 3 and/or 3' (in formula I) after modification of said positions with an amine (two peptides may be linked at the same position).
• the peptide can be linked to the amine, either directly via the C-terminus of the peptide, or the peptide can be linked via the N-terminus of the peptide by use of a linker.
• the linker can be any linker that allows linking of two amine residues.
• the linker is a dialdehyde such as glutaraldehyde or an anhydride such as succinicanhydride.
• Other possible linkers can also supplied by Pierce (Rockford, IL, USA). Examples of the basic reaction for linking the peptide to the 1,4-benzodiazepine by use of a linker are shown in figures 9 and 10.
• the polypeptide can also be linked to positions 1 and/or 4 (in formula I).
• the peptide is linked either via the C-terminus of the peptide or the peptide is linked via the N- terminus after additional modification of respectively position 1 and/or 4 with carboxymethyl (- CH 2 COOH).
• the peptide is linked to position 1 and/or 3 of 1,4- benzodiazepine substituted with a phenyl at position 5.
• One peptide, R can be linked via its C- terminal to position 3 of said 1,4-benzodiazepine after modification of position 3 with amine, or one peptide, R', can be linked via its N-terminal to position 1 of said 1,4-benzodiazepine after modification of position 1 with carboxymethyl, or two peptides, R and R', are linked to respectively positions 3 and 1 of said 1,4-benzodiazepine.
• the present invention relates to said 1,4-benzodiazepine derivatized with one or two peptides, R and/or R', such that R, R' and/or R + R' represent a peptide containing an annexin binding epitope of an annexin binding protein or part thereof.
• the 1,4-benzodiazepine then has a structure as shown in formula IV (figure 4), wherein:
• R, R' and/or R + R' constitute a peptide containing an annexin binding epitope of an annexin binding protein or part thereof.
• position 1 is methylated or carboxymethylated.
• R' is a peptide, position 3 contains amine;
• * X represents the N-terminus of the peptide which may be modified with -COCH 3 ;
• Y represents the C-terminus of the peptide which may be modified with -NH 2 or -Lysine- Biotine.
• a building block that can be used for the synthesis of said 1,4-benzodiazepine derivative by Fmoc based peptide synthesis can be obtained from Neosystem (Strasbourg, France).
• the peptide, R is linked via its N-terminal to position 3 of said 1,4-benzodiazepine (substituted with phenyl at position 5), by use of a linker as described above.
• the nitrogen atom at position 1 (formula I) is methylated or carboxymethylated.
• the present invention relates to a compound according to formula V derivatized with a peptide, R, containing an annexin binding epitope of an annexin binding protein or part thereof.
• the polypeptide linked to the 1,4-benzodiazepine or 1,4-benzothiazepine can be any polypeptide that comprises an annexin binding epitope derived from a protein known to bind annexin.
• annexin binding protein refers to any protein that is able to form a complex with annexin.
• annexin binding epitope refers to that domain of the annexin binding protein that specifically interacts with annexin to form the annexin binding protein - annexin complex.
• the "annexin binding epitope” comprises at least 2, preferably 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35 or more amino acids of the annexin binding protein. Epitopes can be determined by any of the techniques known in the art or may be predicted by a variety of computer prediction models known in the art.
• polypeptide or “peptide” are used interchangeable and refer to a polymer of amino acids which is equal or similar to a part of the protein where it is derived from.
• the polypeptide can consist of any number of amino acids, preferably the polypeptide consists of less than 13 amino acids, preferably of 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acids such that an annexin binding epitope is contained within the polypeptide.
• the compound of the invention has a structure according to formula IV, it is also possible that the peptide R or R' consists of only 1 amino acids and/or that the annexin binding epitope is contained within the polypeptide that represents R+R'.
• polypeptides which are derived from the annexin binding protein and which form a binding region to an annexin might be part of a conformational binding region and should thus not be composed of a contiguous amino acid sequence of the annexin binding protein.
• polypeptide can be prepared by any method known in the art such as classical chemical synthesis, as described by Houbenweyl (1974) and Atherton and Shepard (1989), or by means of recombinant DNA techniques as described by Maniatis et al. (1982).
• polypeptide does not refer to, nor does it exclude, post-translational modifications of the polypeptide such as glycosylation, acetylation, phosphorylation, modifications with fatty acids and the like.
• polypeptides containing one or more analogues of an amino acid including unnatural amino acids or D-isoforms
• polypeptides with substituted linkages for example, peptide variants of the HBsAg protein of HBV, corresponding to the genotypes A to F of HBV, as indicated above
• polypeptides containing disulphide bounds between cysteine residues for example, reverso-inverso polypeptides (such as described by Guichard et al., 1994), as well as other modifications known in the art.
• polypeptide linked to the 1,4-benzodiazepine or 1,4- benzothiazepine is derived from a viral protein that is known to bind annexin.
• polypeptide derived from a viral protein refers to a polymer of amino acids which is equal or similar to a part of the viral protein where it is derived from and which contains an annexin binding epitope of the viral protein.
• the polypeptide linked to the 1,4-benzodiazepine or 1,4- benzothiazepine is derived from the HBsAg protein of HBV, which is a known antigen (Heerman et al., 1984; Robinson et al., 1987).
• the term "polypeptide derived from the HBsAg protein of HBV” refers to a polypeptide having an amino acid sequence which is equal or similar to a part of the amino acid sequence of "small" HBsAg and which contains an annexin binding epitope of HBsAg. It should be clear that the "polypeptides derived from the HBsAg protein of HBV” can be derived from any genotype of HBV (genotype A to F, Magnius and Norder 1995).
• the polypeptide linked to the 1,4- benzodiazepine or 1,4-benzothiazepine is derived from glycoprotein B of the cytomegalovirus.
• glycoprotein B refers to a polymer of amino acids which is equal or similar to a part of glycoprotein B of the cytomegalovirus and which contains an annexin binding epitope of glycoprotein B.
• the polypeptide linked to the 1,4- benzodiazepine or 1,4-benzothiazepine is derived from a protein from any influenza strain on the condition that this influenza protein binds annexin V.
• polypeptide derived from a protein from an influenza strain refers to a polymer of amino acids which is equal or similar to a part of the protein from the influenza strain and which contains an annexin binding epitope of the influenza protein.
• the present invention aims at providing a 1,4-benzodiazepine or 1,4- benzothiazepine with a peptide derivation comprising any combination of peptides as defined above or derived of any mutated strain of HBV and/or HDV, cytomegalovirus or influenza virus.
• the present invention provides a 1,4-benzodiazepine or 1,4- benzothiazepine molecule derivatized with a peptide that comprises less than 13 amino acids containing an annexin binding epitope of one of the following regions of the HBV surface antigen:
• polypeptide linked to the 1,4-benzodiazepine or 1,4-benzothiazepine derivative of the present invention relates to polypeptides comprising 2-10 amino acids derived from the sequence spanning amino acid positions 122 to 131 of the HBsAg of HBV, shown above as SEQ ID NO 1.
• peptides comprising or consisting of the following amino acid (aa) sequences: aa 122-123, aa 123-124, aa 124-125, aa 125-126, aa 126-127, aa 127-128, aa 128-129, aa 129-130, aa 130-131, aa 122-124, aa 123-125, aa 124-126, aa 125-127, aa 126- 128, aa 127-129, aa 128-130, aa 129-131, aa 122-125, aa 123-126, aa 124-127, aa 125-128, aa 126-129, aa 127-130, aa 128-131, aa 122-126, aa 123-127, aa 124-128, aa 125-129, aa 126-130, aa 127-130,
• polypeptide linked to the 1,4-benzodiazepine or 1,4-benzothiazepine derivative of the present invention specifically relates to the polypeptides comprising 2 to 12 amino acids derived from the sequence spanning amino acid positions 158 to 169 of the HBsAg of HBV, shown above as SEQ ID NO 2.
• peptides comprising or consisting of the following amino acid (aa) sequences: aa 158-159, aa 159-160, aa 160-161, aa 161-162, aa 162-163, aa 163-164, aa 164-165, aa 165-166, aa 166-167, aa 167-168, aa 168-169, aa 158-160, aa 159-161, aa 160-162, aa 161-163, aa 162-164, aa 163-165, aa 164-166, aa 165-167, aa 166-
• the invention relates to a 1,4-benzodiazepine derivatized with one of the following peptides or part thereof:
• FAKYLWEWASVR 2 -KKGK(bio)GA (SEQ ID NO 4) FAKYLW-K(bio) (SEQ ID NO 5) FAKYLWEW-K(bio) (SEQ ID NO 6) FAKYLWEWAS-K(bio) (SEQ ID NO 7) FAKYLWEWASVR-K(bio) (SEQ ID NO 8) FAKYLW (SEQ ID NO 9) FAKYLWEW (SEQ ID NO 10) FAKYLWEWAS (SEQ ID NO 11) FAKYLWEWASVR (SEQ ID NO 2)
• Linking IGP 1362 or parts thereof to BDA 250 or BDA 452 or any derivatives thereof yields a compound with an improved specificity in blocking the interaction of annexin V with HBsAg. It should be noted that not any sequence yields the same result. For instance, the sequence presented by peptide IGP 1363, which is also derived from HBsAg (aa 115-125), does not inhibit the binding of annexin V to HBsAg.
• the 1,4-benzodiazepine or 1,4- benzothiazepine is derivatized with a polypeptide that consists of the following amino acid sequence or part thereof:
• the above mentioned peptides or parts thereof can be linked to the 1,4-benzodiazepine or 1,4-benzothiazepine at positions 1, 2, 2', 3, 3' and/or 4 such as described above.
• the peptides are linked at positions 1 (R') and/or 3 (R) of the 1,4-benzodiazepine according to formula IV.
• R+R' FAKYLWEWASVR (SEQ ID NO 2)
• R and/or R' may be linked to the 1,4-benzodiazepine (Bdz) in various ways:
• R is linked at position 3 via the C-terminus of the peptide after modification of position 3 with amine and/or R' is linked at position 1 via the N-terminus of the peptide after modification of position 1 with carboxymethyl.
• position 1 is methylated or carboxymethylated.
• R' is a peptide
• position 3 contains amine. Examples of these compounds are shown in figure 8.
• the 1,4-benzodiazepine can also be linked to only part of the above mentioned peptides.
• the 1,4-benzodiazepine can take any position within the above mentioned peptides with deletion of one or more amino acids.
• R+R' FAKYLWEWASVR (SEQ ID NO 2)
• part thereof may be linked to the 1,4- benzodiazepine (Bdz) in various ways such as for example.
• the peptides as described above or part thereof can also be linked to position 3 via their N-terminus by use of a linker as described above. Examples of such compounds are shown in figure 11.
• the present invention further relates to a polypeptide consisting of one of the following amino acids sequences or part thereof:
• FAKYLW-K(bio) (SEQ ID NO 5) FAKYLWEW-K(bio) (SEQ ID NO 6) FAKYLWEWAS-K(bio) (SEQ ID NO 7) FAKYLWEWASVR-K(bio) (SEQ ID NO 8) FARFLWEWASVR-K(bio) (SEQ ID NO 12) FGKFLWEWASAR-K(bio) (SEQ ID NO 13) LGKYLWEWASAR-K(bio) (SEQ ID NO 14) FAKFLWEWASVR-K(bio) (SEQ ID NO 15) KYGW-K(bio) (SEQ ID NO 16) KFGW-K(bio) (SEQ ID NO 17) RFGW-K(bio) (SEQ ID NO 18) AYLW-K(bio) (SEQ ID NO 19) AFLW-K(bio) (SEQ ID NO 20) KYLW-K(bio) (SEQ ID NO 21) RFLW-K(bio) (SEQ ID
• peptides are natural or homology variants of the annexin binding epitope of the HBsAg of HBV genotype A/B. Surprisingly, they were also found to inhibit the interaction between annexin V and the HBsAg of HBV.
• polypeptides used in the compounds of the present invention may consist of amino acids under the form of L, under the form of D, or under a mixed form.
• the compound of the invention is characterised that all amino acids are under the L from.
• the compound of the invention is characterised that all amino acids are under the D form.
• Also included in the present invention are compounds comprising or consisting of reverso- inverso polypeptides (such as described by Guichard et al., 1994) of the polypeptides as described above. It should also be noted that all compounds mentioned may be lead compounds, which upon additional derivation of either the 1,4-benzodiazepine/ 1,4-benzothiazepine core or the peptide ligand may be further improved
• the present invention also relates to a combination of a polypeptide as defined above and a negatively charged phospholipid such as phosphatidylserine
• a negatively charged phospholipid such as phosphatidylserine
• the interaction between phosphatidylserine and annexin was already demonstrated in WO 97/07268
• the combination of said polypeptide and said negatively charged phospholipid component may be in any possible way known in the art such as for instance in the form of covalently or non-covalently coupled molecules or in the form of liposomes, etc
• the present invention further relates to a method for the production of the above mentioned 1,4-benzodiazepines or 1,4-benzothiazepines derivatives
• Methods for the production of 1,4-benzodiazepines or 1,4-benzothiazepines are well known in the art They include but are not limited to the methods described by Sternbach et al (1963), US 3109843,US 3116203, US 3121114, US 3123529 and US 3203990 1,4-benzodiazepines and 1,4-benzothiazepines are also commercially available from for example Sigma (Dierinhofen, Germany) or Neosystem (Strasbourg, France)
• the polypeptides derived from the annexin binding proteins more particularly, from the viral proteins that bind annexins, more particularly from the HBsAg of the HBV and/or HDV, from the glycoprotein B of the cytomegalovirus or from an annexin binding protein of any influenza virus are linked to the 1,4-benzodiazepines or
• the present invention also relates to the use of the compounds of the invention for the preparation of a medicament to prevent or treat any disease in which interactions between annexin family members and annexin binding proteins are involved
• the present invention relates to the use of the compounds of the invention for the preparation of a medicament to prevent or treat any viral disease in which interactions between viral proteins and annexin family members are involved.
• the present invention relates to the use of compounds of the invention for the preparation of a medicament to interfere with the life cycle of HBV and/or HDV by inhibiting the interaction of HBV and/or HDV with annexin V, and consequently, to prevent or treat HBV and/or HDV infections.
• HBV and/or HDV indicate that an infection with HBV can occur solely or can be accompanied by a superinfection with HDV.
• an infection with HDV does not occur solely.
• the molecules of the present invention can be used for the preparation of a medicament to prevent or treat a HBV infection solely or a mixed HBV/HDV infection.
• the present invention further relates to the use of compounds of the invention for the preparation of a medicament to interfere with the life cycle of the cytomegalovirus by inhibiting the interaction of the cytomegalovirus with annexin II, and consequently, to prevent or treat cytomegalovirus infections.
• the present invention further relates to the use of the compounds of the invention for the preparation of a medicament to interfere with the life cycle of influenza virus by inhibiting the interaction of influenza virus with annexin V, and consequently, to prevent or treat influenza virus infections.
• the present invention relates to the use of the compounds of the invention for the preparation of a medicament to prevent or treat any disease in which protein interactions with annexin family members are involved, more particularly to prevent or treat any viral disease in which interactions between viral proteins and host annexin family members are involved, more particularly to prevent or treat HBV and/or HDV infections, cytomegalovirus infections, or influenza virus infections.
• the present invention further relates to the use of BDA753, a compound according to formula II, or a compound according to formula III for the preparation of a medicament to prevent or treat any disease in which protein interactions with annexin family members are involved, more particularly to prevent or treat any viral disease in which interactions between viral proteins and host annexin family members are involved, more particularly to prevent or treat HBV and/or HDV infections, cytomegalovirus infections, or influenza virus infections.
• BDA753 is specifically used to inhibit the interaction of HBV with annexin V.
• annexin V binding of 1,4-benzodiazepines and 1,4-benzodiazepine derivatized with AYGW has been demonstrated (Hoffman et al., 1998), 1,4-benzodiazepines without a peptide derivation or derivatized with only one amino acid at position 3 were not able to inhibit the interaction of the HBsAg of HBV to annexin V.
• derivation of the 1,4-benzodiazepine with the peptide AYGW resulted in a compound that was able to inhibit the interaction of the HBsAg of HBV to annexin V.
• the present invention also relates to the use of a peptide containing the following amino acid sequence or part thereof: AYGW (SEQ ID NO 3) for the preparation of a medicament to prevent or treat any disease in which protein interactions with annexin family members are involved, more particularly to prevent or treat any viral disease in which interactions between viral proteins and host annexin family members are involved, more particularly to prevent or treat HBV and/or HDV infections, cytomegalovirus infections, or influenza virus infections.
• AYGW SEQ ID NO 3
• the present invention further relates to a pharmaceutical composition or medicament (both terms can be used interchangeably) comprising at least a compound of the invention, at least BDA 753, at least a compound according to formula II, at least a compound according to formula III or at least the peptide AYGW (SEQ ID NO 3) and possibly, a pharmaceutically acceptable carrier or excipient (both terms can be used interchangeably) to prevent or treat any disease in which protein interactions with annexin family members are involved, more particularly to prevent or treat any viral disease in which interactions between viral proteins and annexin family members are involved, more particularly to prevent or treat HBV and/or HDV infections, cytomegalovirus infections, influenza virus infections, or infections by any strain mutated thereof.
• a pharmaceutical composition or medicament both terms can be used interchangeably
• a pharmaceutical composition or medicament comprising at least a compound of the invention, at least BDA 753, at least a compound according to formula II, at least a compound according to formula III or at least the peptide AYGW (SEQ ID NO
• the composition may contain other therapeutic agents useful against the above mentioned infections.
• therapeutic agents include but are not limited to adefovir, BMS 200475, famciclovir, foscarnet, fiacitabine, fialuridine, (-)-FTC, ganciclivor, GEM 132, interferon, lamivudine, L- FMAU, lobucavir, n-docosanol, ribavirin, sorivudine, vidarabine or compounds mentioned in WO 98/18818 for HBV; acyclovir, adefovir, cidofovir, cyclic HPMPC, fiacitabine, fomivirsen, ganciclovir, I263W94, lobucavir, ribavirin, valaciclovir or vidarabine for cytomegalovirus; amantadine, GG167, GS4104, ribavirin or rimantadine for influenza.
• the “medicament” may be administered by any suitable method within the knowledge of the skilled man.
• the mode of administration can be enteral or parenteral.
• the composition of the present invention may take the form of any of the known pharmaceutical compositions for such methods of administration.
• the compositions may be formulated in a manner known to those skilled in the art, to give a controlled release, for example rapid release or sustained release of the active compound.
• the active substances of these pharmaceutical compositions may also be administered alone, without a carrier vehicle. However, they may also be administered with pharmaceutically acceptable non-toxic carriers or diluents, the proportions of which are determined by the suitability and chemical nature of the particular carrier.
• Suitable carriers or excipients known to the skilled man are saline, Ringer's solution, dextrose solution, Hank's solution, fixed oils, ethyl oleate, 5% dextrose in saline, substances that enhance isotonicity and chemical stability, buffers and preservatives.
• the medicament of this invention will be formulated in a unit dosage injectable form such as a solution, suspension or emulsion, in association with the pharmaceutically acceptable excipients as defined above.
• the dosage and mode of administration will depend on the individual.
• the medicament is administered so that the compound of the present invention is given at a dose between 1 ⁇ g/kg and 100 mg/kg, more preferably between 10 ⁇ g/kg and 20 mg/kg, most preferably between 0.1 and 2 mg/kg.
• it is given as a bolus dose.
• Continuous infusion may also be used.
• the medicament may be infused at a dose between 1 and 100 ⁇ g/kg/minute, more preferably between 5 and 20 ⁇ g/kg/minute.
• Dosage forms suitable for oral administration include but are not limited to tablets, pills, capsules, caplets, granules, powders, elixirs, syrups, solutions and aqueous or oil suspensions.
• a suitable daily dose of the active compound for administration to human beings is generally from about 1 mg to about 5000 mg, more usually from about 5 mg to about 1000 mg, given in a single dose or in divided doses at one or more times during the day.
• the present invention finally relates to the use of a compound as described above in a method to screen for molecules that block the binding between annexin and any protein which is interacting with annexin, more particularly, any viral protein which is interacting with annexin, more particularly, the HBsAg protein of HBV, glycoprotein B of cytomegalovirus or annexin binding proteins of any influenza virus.
• a method to screen for molecules refers to any assay known in the art suitable for molecule screening. In particular, the term refers to the assay described in example 1 of the present invention.
• Table 1 provides sequence information concerning the region of amino acids 99 to 169 of "small" HBsAg that determines 6 genotypes of HBsAg (A, B ,C, D, E, F) and regarding the polypeptides which were examined for binding to annexin V.
• Figure 1 shows the basic structure formula (formula I) of the 1,4-benzodiazepines and 1,4- benzothiazepines wherein:
• modification can be H, alkyl, phenyl, -COZ in which Z stands for H, alkyl, phenyl or substituted phenyl;
• positions 1, 2, 3, 4, 5 may form double bonds with adjacent positions; if this is the case the side chains R 2 , R , R 5 or R 7 are non-existing;
• Ri and/or R 2 is amine, this may be further substituted with a peptide defined in the claims as "a peptide containing an annexin binding epitope of an annexin binding protein" or part thereof, either directly linked via the C-terminus of the peptide or linked to the N-terminus of the peptide via a linker such as glutaraldehyde or succinicanhydride;
• R 3 and/or R4 may represent at position 3 and/or 3' H, amine or alkyl.
• R 3 and/or R is amine, this may be further substituted with a peptide defined in the claims as "a peptide containing an annexin binding epitope of an annexin binding protein" or part thereof, either directly linked via the C-terminus of the peptide or linked to the N-terminus of the peptide via a linker such as glutaraldehyde or succinicanhydride;
• R 5 may represent at position 4 a side chain defined in the claims as "a peptide containing an annexin binding epitope of an annexin binding protein", or part thereof, either directly linked via the C-terminus of the peptide or linked to the N-terminus of the peptide after additional modification of position 4 with carboxymethyl (-CH 2 COOH).
• R 5 may represent H, alkyl, CO-R ⁇ 2 or 3-(l-(4-benzyl)piperidinyl)propionyl);
• R 7 represent H or alkyl or phenyl, possibly further substituted with alkyl, cyano, halo, nitro, alkylalkoxy, alkanoyl, carboxy, alkanoylalkoxy, carbamoyl;
• R 8 , R9, Rio and/or R represent H, alkyl, cyano, halo, nitro, alkylalkoxy, alkanoyl, carboxy, alkanoylalkoxy, carbamoyl;
• R 12 represents H or alkyl or phenyl possibly further substituted with alkyl, cyano, halo, nitro, alkylalkoxy, alkanoyl, carboxy, alkanoylalkoxy, carbamoyl.
• FIG. 2 shows the structure of formula II wherein (FR 2479818):
• R represents an amino acid or a peptide consisting of two or three amino acids
• Figure 3 shows the structure of formula III wherein position 3 of the 1,4-benzodiazepine is substituted with the peptide ARPYN and position 1 is substituted with a L residue (Nachman et al , 1998)
• R, R' and/or R + R' constitute a peptide containing an annexin binding epitope of an annexin binding protein or part thereof In case only R is a peptide, position 1 is methylated In case only R' is a peptide, position 3 contains amine,
• X represents the N-terminus of the peptide which may be modified with -COCH 3 ,
• Y represents the C-terminus of the peptide which may be modified with -NH 2 or -Lysine- Biotine
• Figure 5 shows the structure of formula V wherein the peptide R is linked via its N-terminus by a linker (SPC)
• Figure 6 shows the structures of 1,4-benzodiazepines derivatives described in example 1 BDA250 l,3-dihydro-l-methyl-5-phenyl-2H-l,4-benzodiazepine-2-one, BDA452 3-(R,S)-(L- tryptophanyl)-l,3-dihydro-l-methyl-5-phenyl-2H-l,4-benzodiazepine-2-one, BDA753 3-(R,S)- all-L-(NH-Trp-Gly-Tyr-Ala-H)- 1 ,3-dihydro- 1 -methyl-5-phenyl-2H- 1 ,4-benzodiazepine-2-one
• Figure 7 shows the structure of the Fmoc block (R,S)-Fmoc-3-amino-N-l-carboxymethyl-2-oxo- 5 -cyclohexyl- 1,4-benzodiazepine (FB03601, Neosystem, France) used for the synthesis of the 1,4-benzodiazepines derivatives
• FIG. 8 shows some examples of 1,4-benzodiazepines derivatives according to formula IV
• Figure 9 shows the basic reaction for the synthesis of a 1 ,4-benzodiazepine derivative according to formula V by use of glutaraldehyde as a linker
• Figure 10 shows the basic reaction for the synthesis of a 1,4-benzodiazepine derivative according to formula V by use of succinicanhydride as a linker.
• Figure 11 shows some examples of 1,4-benzodiazepines derivatives according to formula V.
• Figure 12 demonstrates the differential inhibition of the interaction between annexin V and HBsAg by different compounds: BDA250, BDA452 and BDA753 are described in figure 6; IGP 1362 and IGP 1363 are peptides derived from respectively amino acid positions 158-169 and amino acid positions 115-125 of the HBsAg of HBV and are shown in table 1.
• BDA 753 was able to inhibit the interaction of annexin V with HBsAg while a compound containing only the benzodiazepine part of the molecule (BDA250) did not.
• Figure 13 demonstrates the differential inhibition of the interaction between annexin V and HBsAg by different peptides representing natural variants of the amino acids 158-169 of HBsAg or representing a peptide without additional modification of the C-terminus with a biotinylated lysine (IGP 1623) (table 1). The results have been normalised to the inhibition potency of IGP 1481.
• Figure 14 demonstrates the differential inhibition of the interaction between annexin V and HBsAg by different peptides representing C-terminal truncated variants of IGP 1481 (table 1). The results have been normalised to the inhibition potency of IGP 1481.
• Figure 15 demonstrates the differential inhibition of the interaction between annexin V and HBsAg by different peptides representing non-natural mutants of IGP 1481 (table 1). The results have been normalised to the inhibition potency of IGP 1481. Peptides marked with an asterisks showed no inhibition at the highest concentration tested, in theses cases the bars represent the highest relative concentration tested.
• Example 1 Influence of 1,4 benzodiazepine and derivatives on the binding of annexin V to HBsAg
• annexin V binding molecules were allowed to compete with HBsAg in binding annexin V. Finally labelled annexin V bound to HBsAg was measured. Briefly, recombinant HBsAg was coated on microtiterplates (2 ⁇ g/ml, overnight 4°C in TBS, supplemented with ImM CaCl 2 and ImM MgCl 2 ).
• 125 I-labelled annexin V 50ng was added in the presence of an excess of peptide or compound and allowed to interact (2h, 37°C, in TBS, supplemented with ImM CaCl 2 and ImM MgCl 2 and 0.3% cold fish gelatine). After this incubation the plates were washed three times with TBS, supplemented with ImM CaCl 2 and ImM MgCl 2 and 0.05% Tween-20. The experiment was conducted in six-fold and the results expressed are mean values (standard deviation is presented by lines on top of the mean). The molar excess, versus HBsAg, for each compound or peptide used is as follows:
• BDA 753 is composed of a 1,4-benzodiazepine derivatized with a peptide chain of 4 amino acids.
• the benzodiazepine part is equal to BDA 250, which binds annexins but was not able to inhibit the binding of HBsAg to annexin V. On the contrary, BDA250 rather enhanced binding.
• the molecule BDA 452 which has a similar core structure as BDA 250 but which carries an aminated tryptophan did not influence the binding of HBsAg to annexin V at all, while the molecule BDA 753 did inhibit the binding of HBsAg to annexin V. From these results it may be concluded that the three additional amino acids present on BDA 753 and not present on BDA 452 were responsible for the observed competition. Most likely these amino acids bound at the same site on annexin V as did HBsAg. The aminated tryptophan residue already present in BDA 452 probably also interfered partially with the HBsAg binding since the additional tryptophan residue did abolish the binding enhancement caused by BDA 250.
• BDA 452 may be further modified to obtain a better specificity to inhibit the binding of annexin V to HBsAg.
• the peptide IGP 1362 which contains aa 158-169 of HBsAg, was also able to inhibit binding of HBsAg to annexin V (figure 12) and most likely binds to the same site as does BDA 753. It should be noted that not any peptide sequence yields the same result as the sequence presented by peptide IGP1363, which is also derived from HBsAg (amino acids 115-125), did not inhibit the binding of annexin V to HBsAg. It may be concluded that the sequences present in this peptide did not bind to the HBsAg binding domain of annexin V.
• IGP1362 or parts thereof are linked to 1,4-benzodiazepines, BDA250 or BDA452 or any derivative thereof.
• Such molecules can be made by Fmoc based peptide synthesis using (R,S)-Fmoc-3-amino-N-l-carboxymethyl-2-oxo-5-cyclohexyl-l,4-benzodiazepine
• the peptide IGP 1362 is a branched peptide presenting two branches of the amino acids 158-169 of small HBsAg on a lysine core which is biotinylated (table 1). This sequence was also synthesised in a monomeric form with a C-terminal lysine carrying a biotin on the epsilon amine function, or with an amidated C-terminus (IGP 1481 and 1623, see table 1).
• peptides were made representing the same domain of HBsAg (amino acids 158-169) but derived from other genotypes of HBV: IGP 1624 and 1625 representing the major sequences found in genotype C, IGP 1618 representing the major sequence found in genotypes D/E and IGP 1619 representing the major sequence found in genotype F, while IGP 1481 represents the major sequence found in genotypes A/B (table 1). In total the sequences of the peptides IGP 1481, 1624, 1625, 1618 and 1619 represent 90% of 460 HBsAg sequences available on public sequence databanks in October 1998.
• All peptides were purified by reversed phase chromatography and fractions containing peptide were analysed by mass spectrometry in order to confirm the presence of the desired peptide.
• the purified peptides were assayed in the annexin V/HBsAg binding assay and the concentration resulting in a 50% competition was calculated as molar excess to the coated recombinant HBsAg.
• the molar excess to reach the 50% competition level is 17.5 for IGP 1481, which is a sequence homologous to the coated HBsAg.
• the results of the other peptides are shown in figure 13 and have been normalised to the value obtained with IGP 1481 (molar excess of IGP 1481 to reach 50% competition is set to 1).
• IGP1556, 1557 or 1558 or parts thereof are linked to 1,4-benzodiazepines, BDA250, BDA452 or any derivatives thereof.
• the resulting 1,4-benzodiazepines derivatives are tested in the competition experiment to demonstrate their improved specificity in blocking the interaction of annexin V with HBsAg.
• Example 4 Identification of compounds with improved specificity in blocking the interaction of annexin V with HBsAg.
• Bdz stands for the compound BDA250 (figure 6) in which position 3 is modified with an amine to which the peptides are coupled via their C-terminus and position 1 may be modified to carboxymethyl.
• Example 5 Identification of amino acids in the region 158-169 of HBsAg of HBV, important for high specificity in blocking the interaction of annexin V with HBsAg.
• IGP 1363 (TTSTGPCKTCT) 2 -KKGK(bio)GA (SEQ ID NO 60) IGP 1362 (FAKYLWEWASVR) 2 -KKGK(bio)GA (SEQ ID NO 4 IGP 1481 FAKYLWEWASVR-K (bio) (SEQ ID NO 8) IGP 1623 FAKYLWEWASVR (SEQ ID NO 2 ) IGP 1624 FARFLWEWASVR-K(bio) (SEQ ID NO 12) IGP 1618 FGKFLWEWASAR-K(bio) (SEQ ID NO 13) IGP 1619 LGKYLWEWASAR-K(bio) (SEQ ID NO 14) IGP 1625 FAKFLWEWASVR-K(bio) (SEQ ID NO 15) IGP 1556 FAKYLWEWAS-K(bio) (SEQ ID NO 7) IGP 1557 FAKYLWEW-K(bio) (SEQ ID NO 6 ) IGP 1558 FAKYLW-K
• Endonexin II present on human liver plasma membranes, is a specific binding protein of small hepatitis B virus (HBV) envelope protein.
• HBV small hepatitis B virus envelope protein.

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