WO2015014830A1 - Lipopeptides convenant au traitement d'affections hépatiques et d'affections cardiovasculaires - Google Patents

Lipopeptides convenant au traitement d'affections hépatiques et d'affections cardiovasculaires Download PDF

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Publication number
WO2015014830A1
WO2015014830A1 PCT/EP2014/066262 EP2014066262W WO2015014830A1 WO 2015014830 A1 WO2015014830 A1 WO 2015014830A1 EP 2014066262 W EP2014066262 W EP 2014066262W WO 2015014830 A1 WO2015014830 A1 WO 2015014830A1
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Prior art keywords
lipopeptide
based compound
ntcp
liver
amino acid
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PCT/EP2014/066262
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English (en)
Inventor
Volker CLEEVES
Stephan Urban
Ralf KUBITZ
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Rupprecht-Karls-Universität Heidelberg
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Priority claimed from PCT/EP2013/073600 external-priority patent/WO2014072524A1/fr
Application filed by Rupprecht-Karls-Universität Heidelberg filed Critical Rupprecht-Karls-Universität Heidelberg
Publication of WO2015014830A1 publication Critical patent/WO2015014830A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/162—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from virus
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5067—Liver cells
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/044—Hyperlipemia or hypolipemia, e.g. dyslipidaemia, obesity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/08—Hepato-biliairy disorders other than hepatitis
    • G01N2800/085—Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders

Definitions

  • Lipopetides for use in treating liver diseases and cardiovascular diseases Lipopetides for use in treating liver diseases and cardiovascular diseases
  • the present invention relates to lipopeptide-based compounds for use in the diagnosis, prevention and/or treatment of a liver disease or condition, preferably liver involved metabolic diseases, as well as in the control or modification of the cholesterol level or cholesterol uptake and, thus, diagnosis, prevention and/or treatment of a cardiovascular disease.
  • the present invention further relates to the sodium taurocholate cotransporter polypeptide ( TCP) for use in the prevention and/or treatment of certain liver diseases or conditions.
  • TCP sodium taurocholate cotransporter polypeptide
  • the present invention furthermore relates to an in vitro or in vivo assay or method for testing or measuring the NTCP-mediated transport of test compound(s).
  • the present invention furthermore relates to a method for the diagnosis, prevention and/or treatment of a liver disease or condition, comprising administering a therapeutically effective amount of a lipopeptide-based compound to a patient.
  • the present invention furthermore relates to a method for the diagnosis, prevention and/or treatment of a cardiovascular disease.
  • HBV human hepatitis B virus
  • M middle
  • S small
  • L viral envelope proteins
  • M middle
  • S small
  • M contains an N-terminal hydrophilic extension of 55 amino acids (preS2), while L is further extended by 107, 117 or 118 amino acids (genotype-dependent) termed preSl (Urban 2008).
  • the hepatitis D virus is a satellite virusoid utilizing the HBV envelope proteins for entry into hepatocytes.
  • the myristoylated preSl -domain of L is known to play the key role in HBV and HDV infectivity.
  • HBV L-protein derived lipopeptides that block HBV and HDV infection of PHH and HepaRG cells (Gripon et al, 2005, Schulze et al, 2010, WO 2009/092611 Al). They represent the N-terminal 47 amino acids of the preSl-domain of HBV (HBVpreS/2-48 myr ) and include the naturally occurring modification with myristic acid.
  • HBVpreS/2-48 myr the preSl-domain of HBV
  • WO 2009/092612 and WO 2012/107579 whose contents are incorporated herewith by reference in its entirety, the inventors describe hydrophobic modified preS-derived peptides of HBV and their use as vehicles for the specific delivery of compounds to the liver.
  • NTCP/SLC10A1 sodium taurocholate co-transporting polypeptide
  • US Provisional application 61/725,144, filed November 12, 2012 NTCP is an integral transmembrane protein, not expressed in HepG2, HuH7, induced in HepaRG cells after DMSO treatment (Kotani et al, 2012) and down-modulated in primary hepatocytes during de-differentiation (Doring et al., 2012).
  • HBV preSl -specific receptor playing a key role in Hepatitis B virus (HBV) and/or Hepatitis D virus (HDV) infection, the human sodium taurocholate cotransporter polypeptide NTCP/SLC10A1.
  • HBV Hepatitis B virus
  • HDV Hepatitis D virus
  • Expression of this receptor or of certain non-human counterparts allows to transform cells that were previously unable to bind HBV and/or HDV and/or non-susceptible to HBV and/or HDV infection into cells that are HBV and/or HDV binding-competent and/or susceptible to HBV and/or HDV infection.
  • Cells that are already susceptible to HBV and/or HDV infection show a significantly increased susceptibility upon expression of NTCP.
  • NTCP/SLC10A1 as a preS-specific receptor in primary Tupaia hepatocytes (PTH) and demonstrate that human (h) NTCP promotes HBV/HDV entry into hepatoma cells.
  • the liver plays a predominant role in drug biotransformation and disposition from the body. In view of its barrier function between the gastrointestinal tract and systemic blood, it is constantly exposed to ingested xenobiotics entering the portal circulation. Drug-induced liver injury accounts for up to 7% of all reports of adverse drug effects voluntarily reported to pharmacovigilance registries. Drugs cause direct damage to hepatocytes, bile ducts or vascular structures or may interfere with bile flow. The phenotypes commonly encountered thus include hepatitis, cholestasis, steatosis, cirrhosis, vascular and neoplastic lesions and even fulminant hepatic failure.
  • liver to injury Almost every drug has the potential to cause hepatic injury, be it through direct toxicity of the agent or through an idiosyncratic response of the individual.
  • the susceptibility of the liver to injury by drugs is influenced by various factors such as age, sex, pregnancy, comedication, renal function and genetic factors (Kullak-Ublick, 2000).
  • Drug induced cholestatic liver disease is a subtype of liver injury that is characterized by predominant elevations of alkaline phosphatase and bilirubin secondary to the administration of a hepatotoxic agent. It can manifest itself as a cholestatic hepatitis or as bland cholestasis, depending upon the causative agent and the mechanism of injury. Drugs that typically cause cholestasis with hepatitis include psychotropic agents, antibiotics and nonsteroidal antiinflammatory drugs (NSAIDs). The mechanism is immunoallergic and results from hypersensitivity.
  • NSAIDs nonsteroidal antiinflammatory drugs
  • cholestasis without hepatitis is observed most frequently with contraceptive and 17a-alkylated androgenic steroids and the mechanism most likely involves interference with hepatocyte canalicular efflux systems for bile salts, organic anions and phospholipids.
  • the rate-limiting step in bile formation is considered to be the bile salt export pump (BSEP) mediated translocation of bile salts across the canalicular hepatocyte membrane.
  • BSEP bile salt export pump
  • Inhibition of BSEP function by metabolites of cyclosporine A, troglitazone, bosentan, rifampicin and sex steroids is an important cause of drug induced cholestasis (Kullak-Ublick, 2000).
  • liver involved metabolic diseases drug induced toxicity and cholestatic liver diseases, as well as cardiovascular diseases.
  • this object is solved by providing a lipopeptide-based compound for use in the diagnosis, prevention and/or treatment of a liver disease or condition, wherein said liver disease or condition is related to sodium taurocholate cotransporter polypeptide (NTCP)-mediated transport of compounds into hepatocytes.
  • NTCP sodium taurocholate cotransporter polypeptide
  • this object is solved by providing a lipopeptide-based compound for use in the diagnosis, prevention and/or treatment of a cardiovascular disease.
  • this object is solved by providing the sodium taurocholate cotransporter polypeptide (NTCP) for use in the diagnosis, prevention and/or treatment of: - hyperlipidemia,
  • hypercholesterolemia in particular hypercholesterolemia
  • NAFLD non-alcoholic fatty liver disease
  • this object is solved by an in vitro or in vivo assay or method for testing or measuring the NTCP-mediated transport of test compound(s), comprising the steps of
  • step (d) determining whether the test compound(s) are transported via NTCP by comparing the results of step (b) and (c) each with or without the addition of the lipopeptide-based compound,
  • test compound is considered being transported via NTCP when the compound(s) decrease, block or inhibit bile salt transport by NTCP (competitive transport) or when the transport of the compound(s) can be decreased, blocked or inhibited by the addition of the lipopeptide-based compound.
  • this object is solved by a method for the diagnosis, prevention and/or treatment of a liver disease or condition
  • liver disease or condition is related to sodium taurocholate cotransporter polypeptide (NTCP)-mediated transport of compounds into hepatocytes
  • a lipopeptide-based compound comprising administering a therapeutically effective amount of a lipopeptide-based compound to a patient.
  • this object is solved by a method for the diagnosis, prevention and/or treatment of a cardiovascular disease. According to the present invention this object is solved by a method for the control or modification of the cholesterol level or cholesterol uptake, comprising administering a therapeutically effective amount of a lipopeptide-based compound to a patient.
  • a numerical range of "at least 1 amino acid, preferably 1 to 78" should be interpreted to include not only the explicitly recited values of 1 to 78, but also include individual values and sub-ranges within the indicated range.
  • individual values such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ... 75, 76, 77, 78, and sub-ranges such as from 10 to 50, from 15 to 40, from 8 to 35, from 30 to 50, and from 20 to 40, etc.
  • This same principle applies to ranges reciting only one numerical value. FurtheiTnore, such an interpretation should apply regardless of the breadth of the range or the characteristics being described.
  • the present invention provides a lipopeptide-based compound for use in the diagnosis, prevention and/or treatment of a liver disease or condition.
  • Said liver disease or condition is related to sodium taurocholate cotransporter polypeptide (NTCP)-mediated transport of compounds into hepatocytes.
  • NTCP sodium taurocholate cotransporter polypeptide
  • said liver disease or condition that is related to NTCP-mediated transport of compounds into hepatocytes is a liver involved metabolic disease selected from
  • liver toxins poisoning of the liver (by liver toxins) / hepatotoxicity
  • the lipopeptide-based compound preferably comprises:
  • the peptide or amino acid sequence (a) has or comprises the general formula
  • P is the amino acid sequence NPLGFJfoaP SEQ. ID NO: 1 ,
  • Xaa is an arbitrary amino acid; preferably F or L, more preferably F
  • P is preferably NPLGFFP or NPLGFLP
  • X is an amino acid sequence having a length of m amino acids, wherein m is at least 4;
  • Y is an amino sequence having a length of n amino acids
  • n is 0 or at least 1 ;
  • the peptide or amino acid sequence (a) (having the general formual X-P-Y) is preferably derived from the preS domain of hepatitis B virus (HBV) (also designated "preS- peptide").
  • HBV hepatitis B virus
  • the envelope of HBV encloses three proteins termed L (large), M (middle) and S (small). They share the C-terminal S-domain with four transmembrane regions.
  • the M-and L-protein carry additional N-terminal extensions of 55 and, genotype-dependent, 107 or 118 amino acids (preS2- and preSl).
  • a peptide or amino acid sequence (a) preferably refers to a peptide with an amino acid sequence that corresponds to or is based on the N-terminal extensions of the L-protein of HBV, preSl, preferably of genotypes A to H as well as of woolly monkey (WMHBV), orangutan, chimpanzee and gorilla hepatitis B viruses, but it also refers to variants thereof, preferably C-terminally truncated variants, amino acid substitution variants.
  • WHBV woolly monkey
  • amino acid residues being important for the binding of the lipopeptide-based compounds of the present invention to NTCP as set out in SEQ ID NO: 1 (NPLGFXaaP) are present in the peptide/amino acid sequence (a) of the lipopeptide-based compounds of the invention.
  • peptides are based on the following sequences (amino acids in single letter code; essential domain underlined).
  • NPLGFXP (wherein X or Xaa is an arbitrary amino acid, preferably F or L, more preferably F) preS HBV-A (ID: M57663; SEQ ID NO:2):
  • HBV Gibbon (ID: AJ131572, SEQ ID NO: 10)
  • HBV-H (ID: Q8JMY6, SEQ ID NO: 11)
  • HBV Woolly Monkey (ID: NC 001896, SEQ ID NO: 13)
  • HBV preS consensus sequence for amino acid positions (-11) to 48) (SEQ ID NO: 14 ):
  • Variants are preferably N-terminally and/or C-terminally truncated variants, amino acid substitution or deletion variants, or prolonged variants of the sequences of SEQ ID NOs: 2-14, carrying a hydrophobic modification and wherein, optionally, one or more further moiety or moieties is/are coupled to one or amino acid(s) N- or C-terminal of the essential domain.
  • Variants comprise furthermore an amino acid sequence comprising modified amino acid(s), unnatural amino acid(s) or peptidomimetic(s) or further compounds which can mimic a peptide backbone/structure.
  • variants are selected from C-terminally truncated variants of SEQ ID NOs. 2 to 14; amino acid substitution or deletion variants; variants comprising modified amino acid(s), unnatural amino acid(s) or peptidomimetic(s) or further compounds which can mimic a peptide backbone/structure.
  • the peptide or amino acid sequences are preferably L-amino acid sequences, but can also comprise D-amino acids or are D-amino acid sequences.
  • the peptide of the lipopeptide-based compound comprises at least the amino acids having the sequence of SEQ ID NO: 1 and can consist of 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,
  • N-terminally and/or C-terminally truncated variants comprise preferably at least 18 consecutive amino acids, more preferably at least 19 consecutive amino acids, even more preferably at least 20 and just even more preferably at least 21 consecutive amino acids of SEQ ID NOs. 2 to 14 or variants thereof.
  • the N-terminal sequence X of the peptide having a length of m amino acids comprises at least 4 amino acids (i.e. m is at least 4).
  • the N-terminal sequence X can consist of
  • m may be 4 to 19.
  • one or amino acid(s) of X have an amino group in a side chain, which is/are preferably selected from lysine, a-amino glycine, ⁇ , ⁇ -diaminobutyric acid, ormthine, ⁇ , ⁇ -diaminopropionic acid, more preferably lysine.
  • the amino acid(s) of X having an amino group in a side chain is/are preferably is/are located at the N-terminus of X, wherein one to eleven (1-11), preferably one to three (1 - 3), amino acids having an amino group in a side chain are located at the N-terminus of X.
  • the N-terminal sequence X preferably comprises the sequence NX 1 SX 2 X 3 (SEQ ID NO: 15), wherein X l3 X 2 and, X 3 may be arbitrary amino acids.
  • ⁇ ⁇ of SEQ ID NO: 15 is L, I or Q, more preferably L.
  • X 2 of SEQ ID NO: 15 is T, V, A or is not present, preferably T or V, more preferably T.
  • X 3 of SEQ ID NO: 15 is P,
  • the sequence NXiSX ⁇ (SEQ ID NO: 15) is directly attached to the N-terminus of the amino acid sequence P (SEQ. ID NO: 1 ; NPLGFXaaP), resulting in a peptide comprising the sequence NX t SX ⁇ NPLGFXaaP, wherein Xi, X 2 , X 3 and Xaa are defined as above.
  • the C-terminal sequence Y can consist of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 or 93 amino acids.
  • n may be 0 to 93.
  • the C-terminal sequence Y consists of at least 4 amino acids (i.e. n is at least 4), which preferably has the sequence X 4 HQLDP (SEQ ID NO: 16), wherein 3 ⁇ 4 is an arbitrary amino acid.
  • X 4 of SEQ ID NO: 16 is D, E or S, more preferably D or E, even more preferably D.
  • the sequence 3 ⁇ 4HQLDP (SEQ ID NO: 16) is directly attached to the C-terminus of the amino acid sequence P (SEQ. ID NO: 1; NPLGFXaaP), resulting in a peptide comprising the sequence NPLGFXaaPX 4 HQLDP, wherein X 4 and Xaa are defined as above.
  • the peptide of the lipopeptide-based compound of the present invention comprises a peptide encoded by the amino acid sequence NXiSX 2 X 3 NPLGFXaaP X 4 HQLDP (SEQ ID NO: 17), wherein X b X 2 , X 3 , X and Xaa are defined as above.
  • variant also refers to the homologous sequences found in the different viral species, strains or subtypes of the hepadnavirus genus, such as HBV strain alpha, HBV strain LSH (chimpanzee isolate), woolly monkey HBV (WMHBV), or strains selected from the group consisting of the HBV genotypes A to H (see SEQ ID NO: 2-13).
  • variant also refers to homologous sequences which show at least 50% sequence identity to an amino acid sequence comprising the invariant NPLGFXaaP-domain and the adjacent sequences of SEQ ID NO. 2-14 or any other amino acid sequence disclosed herein, preferably 70%, more preferably 80%, even more preferably 90% or 95%.
  • a preferred peptide/amino acid sequence (a) comprises a variant of SEQ ID NOs. 2 to 14 with an amino acid sequence of the different viral species, strains or subtypes, preferably of the genotypes of HBV or woolly monkey HBV (WMHBV) or variants thereof.
  • “Variants” of SEQ ID NOS. 2 to 14 also comprise variants or "analogues” comprising amino acid deletions, amino acid substitutions, such as conservative or non-conservative replacement by other amino acids or by isosteres (modified amino acids that bear close structural and spatial similarity to protein amino acids), amino acid additions or isostere additions, as long as the sequence still binds to NTCP.
  • Conservative amino acid substitutions typically relate to substitutions among amino acids of the same class. These classes include, for example,
  • amino acids having basic side chains such as lysine, arginine, and histidine ;
  • - amino acids having nonpolar side chains such as glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and cysteine.
  • the peptide or amino acid sequences (a) are preferably L-amino acid sequences, but can also comprise D-amino acids or are D-amino acid sequences.
  • the peptide or amino acid sequence (a), X - P - Y is selected from a peptide comprising an amino acid sequence selected from
  • the lipopeptide is Myrcludex B: (a) having the amino acid sequence of HBV preS/2-48 (genotype C) with SEQ ID NO. 18.
  • the lipopeptide-based compound according to the invention preferably comprises:
  • the peptide comprises a hydrophobic or lipid-modification (b), such as at the N- terminus, the C-teraiinus or at an amino acid side chain.
  • a hydrophobic or lipid-modification such as at the N- terminus, the C-teraiinus or at an amino acid side chain.
  • the peptide is modified with at least one hydrophobic moiety or group.
  • the peptide is modified with 1, 2, 3, 4 or more hydrophobic moiety/ies or group(s). That is, the peptide can be modified with more than one hydrophobic moiety or group, such as 2.
  • the hydrophobic moieties or groups can be the same or different to each other.
  • the hydrophobic modification is preferably selected from: acylation and/or addition of hydrophobic moieties.
  • the peptide comprises an N or C-terminal hydrophobic modification (b).
  • An N-terminal hydrophobic modification is preferred.
  • N-terminal refers to the N-terminus of a peptide, thus in a peptide with the general formula X-P-Y, it refers to the N-terminus of X, i.e. the respective first amino acid residue, but comprises also the hydrophobic modification in close proximity to the N-terminus, such as respective amino acid residues (-4), (-3), (-2), (-1), 1, 2 or 3 or 4.
  • the coupling of the hydrophobic modification can furthermore be obtained by an attachment of a hydrophobic moiety at a site close to the N-terminus of X.
  • the hydrophobic modification of the lipopeptide-based compound according to the present invention adds a hydrophobic moiety, preferably to the peptide/amino acid sequence.
  • Acylation is preferably selected from acylation with carboxylic acids, fatty acids, amino acids with lipophilic side chains.
  • Preferred fatty acids are saturated or unsaturated fatty acids, branched or unbranched fatty acids, preferably with 8 to 22 carbon atoms (C 8 to C 22).
  • the hydrophobic modification by acylation is selected from acylation with myristoyl (C 14), palmitoyl (C 16) or stearoyl (C 18). Modification by myristoylation is preferred in in vivo and medicinal applications due to its higher safety, e.g. not showing the adverse effects of the stearoyl group (imiate immune response etc).
  • hydrophobic moieties is preferably selected from addition of cholesterol, derivatives of cholesterol, phospholipids, glycolipids, glycerol esters, steroids, ceramids, isoprene derivatives, adamantane, farnesol, aliphatic groups, polyaromatic compounds, oleic acid, bile salts or bile salt conjugates, more preferably oleic acid, cholesterol, bile salts or bile salt conjugates.
  • the attachment of the hydrophobic moieties is preferably by covalent binding, which can be achieved via carbamate, amide, ether, disulfide or any other linkage that is within the skill of the person skilled in the art.
  • the peptide/amino acid sequences (a) are preferably hydrophobically modified, preferably acylated and, thus, preferably lipopeptides due to their lipophilic or hydrophobic group/moiety.
  • the peptide or amino acid sequence (a) has or comprises the general formula
  • R is a C-terminal modification of said hydrophobic modified peptide
  • amide which is preferably a moiety that protects from degradation selected from amide, D- amino acid, modified amino acid, cyclic amino acid, albumin, natural and synthetic polymer, such as PEG, glycane,
  • o 0 or at least 1.
  • the C-terminal modification (R) of Y is preferably a modification with a moiety that protects from degradation, such as in vivo degradation.
  • C-terminal refers to the modification at the C-terminus, i.e. the respective last amino acid residue, but comprises also the modification in close proximity to the C-terminus, such as the last but one amino acid residue, the last but two amino acid residue or more amino acid residues (e.g. introduction of one D-amino acid that protects the carrier from enzymatic degradation e.g. by the action of carboxypeptidases).
  • the skilled artisan will be able to select the respective suitable moiety(s) depending on the respective application.
  • Preferred moieties that protect from degradation are selected from amides, D-amino acids, modified amino acids, cyclic amino acids, albumin, natural and synthetic polymers, such as PEG, glycane.
  • o is 0 or at least 1 , i.e. the C-terminal modification (R) is optional.
  • R the C-terminal modification
  • o is 1.
  • o is 1, 2, 3, 4 or more. That is, the C-terminus or its proximity can be modified with more than one moiety or group, such as 2.
  • the moieties or groups can be the same or different to each other.
  • the preferred C-terminal modification is an amide.
  • the hydrophobic modification and/or R are linked to the peptide via a linker or spacer.
  • Linker or spacer are known to the skilled artisan, such as polyalanine, polyglycin, carbohydrates, (CHa)n groups. The skilled artisan will, thus, be able to select the respective suitable linker(s) or spacer(s) depending on the respective application.
  • the lipopeptide is Myrcludex B having
  • the lipopeptide-based compound according to the invention comprises (c) a further moiety or moieties.
  • tag(s) such as fluorescent dye(s), radioisotope(s) and contrast agent(s); recombinant virus(s) or derivative(s) thereof;
  • hormones peptide hormones, steroid hormones, monoamines, amino acid derivatives, eicosanoids.
  • the further moiety or moieties are covalently attached to the lipopeptide- compound (preferably to the peptide), such as via linker, spacer and/or anchor group(s).
  • the lipopeptide-based compounds can further contain anchor group(s) that can serve as an additional point(s) of attachment for further moieties (such as compound, tag, label) and can be located at an amino acid of Y.
  • An anchor group can be at an amino acid side chain or can be the amino acid side chain itself, i.e. the anchor group can be a side chain itself or a modified side chain.
  • the anchor group can also be a modified amino acid residue which was introduced into the amino acid sequence of the lipopeptide to serve as an anchor group.
  • the anchor group A is attached to the hydrophobic modification and/or the C-terminal modification R.
  • Preferred anchor groups are selected from ester, ether, disulfide, amide, thiol, thioester.
  • the skilled artisan will be able to select the respective suitable anchor group(s) depending on the respective further moiety to be attached.
  • the anchor group can furthermore be suitable for attaching a complex-forming component, such as of the biotin/avidin, polyarginine/oligonucleotide (e.g. siRNA) complex.
  • a complex-forming component such as of the biotin/avidin, polyarginine/oligonucleotide (e.g. siRNA) complex.
  • there are more than one anchor group such as 2, 3, 4 or more, such as 2.
  • the anchor groups can be the same or different to each other, allowing the attachment of several further moieties.
  • the further moiety/moieties is/are contrast agent(s) which are coupled via a chelating agent.
  • the contrast agent is bound/coupled in the form of a complex with a chelating agent being able to form complexes with the respective contrast agent.
  • a chelating agent can be 1,4,7, 10-tetraazacyclododecane-N,N',N,N'- tetraacetic acid (DOT A), ethylenediaminetetraacetic acid (EDTA), 1,4,7-triazacyclononane- 1,4,7-triacetic acid (NOTA), triethylenetetramine (TETA), immodiacetic acid, Diethylenetriamine- ⁇ , ⁇ , ⁇ ', ⁇ ', ⁇ ''-pentaacetic acid (DTP A) and 6-Hydrazinopyridine-3- carboxylic acid (HYNIC), such as preferably DOTA.
  • DOTA Diethylenetriamine- ⁇ , ⁇ , ⁇ ', ⁇ ', ⁇ ''-pentaacetic acid
  • HYNIC 6-Hydrazinopyridine-3- carboxylic acid
  • contrast agents examples include paramagnetic agents, e.g. Gd, Eu, W and Mn, preferably complexed with a chelating agent.
  • a chelating agent e.g. Licosin, EDTA, EDTA, EDTA, EDTA, EDTA, EDTA, EDTA, EDTA, EDTA, EDTA, EDTA, EDTA, EDTA, EDTA, EDTA, EDTA, EDTA, EDTA, EDTA, EDTA, EDTA, adenodine for computer tomography (CT), microbubbles (such as for contrast enhanced ultrasound (CEUS)) and carriers such as liposomes that contain these contrast agents.
  • CT computer tomography
  • CEUS contrast enhanced ultrasound
  • the peptides of the invention can be prepared by a variety of procedures readily known to those skilled in the art, in general by synthetic chemical procedures and/or genetic engineering procedures. Synthetic chemical procedures include more particularly the solid phase sequential and block synthesis. More details can be taken from WO 2009/092612.
  • Sodium/bile acid cotransporter also known as the sodium/Na + -taurocholate cotransporting polypeptide (NTCP) is a protein that in humans is encoded by the SLC10A1 (solute carrier family 10 member 1) gene.
  • Sodium/bile acid cotransporters are integral membrane glycoproteins that participate in the enterohepatic circulation of bile acids. Two homologous transporters are involved in the reabsorption of bile acids, one absorbing from the intestinal lumen, the bile duct, and the kidney with an apical localization (SLC10A2), and the other sodium-dependent cotransporter being found in the basolateral membranes of hepatocytes (SLC10A1).
  • Bile formation is an important function of the liver. Bile salts are a major constituent of bile and are secreted by hepatocytes into bile and delivered into the small intestine, where they assist in fat digestion. In the liver, hepatocytes take up bile salts (mainly via NTCP) and secrete them again into bile (mainly via the bile salt export pump (BSEP)) for ongoing enterohepatic circulation. Uptake of bile salts into hepatocytes occurs largely in a sodium- dependent manner by the sodium taurocholate cotransporting polypeptide NTCP. The transport properties of NTCP have been extensively characterized.
  • NTCP For NTCP a large range of substrates could be detected, it transports unconjugated as well as taurine-conjugated and glycine-conjugated bile acids (Hagenbuch & Meier, 1994), also sulfated bile acids and, in contrast to the apical sodium dependent bile acid transporter (ASBT), also steroid sulfates (Craddock et al 1998; Kramer et al, 1999; Schroeder et al 1998), and thyroid hormones (Friesema et al, 1999).
  • ABT apical sodium dependent bile acid transporter
  • the compounds which are transported into hepatocytes via NTCP are those which are transported into hepatocytes via NTCP.
  • taurine- or glycine conjugated bile acids and salts thereof taurine- or glycine conjugated bile acids and salts thereof
  • estrogen conjugates e.g. estrone-3 -sulfate, 17a-ethinylestradiol-3-0- sulfate
  • taurocholate e.g. chlorambucil- taurocholate
  • antifungal e.g. micafungin
  • antihyperlipidemic e.g. simvastatin, rosuvastatin, pitavastatin, fluvastatin, atorvastatin
  • immunosuppressives e.g. cyclosporine A
  • immunosuppressives e.g. cyclosporine A
  • said liver disease or condition that is related to NTCP-mediated transport of compounds into hepatocytes is a liver involved metabolic disease selected from
  • liver toxins poisoning of the liver (by liver toxins) / hepatotoxicity
  • NAFLD non-alcoholic fatty liver disease
  • a Treating involved metabolic disease when used herein refers to metabolic disorders including visceral obesity, diabetes mellitus and dyslipidemia which are influenced by the liver metabolism of lipids and bile acids.
  • cholestasis is a condition where bile constituents cannot be secreted from hepatocytes into the biliary tree or where bile cannot flow from the liver to the duodenum, resulting in hepatocyte bile acid accumulation within hepatocytes.
  • Cholestasis or “intrahepatic cholestasis” when used herein refers to intrahepatic toxic effects of hepatocyte bile acid accumulation related to an insufficient expression and/or activity of bile salt pumps (like BSEP or MRP) in the canalicular membrane.
  • BSEP bile salt pumps
  • Posthepatic cholestasis when used herein refers to a cholestatic liver disease due to obstruction of the large bile ducts.
  • “Poisoning of the liver” or “hepatotoxicity” or “toxic liver disease” when used herein refer to toxic effects of drugs independent of bile acid accumulation. These drugs penetrate the hepatocytes via the NTCP-mediated transport and cause several direct toxic effects, by damaging the mitochondria or by activating enzymes in the cytochrome P-450 system leading to oxidative stress.
  • Drug-induced cholestatic liver disease when used herein refers to inhibition of the export of bile acids from hepatocytes due to drug effects on bile salt export pump (BSEP).
  • BSEP bile salt export pump
  • Drug-induced cholestasis may be caused by several drugs which inhibit BSEP, such as rifampicin, cyclosporine A, rifamycin SV, bosentan, troglitazone, erythromycin estolate, and glibenclamide (Fattinger et al., 2001; Funk et al, 2001; Funk et al., 2001; Stieger et al., 2000; Dawson et al., 2012; Morgan et al., 2010; Ogimura et al., 2011).
  • rifampicin cyclosporine A
  • rifamycin SV bosentan
  • troglitazone erythromycin estolate
  • glibenclamide glibenclamide
  • BSEP is a member of the ATP-binding cassette (ABC) family of transporters (BSEP is also identified as ABCB11) and it is involved in the process of exporting bile acids out of hepatocytes, thus reducing their toxicity to these cells.
  • ABC ATP-binding cassette
  • the above mentioned drugs cause the toxic effects of excess bile acid accumulation because the excretion of bile acid via BSEP is disabled.
  • Inhibition of NTCP- mediated bile acid uptake via the lipopeptide-based compound (such as MyrB) and NTCP counterbalances BSEP inhibition, and thereby prevents hepatotoxicity or is suitable for treatment and/or diagnosis.
  • Hyperlipidemia involves abnormally elevated levels of any or all lipids and/or lipoproteins in the blood. Hyperlipidemias are divided in primary and secondary subtypes. Primary hyperlipidemia is usually due to genetic causes (such as a mutation in a receptor protein), while secondary hyperlipidemia arises due to other underlying causes such as diabetes. Lipid and lipoprotein abnormalities are common in the general population, and are regarded as a modifiable risk factor for cardiovascular disease due to their influence on atherosclerosis.
  • “Hypercholesterolemia” (or hypercholesterolaemia) is the presence of high levels of cholesterol in the blood. It is a form of "hyperlipidemia”.
  • “Hyperlipidemia” when used herein preferably refers to hypercholesterolemia which includes elevated LDL cholesterol, reduced HDL cholesterol, elevated triglycerides, clogged arteries leading to high blood pressure, cardiovascular disease (CVD), heart attacks and strokes.
  • CVD cardiovascular disease
  • Metabolic syndrome refers to a disorder of energy utilization and storage, diagnosed by a co-occurrence of three out of five of the following medical conditions: abdominal (central) obesity, elevated blood pressure, elevated fasting plasma glucose, high serum triglycerides, and low high-density cholesterol (HDL) levels. Metabolic syndrome increases the risk of developing cardiovascular disease, particularly heart failure, and diabetes. Metabolic syndrome is also known as metabolic syndrome X, cardiometabolic syndrome, syndrome X, insulin resistance syndrome, Reaven's syndrome, and CHAOS.
  • Non-alcoholic fatty liver disease refers to one cause of a fatty liver, occurring when fat is deposited (steatosis) in the liver not due to excessive alcohol use. It is related to insulin resistance and the metabolic syndrome.
  • Non-alcoholic steatohepatitis is the most extreme form of NAFLD, and is regarded as a major cause of cirrhosis of the liver of unknown cause.
  • the NTCP-mediated transport is decreased or blocked by the lipopeptide-based compound.
  • the inventors have found that the lipopeptide MyrB interferes with NTCP-mediated bile salt transport. In particular, MyrB inhibits NTCP-mediated bile salt transport.
  • the Kj for transporter inactivation (3 ⁇ 4 for rNTCP ⁇ 4nM) is much higher compared to the IC 50 observed for HBV/HDV infection inhibition (80 pM), which coincides with the finding that HBV infection can already been blocked at concentrations below receptor saturation (Schulze et al., 2010).
  • a plausible explanation is the assumption that similar to other viruses the L-protein/h TCP complex has to multimerize. Binding of MyrB to a single subunit could abrogate virus entry whereas substrate transport may continue. This assumption is supported by reports demonstrating oligomerization of NTCP (Doring et al., 2012).
  • the lipopeptide-based compound is administered in a therapeutically effective amount.
  • a “therapeutically effective amount” of a lipopeptide-based compound of this invention refers to the amount that is sufficient to block or inhibit the NTCP-mediated bile salt transport.
  • a “therapeutically effective amount” of a lipopeptide-based compound of this invention further refers to the amount that is sufficient to diagnose, prevent and/or treat the respective liver disease or disorder.
  • the preferred therapeutically effective amount depends on the respective compound that is to be delivered and its respective therapeutic potential.
  • the lipopeptide-based compound is preferably used in a concentration such that a K, of about 1 to 10 nM is reached at the target site, i.e. NTCP site (hepatocytes).
  • the lipopeptide-based compound in order to inhibit substrate transport is preferably used in a dose such that the concentration at the target site is above the 3 ⁇ 4 of about 1 to 10 nM.
  • a preferred therapeutically effective amount is about 100 ⁇ g per kg body weight or in the range of 1 to 5 mg per patient.
  • the preferred therapeutically effective amount in the range of 1 to 5 mg per patient can be administered once a day or in other embodiments only once every 2-3 days, depending on stability and metabolism of the compound used and the turnover of the complex of NTCP/compound.
  • the route of administration or application of the present invention is selected from subcutaneous, intravenous, oral, nasal, intramuscular, transdermal, inhalative, by suppository.
  • a preferred embodiment for nasal administration or application is a nasal spray.
  • the lipopeptide-based compound of the present invention is dissolved in serum from the patient and is applied via injection.
  • the preferred therapeutically effective amount depends on the respective application and desired outcome of inhibition, diagnosis, prevention and/or treatment.
  • the lipopeptide-based compounds can be administered/applied in form of pharmaceutical compositions comprising:
  • compositions are very well suited for all the uses and methods described herein.
  • a “pharmaceutically acceptable carrier or excipient” refers to any vehicle wherein or with which the pharmaceutical compositions may be formulated. It includes a saline solution such as phosphate buffer saline. In general, a diluent or carrier is selected on the basis of the mode and route of administration, and standard pharmaceutical practice.
  • Lipopeptides for use in the control of the cholesterol level and in cardiovascular diseases provides a lipopeptide-based compound for use in the control or modification of the cholesterol level or cholesterol uptake.
  • the cholesterol level or uptake is controlled or modified by decreasing or blocking the NCTP- mediated bile salt transport by the lipopeptide-based compound as defined in this application.
  • the present invention provides a lipopeptide-based compound for use in the diagnosis, prevention and/or treatment of a cardiovascular disease (CVD).
  • CVD cardiovascular disease
  • Said uses comprises the control or modification of the cholesterol level or cholesterol uptake, wherein the cholesterol level or uptake is controlled or modified by decreasing or blocking the NCTP-mediated bile salt transport by the lipopeptide-based compound as defined in this application.
  • Cardiovascular diseases are the major cause of morbidity and death in the western world. High levels of cholesterol have been associated with CVD as one of the rise factors. Of particular importance clinically is the abnormal deposition of cholesterol and cholesterol-rich lipoproteins in the coronary arteries. Such deposition, eventually leading to atherosclerosis, is the leading contributory factor in diseases of the coronary arteries. In this case the management of CVD is critical dependent on lipid-lowering therapies. Different classes of drugs are available for this purpose, such as statins, cholesterol absorption inhibitors, bile acid resins, fibrates and nicotinic acid that act by reducing the levels of cholesterol by distinct pathways (Schmitz & Langmann, 2006). These drugs have several side effects and depend on the relative levels of the metabolizing enzymes and transporters that act on cardiovascular drugs.
  • the main control of cholesterol metabolism is caused by bile acid as an important regulator of cholesterol homeostasis.
  • the levels of bile acid and cholesterol are linked by the regulation of cholesterol metabolism and absorption.
  • the synthesis of the bile acids is the major pathway of cholesterol catabolism in mammals, because the end products of cholesterol utilization are the bile acids.
  • the major pathway for the synthesis of the bile acids is initiated via hydroxylation of cholesterol at the 7 position via the action of cholesterol 7a-hydroxylase (CYP7A1).
  • the lipopeptide-based compounds are suitable for lipid- lowering therapies to prevent CVD.
  • NTCP as target for control or modification of the bile acid / cholesterol metabolism
  • the present invention provides the sodium taurocholate cotransporter polypeptide (NTCP) as target.
  • NTCP sodium taurocholate cotransporter polypeptide
  • NTCP is a target for the control or modification of the bile acid / cholesterol metabolism.
  • NTCP is a suitable target and/or can be provided for use in the prevention and/or treatment of:
  • hypercholesterolemia in particular hypercholesterolemia
  • NAFLD non-alcoholic fatty liver disease
  • hyperlipidemia in particular hypercholesterolemia and cardiovascular disease have been discussed herein.
  • metabolic syndrome refers to a disorder of energy utilization and storage, diagnosed by a co-occurrence of three out of five of the following medical conditions: abdominal (central) obesity, elevated blood pressure, elevated fasting plasma glucose, high serum triglycerides, and low high-density cholesterol (HDL) levels. Metabolic syndrome increases the risk of developing cardiovascular disease, particularly heart failure, and diabetes. Metabolic syndrome is also known as metabolic syndrome X, cardiometabolic syndrome, syndrome X, insulin resistance syndrome, Reaven's syndrome, and CHAOS.
  • NAFLD non-alcoholic fatty liver disease
  • Non-alcoholic steatohepatitis is the most extreme form of NAFLD, and is regarded as a major cause of cirrhosis of the liver of unknown cause.
  • NTCP sequences are as disclosed in US Provisional application 61/725,144 filed November 12, 2012 and WO 2014/072526.
  • SEQ ID Nos.: 23 to 32 refer to the following sequences:
  • SEQ ID NO. 23 is the human sodium taurocholate cotransporter polypeptide NTCP/SLC10A1 (human NTCP).
  • SEQ ID NO. 24 amino acids 265 to 291 of human NTCP.
  • SEQ ID NO. 25 chimpanzee NTCP.
  • SEQ ID NO. 26 orang-utan NTCP.
  • SEQ ID NO. 27 tupaia belangeri / tree shrew NTCP.
  • SEQ ID NO. 28 mouse NTCP.
  • SEQ ID NO. 29 rat NTCP.
  • SEQ ID NO. 30 dog NTCP.
  • SEQ ID NO. 31 cynomolgus NTCP.
  • SEQ ID NO. 32 pig NTCP.
  • HBV myr-preSl The initial attachment of HBV to hepatocytes occurs at the basolateral membrane and is mediated mainly by low-affinity binding between the antigenic loop of the small hepatitis B surface protein domain and heparan-sulfate proteoglycans. Thereafter, high-affinity interaction between HBV myr-preSl and NTCP is regarded as initial step for HBV uptake and infection. Amino acids 157-165 of NTCP located at the N-terminal end of transmembran domain 5 (TMD5) as well as amino acids 84-87 in the extracellular loop upstream of TMD3 were shown to be involved in this interaction (Yan et al., 2012; Yan et al, 2013; Ni et al, 2013).
  • the NTCP binding site interacts with the NPLGFFP binding site of the preS.
  • the present invention provides an in vitro and in vivo assay or method for testing or measuring the NTCP-mediated transport of test compound(s).
  • Said in vitro and in vivo assay or method comprises the steps of
  • step (d) determining whether the test compound(s) are transported via NTCP by comparing the results of step (b) and (c) each with or without the addition of the lipopeptide-based compound,
  • test compound is considered being transported via NTCP when the compound(s) decrease, block or inhibit bile salt transport by NTCP (competitive transport) or when the transport of the compound(s) can be decreased, blocked or inhibited by the addition of the lipopeptide-based compound.
  • Such a suitable test system comprises the functional and selective NTCP expression and thus a functional NTCP transport, which can selectively be blocked/inhibited by a lipopeptide- based compound of the invention (such as MyrB). It can be one or more of the following
  • step (c) bile acid(s) can be added to the NTCP test system, if necessary.
  • the present invention provides a method for the diagnosis,, prevention and/or treatment of a liver disease or condition.
  • Said liver disease or condition is related to sodium taurocholate cotransporting polypeptide (NTCP)-mediated transport of compounds into hepatocytes.
  • NTCP sodium taurocholate cotransporting polypeptide
  • the method of the invention comprises the step of administering a therapeutically effective amount of a lipopeptide-based compound to a patient.
  • the lipopeptide-based compound is preferably as defined in this application.
  • said liver disease or condition that is related to NTCP- mediated transport of compounds into hepatocytes is a liver involved metabolic disease selected from
  • liver toxins poisoning of the liver (by liver toxins) / hepatotoxicity
  • NAFLD non-alcoholic fatty liver disease
  • the compounds which are transported into hepatocytes via NTCP are those which are transported into hepatocytes via NTCP.
  • taurine- or glycine conjugated bile acids and salts thereof taurine- or glycine conjugated bile acids and salts thereof
  • estrogen conjugates e.g. estrone-3-sulfate, 17a-ethinylestradiol-3-0- sulfate
  • taurocholate e.g. chlorambucil- taurocholate
  • antifungal e.g. micafungin
  • antihyperlipidemic e.g. simvastatin, rosuvastatin, pitavastatin, fluvastatin, atorvastatin
  • immunosuppressives e.g. cyclosporine A
  • immunosuppressives e.g. cyclosporine A
  • the NCTP-mediated transport is decreased or blocked by the lipopeptide-based compound.
  • the therapeutically effective amount of the lipopeptide- based compound is in the range of from about 0.1 mg to about 50 mg per patient and per day, preferably from about 1 mg to about 20 mg per patient per day.
  • the present invention provides a method for the control or modification of the cholesterol level or cholesterol uptake.
  • the cholesterol level or uptake is controlled or modified by decreasing or blocking the NCTP- mediated bile salt transport (by the lipopeptide-based compound).
  • the method of the invention comprises the step of administering a therapeutically effective amount of a lipopeptide-based compound to a patient.
  • the lipopeptide-based compound is preferably as defined in this application.
  • the present invention provides a method for the diagnosis, prevention and/or treatment of a cardiovascular disease (CVD),
  • the cholesterol level or uptake is preferably controlled or modified by decreasing or blocking the NCTP-mediated bile salt transport by the lipopeptide-based compound as defined in this application.
  • NTCP-mediated blocking bile acid uptake enables to an elevated cholesterol turn over via hepatocytes. Hence LDL cholesterol will be reduced and HDL cholesterol will be elevated. As a consequence the risk of clogged arteries leading to high blood pressure, CVD heart attacks and strokes will be minimized.
  • the inventors show that the lipopeptide Myrcludex B (MyrB) interferes with NTCP-mediated bile salt transport.
  • NTCP/SLCIOA receptor Functional analyses of the NTCP/SLCIOA receptor revealed that: (i) human NTCP (hNTCP) binds MyrB;
  • MyrB is an interesting novel drug to target NTCP, but also to study its function in vivo.
  • Kj for transporter inactivation (K, for rNTCP ⁇ 4nM) is much higher compared to the IC 50 observed for HBV/HDV infection inhibition (80 pM) (Schulze et al., 2010). This coincides with the finding that HBV infection can already been blocked at concentrations below receptor saturation (Schulze et al., 2010).
  • a plausible explanation is the assumption that similar to other viruses the L-protein/hNTCP complex has to multimerize. If only one subunit bound MyrB, entry may be abrogated although substrate transport may progress.
  • Figure 1 hNTCP specifically binds lipopeptide MyrB.
  • hNTCP human NTCP
  • hNTCP expressing human cell lines were incubated with the Atto488-labeled peptide MyrB att0 (green or 488 ⁇ ). Peptide binding was analysed by co-localisation of the peptide with hNTCP-IF using an hNTCP-specific antibody (red) (B) or FACS using the mutant peptide
  • rNtcp-eGFP expressing HepG2 cells were incubated with increasing concentrations of MyrB or mutant MyrB A,al l ⁇ 15 (a mutant with Ala mutations in the region 9-NPLGFFP-15, namely 9-NPAAAAA-15) and 3 H-taurocholate uptake was quantified. Uncompeted uptake was set to 100%.
  • C-D Differentiated HepaRG (B) or HuH7 hNTCP cells (C) were preincubated 2 h before and coincubated during HBV infection with 5, 50 and 500 ⁇ TC, TDC or TCDC and secreted HBeAg was determined d7-9 p.i.. Infection was controlled by addition of MyrB 2h prior to and during infection.
  • HuH7 hNTCP cells were incubated at the indicated bile salt concentrations overnight at 37 °C, trypsinized and incubated in the presence of bile salts with MyrB att0 for further 30 min. Binding was quantified by FACS analysis. Untagged MyrB was used as a control.
  • hNTCP cDNA (Origene, USA) and mNtcp cDNA (Rose et al., 2011) were subcloned into the puromycin co-expressing lentiviral vector pWPI-puro.
  • hNTCP, mNtcp and h/mNtcp chimera were generated by overlapping PCR and introduced into pWPI-GFP.
  • 1.2 Cells Lentiviruses were produced and used to transduce hNTCP into human (HepaRG, HepG2, HuH7), mouse (Hepal-6, Hep56.1D) and the rat hepatoma cell line TC5123. The respective mock transduced cells were used as controls. To generate stable cell lines, selection with 2.5 ⁇ g/ml puromycin was achieved. Differentiation of transduced HepaRG was induced by DMSO as described (Gripon et al, 2002). HepG2-rNTCP and HepG2-rNTCP-eGFP cell line have been described previously for expression of rat Ntcp with or without fused eGFP (Stress et al., 2010). 1.3 Synthesis and labeling of peptides
  • Flow Cytometry Cells were incubated for 30 min at 37°C in medium containing 200 nM MyrB att0 or the MyrB att0 -mutant. Cells were washed (PBS/1% BSA), trypsinized, and suspended in Krebs-Henseleit-Buffer. Flow cytometry was performed on a FACS Canto II (BD Bioscience, Heidelberg, Germany); Flow Jo v7.61 software (Treestar, Ashton, USA) was used for analysis. Compensation was performed using BD Compbeats (BD Bioscience, Heidelberg, Germany).
  • NTCP immune staining was achieved after permeabilisation (10 min/RT) with TritonX 100 using a cc- SCL10A1/NTCP antibody (Sigma, Germany) diluted 1 :750 in PBS/2% BSA (18h at 4°C).
  • a polyclonal rabbit antiserum H863 was used for HBcAg-staining, a polyclonal rabbit antiserum for MRP-2 detection, patient-derived serum (M. Roggendorf, Essen) for H5Ag.
  • secondary antibodies goat anti-rabbit or -human, labelled with either AlexaFluor488 or AlexaFluor546 (Invitrogen) was used as secondary antibodies goat anti-rabbit or -human, labelled with either AlexaFluor488 or AlexaFluor546 (Invitrogen) was used.
  • Actin staining was performed by the addition of atto633 -labelled Phalloidin diluted 1:2000 (ATTO-Tec, Germany) to the second staining step. Images were talien on a Leica DM IRB or Leica SP2 confocal microscope (Leica, Germany), image analysis was performed using ImageJ.
  • HepG2-rNtcp cells were used for studying [3H] TC uptake as described before (Kubitz et al., 2004). Briefly, HepG2-rNtcp cells were cultured for 12 h (in D-MEM/Ham's F12 w. 10% FCS medium containing G418 for selection) were preincubated with increasing concentrations of MyrB for 20 min before addition of TC (150 ⁇ containing 450 cpm/fmol [3FTJTC). Uptake was stopped after 5 min by removing the medium and washing thrice with ice-cold PBS. Cells were lysed (0.2 M NaOH and 0.05% SDS). Radioactivity of cell lysates was measured in a liquid scintillation counter (Packard instruments, Frankfurt, Germany) using Ultima Gold liquid scintillation solution (Perkin Elmer, Rodgau, Germany).
  • HuH7-, HepG2-, HepaRG- and the two mouse hepatoma cells Hepal-6 and Hep56.1D were stably transduced with an hNTCP-encoding lentivirus.
  • hNTCP expression was verified by Western Blot ( Figure 1A).
  • HuH7 hNTCP , HepG2 hNTCP , Hepal-6 hNTCP and Hep56.1D hNTCP express comparable amounts of hNTCP.
  • HepaRG hNTCP -expression was higher for unknown reasons. No hNTCP was detected in mock-transduced cells.
  • HepG2 hNTCP and HepaRG hNTCP cells capable of binding HBVpreS we analysed cell association of atto-dye-labeled MyrB (MyrB alt0 ) by fluorescence microscopy ( Figure IB) and flow cytometry ( Figure 1C). Specificity was controlled through MyrB-competition and the MyrB attoAlal l ⁇ 15 mutant. hNTCP-expression resulted in specific MyrB-binding indicating a valid role of hNTCP as an HBVpreS-specific receptor.
  • Lipopeptide MyrB inhibits the bile salt transporter function of NTCP.
  • MyrB as a specific ligand for some NTCPs suggests that several contact sites are involved in binding.
  • MyrB therefore interferes with the bile salt transporter function of NTCPs.
  • MyrB inhibited rNtcp with an IC 50 of 4 nM ( Figure 2A).
  • the IC 50 s for inhibition of HBV infection ( ⁇ 100pM) and of bile salt transport ( ⁇ 5nM) differ substantially which relates to observations that infection inhibition does not require binding saturation of NTCP (Schulze et al, 2010).
  • mutant MyrB Alal 5 ((a mutant with Ala mutations in the region 9-NPLGFFP-15, namely 9- NPAAAAA-15)) and preS2-78myr (see Figure 2B).
  • mutant MyrB ⁇ 311"15 SEQ ID NO: 21
  • taurocholate TC
  • taurodeoxycholate TDC
  • taurochenodeoxy-cholate TCDC
  • Bile salt export pump inhibitors are associated with bile acid-dependent drug-induced toxicity in sandwich-cultured hepatocytes. Biochem Biophys Res Commun 416: 313-317.
  • Protein kinase C induces endocytosis of the sodium taurocholate cotransporting polypeptide.

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Abstract

L'invention concerne des composés à base de lipopeptides convenant, d'une part au diagnostic, à la prévention, et/ou au traitement d'une affection ou d'un état hépatique, de préférence des affections métaboliques à implication hépatique, mais aussi d'autre part au contrôle ou à la modification de la cholestérolémie ou de l'assimilation du cholestérol, et ainsi au diagnostic, à la prévention, et/ou au traitement d'une affection cardiovasculaire. L'invention concerne également le polypeptide NTCP, qui est le co-transporteur du taurocholate sodique, utilisé dans la prévention et/ou le traitement de certains états ou de certaines affections hépatiques. L'invention concerne en outre un dosage in vitro ou in vivo, ou un procédé de test ou de quantification du transport des composés de test médié par le NTCP. L'invention concerne aussi un procédé convenant au diagnostic, à la prévention, et/ou au traitement d'une affection ou d'un état hépatique, consistant en l'administration à un patient d'une quantité suffisante d'un composé à base de lipopeptide. L'invention concerne enfin un procédé convenant au diagnostic, à la prévention, et/ou au traitement d'une affection cardiovasculaire.
PCT/EP2014/066262 2013-07-29 2014-07-29 Lipopeptides convenant au traitement d'affections hépatiques et d'affections cardiovasculaires WO2015014830A1 (fr)

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PCT/EP2013/073600 WO2014072524A1 (fr) 2012-11-12 2013-11-12 Lipopeptides utilisables dans le cadre du traitement de maladies hépatiques et cardiovasculaires

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3181146A1 (fr) 2015-12-16 2017-06-21 Ruprecht-Karls-Universität Heidelberg Peptides ciblant les ntcp cycliques et leurs utilisations en tant qu'inhibiteurs d'entrées
EP3189850A1 (fr) 2015-12-16 2017-07-12 Ruprecht-Karls-Universität Heidelberg Ciblage hépatique de peptides issus de pres cyclique de hbv
CN108721599A (zh) * 2017-04-18 2018-11-02 Myr有限公司 使用htcp抑制剂的疗法
WO2020190936A1 (fr) * 2019-03-18 2020-09-24 Svenska Vaccinfabriken Produktion Ab Récepteurs antigéniques chimériques dirigés vers des cellules exprimant le récepteur de co-transport de taurocholate de sodium
EP3804750A1 (fr) 2019-10-10 2021-04-14 Universität Heidelberg Composés conjugués pour la prévention et/ou le traitement des infections par le vhb et/ou le vhd, des maladies du foie et pour le ciblage des ntcp
CN115337312A (zh) * 2022-08-19 2022-11-15 金熊药业(珠海横琴)有限公司 胆酸复合物及其制备方法和应用

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3181146A1 (fr) 2015-12-16 2017-06-21 Ruprecht-Karls-Universität Heidelberg Peptides ciblant les ntcp cycliques et leurs utilisations en tant qu'inhibiteurs d'entrées
EP3189850A1 (fr) 2015-12-16 2017-07-12 Ruprecht-Karls-Universität Heidelberg Ciblage hépatique de peptides issus de pres cyclique de hbv
CN108721599A (zh) * 2017-04-18 2018-11-02 Myr有限公司 使用htcp抑制剂的疗法
WO2020190936A1 (fr) * 2019-03-18 2020-09-24 Svenska Vaccinfabriken Produktion Ab Récepteurs antigéniques chimériques dirigés vers des cellules exprimant le récepteur de co-transport de taurocholate de sodium
EP3804750A1 (fr) 2019-10-10 2021-04-14 Universität Heidelberg Composés conjugués pour la prévention et/ou le traitement des infections par le vhb et/ou le vhd, des maladies du foie et pour le ciblage des ntcp
CN115337312A (zh) * 2022-08-19 2022-11-15 金熊药业(珠海横琴)有限公司 胆酸复合物及其制备方法和应用

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