WO2015193669A1 - Diagnostic and therapeutic method involving the autotaxin-lysophosphatidic acid signalling pathway - Google Patents

Diagnostic and therapeutic method involving the autotaxin-lysophosphatidic acid signalling pathway Download PDF

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WO2015193669A1
WO2015193669A1 PCT/GB2015/051777 GB2015051777W WO2015193669A1 WO 2015193669 A1 WO2015193669 A1 WO 2015193669A1 GB 2015051777 W GB2015051777 W GB 2015051777W WO 2015193669 A1 WO2015193669 A1 WO 2015193669A1
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atx
alpha
autotaxin
hif
lpa
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PCT/GB2015/051777
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French (fr)
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Michael WAKELAM
Simon RUDGE
Jane Mckeating
Michelle FARQUHAR
Garrick WILSON
Gary Reynolds
Luke MEREDITH
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Babraham Institute
The University Of Birmingham
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/145—Amines having sulfur, e.g. thiurams (>N—C(S)—S—C(S)—N< and >N—C(S)—S—S—C(S)—N<), Sulfinylamines (—N=SO), Sulfonylamines (—N=SO2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/42—Oxazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/472—Non-condensed isoquinolines, e.g. papaverine
    • A61K31/4725—Non-condensed isoquinolines, e.g. papaverine containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/538—1,4-Oxazines, e.g. morpholine ortho- or peri-condensed with carbocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/69—Boron compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • 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
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • G01N33/5761—Hepatitis B
    • 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
    • G01N2333/4703—Regulators; Modulating activity
    • 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
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2405/00—Assays, e.g. immunoassays or enzyme assays, involving lipids
    • G01N2405/04—Phospholipids, i.e. phosphoglycerides

Definitions

  • the invention relates to a method of diagnosing and treating hepatitis virus infection, such as hepatitis B or C virus infection, or a disease mediated by hepatitis infection, such as hepatitis B or C virus infection.
  • the invention also relates to biomarkers for hepatitis infection, such as hepatitis B or C virus infection, or a disease mediated by hepatitis infection, such as hepatitis B or C virus infection.
  • Hepatitis B virus is the major cause of hepatocellular carcinoma.
  • HBV associated liver disease is a major health problem of global impact. An estimated 14 million European patients suffer with chronic hepatitis B with an associated mortality rate of 36,000 deaths/year.
  • Hepatitis B associated liver disease includes cirrhosis and hepatocellular carcinoma (HCC), and is the leading cause of death among cirrhotic patients (El-Serag (2012) Gastroenterology 142(6), 1264-1273).
  • HCC cirrhosis and hepatocellular carcinoma
  • In 2008 almost 750,000 new HCC cases were reported worldwide and given the growing incidence of HCC (Villanueva (2011) Gastroenterology 140, 1410), the economic burden will increase in Western populations during the next decades.
  • early-stage tumors can be curatively treated with surgical approaches, efficient treatment options for advanced HCC are limited or absent.
  • HBV-induced liver disease is a major challenge for public health.
  • NUCs with a high barrier of resistance have been introduced, drug resistant variants evolve during long-term treatment and cross-resistance to other agents represents a significant limitation to current therapies. Furthermore, the long-term safety of sustained therapy is unknown. It is important to note that 90% of acutely infected adults clear HBV via strong and multispecific T cell responses that eliminate infected cells by cytolytic and non-cytolytic means (Reherman (2005) Nature Rev Immunol 5, 215). In contrast, persistent infection is common amongst immune compromised patients or those who acquired their infection via perinatal transmission, highlighting the important role of host immune responses in resolving HBV.
  • Hepatitis C is an infectious disease affecting primarily the liver, caused by the hepatitis C virus (HCV).
  • HCV hepatitis C virus
  • the infection is often asymptomatic, but chronic infection can lead to scarring of the liver and ultimately to cirrhosis, which is generally apparent after many years. In some cases, those with cirrhosis will go on to develop liver failure, liver cancer, or life-threatening esophageal and gastric varices.
  • HCV is spread primarily by blood-to-blood contact associated with intravenous drug use, poorly sterilized medical equipment, and transfusions. An estimated 150-200 million people worldwide are infected with hepatitis C. The existence of hepatitis C (originally identifiable only as a type of non-A non-B hepatitis) was suggested in the 1970s and proven in 1989. Hepatitis C infects only humans and chimpanzees.
  • the virus persists in the liver in about 85% of those infected.
  • This chronic infection can be treated with medication: the standard therapy is a combination of peginterferon and ribavirin, with either boceprevir or telaprevir added in some cases. Overall, 50-80% of people treated are cured. Those who develop cirrhosis or liver cancer may require a liver transplant. Hepatitis C is the leading reason for liver transplantation, though the virus usually recurs after transplantation. No vaccine against hepatitis C is available.
  • hepatitis infection such as hepatitis B or C virus infection
  • a disease mediated by hepatitis infection such as hepatitis B or C virus infection.
  • an autotaxin-lysophosphatidic acid signalling pathway modulator for use in the prophylaxis or treatment of hepatitis infection or a disease mediated by hepatitis infection.
  • hypoxia inducible factor 1 alpha subunit (HIF-1 alpha), autotaxin (ATX), lysophosphatidylcholine (LPC) or lysophosphatidic acid (LPA) as a biomarker for hepatitis infection or a disease mediated by hepatitis infection.
  • a method of diagnosing hepatitis infection or a disease mediated by hepatitis infection in an individual thereto comprising:
  • a difference in the level of hypoxia inducible factor 1 , alpha subunit (HIF- 1 alpha), autotaxin, lysophosphatidylcholine or lysophosphatidic acid in the biological sample is indicative of hepatitis infection or a disease mediated by hepatitis infection.
  • a method of monitoring efficacy of a therapy in a subject having, suspected of having, hepatitis infection or a disease mediated by hepatitis infection comprising:
  • kits comprising a biosensor capable of detecting and/or quantifying hypoxia inducible factor 1 , alpha subunit (HIF-1 alpha), autotaxin, lysophosphatidylcholine or lysophosphatidic acid for monitoring, prognosing or diagnosing hepatitis infection or a disease mediated by hepatitis infection.
  • a method of screening for an autotaxin-lysophosphatidic acid signalling pathway modulator which comprises the steps of: (a) contacting a virally infected hepatoma cell with a test compound; and
  • an autotaxin-lysophosphatidic acid signalling pathway modulator obtainable by a method as defined herein.
  • FIG. 1 Hepatic ATX expression in viral and non-viral associated liver disease.
  • B Representative immmunohistochemical stains of ATX in normal, HBV and HCV infected liver tissue.
  • D Huh-7 cells were transfected to express human HIF-1a and a luciferase reporter under the control of a hypoxic responsive element (HRE) or empty pCDNA3.1 (Control) and cultured under normoxic conditions for 24h.
  • HIF-1a expression was confirmed by Western blotting and an increase was noted in HRE- transcriptional activity expressed as relative light units (RLU) and ATX mRNA levels.
  • RLU relative light units
  • uPA- SCID mice transplanted with human hepatocytes were infected with HBV (genotype E) or HCV (genotype 2) and sacrificed at 5 and 16 weeks post infection, respectively. Serum viral loads at the time of sacrifice ranged between 26,600 - 248,400 and 9,300 - 34,200 lU/mL for HBV and HCV respectively.
  • Virus infection induced a significant increase in ATX mRNA (E) and hepatocellular protein expression (F). Primers were designed to detect human ATX and no PCR products were obtained from the tissue of untransplanted mice (data not shown).
  • HBV or HCV strain SA13/JFH infection increases ATX mRNA (shown as graph) and protein secreted from HepG2-NTCP or Huh-7 cells (upper panel), respectively.
  • Infection was confirmed by measuring HBeAg and intracellular HBV DNA (6.7 x 105 HBV DNA copies/ng total DNA) or HCV RNA (8.4 x 103 HCV RNA copies/105 cells). Gene expression is expressed relative to control where all samples have been normalized to GAPDH. The median and range of each data set is presented and is representative of three independent PCR amplifications.
  • FIG. 2 Autotaxin plays an essential role in HCV infection.
  • Huh-7.5 cells were serum starved and incubated with increasing concentrations of HA130 for 60 min prior to 24 h infecting with HCVcc strains SA13/JFH or J6/JFH. The effect of increasing doses of HA130 on HCV infection was analysed by one-way ANOVA (trend analysis [[* P ⁇ 0.05, *** P ⁇ 0.001])
  • Serum starved Huh-7.5 cells expressing shControl minus/plus ATX-wt or ATX-T210A ⁇ , shATX minus/plus ATX-wt or ATX-T210A ⁇ were infected with HCVcc SA13/JFH or J6/JFH for 24h.
  • FIG. 3 LPA plays an essential role in HCV infection
  • A Serum starved Huh-7.5 cells pre-incubated for 15 min with LPA and were infected with HCVcc strains SA13/JFH ⁇ or J6/JFH ⁇ for 24h. The effect of increasing doses of LPA on HCV infection was analysed by one-way ANOVA (trend analysis [[* P ⁇ 0.05, *** P ⁇ 0.001]).
  • B Huh-7.5 cells were serum starved and incubated in the presence/absence of Ki 16425 (10 ⁇ ) for 30min prior to 15min incubation in the presence/absence of LPA. Cells were subsequently infected with HCVcc SA13/JFH ⁇ or J6/JFH ⁇ for 24h.
  • FIG. 4 ATX-LPA axis has an essential role in HCV RNA replication.
  • Huh-7.5 cells were serum starved and incubated with HA130 (100nM) for 60 min prior to 24 h infection with HCVpp-H77 ⁇ or HCVpp-1A38 ⁇ . Infectivity is expressed relative to control and represents the mean of three replicate infections.
  • B Serum starved Huh-7 cells expressing the Luc2A-JFH replicon were incubated with HA130 (100nM) for 24h or transfected to express shControl or shATX (24h). Cells were lysed and luciferase detection performed. Infectivity is expressed relative to control and represents the mean of three replicate infections.
  • Huh-7.5 cells were transfected to express GL.UC-SA13. After 24h cells were serum starved and incubated for a further 24h with increasing concentrations of HA130. Cells were lysed and luciferase detection performed. Infectivity is expressed relative to control and represents the mean of three replicate infections. The effect of increasing doses of HA130 on HCV infection was analysed by one-way ANOVA (trend analysis [*** P ⁇ 0.001]).
  • FIG. 5 LPA promotes HCV infection via stabilization of HIF-1 alpha.
  • A Serum starved Huh-7.5 cells were incubated with LPA (10 ⁇ ) in the absence or presence of Ki16425 (10 ⁇ ), U0126 (10 ⁇ ), wortmannin (200nM), BYL-779 (2 ⁇ ) or TGX-221 (50nM) for 24h. Lysates were separated by SDS-PAGE and detection of HIF-1alpha performed.
  • Huh-7.5 cells were serum starved and following pre-incubation with or without Ki16425 (10 ⁇ ), U0126 (10 ⁇ ), wortmannin (200nM), BYL-779 (2 ⁇ ) or TGX-221 (50nM) (15min) incubated in the absence ⁇ or presence ⁇ of LPA (10 ⁇ ) for 15min prior to infection with HCV (J6/JFH) for 24h. Infectivity is expressed relative to control and represents the mean of three replicate infections. Data is presented from a single experiment representative of three independent experiments and compared using students T-test ([* P ⁇ 0.05], [**** P ⁇ 0.0001]).
  • Huh-7.5 cells were transected with HRE-Luc and following serum starvation incubated with LPA (10 ⁇ ) in the absence or presence of Ki16425 (10 ⁇ ) under normoxic (20% oxygen ⁇ ) or hypoxic (3% oxygen ⁇ ) conditions for 24h. Cells were lysed and luciferase activity measured. Data is presented from a single experiment representative of three independent experiments and compared compared using a one-way ANOVA (Holm-Sidak [**** P ⁇ 0.0001]).
  • Huh-7.5 cells were serum starved and incubated with NSC (25 ⁇ ) in the absence ⁇ or presence ⁇ of LPA (10 ⁇ ) for 15min prior to infection with HCV (SA13/JFH) for 24h.
  • HepG2.2.15 cells were serum starved and incubated with HA130 (100nM) for 24h. Supernatants (Extracellular) were harvested and cells lysed (Intracellular) for detection of total HBV DNA. Data is presented as copies of HBV/ug DNA. Data is presented from a single experiment representative of two independent experiments and compared using a one-way ANOVA (Sidak [* P ⁇ 0.05]).
  • B ATX expression in HepG2.2.15 cells transduced with plKO.1 control (shControl) or plKO.1 shATX (shATX) minus/plus ATX-wt or ATX-T210A determined by western blot.
  • FIG. 7 ATX plays a role in HBV infection of naive target cells.
  • Differentiated HepG2-NTCP cells were treated with HA130 (100nM) for 1 h or transduced with shRNA targeting ATX or an irrelevant control prior to infecting with HBV.
  • ATX expression was confirmed by Western blotting.
  • HBV infection was quantified 7 days later by measuring intracellular and extracellular viral DNA and HBV encoded pre-core or e antigen (HBeAg). For all experiments infectivity is expressed relative to control and represents the mean of three replicate infections analysed by Student's t-test (* P ⁇ 0.05, *** P ⁇ 0.001).
  • FIG 8 A role for ATX in HBV persistently infected cells.
  • A Differentiated HBV infected HepG2.2.15 producer cells were cultured without serum for 24h and the effect of HA130 (100nM) on intracellular or extracellular HBV DNA assessed over a 24h period.
  • B To confirm the inhibitory effect of HA130 on HBV particle secretion, differentiated HepG2.2.15 cells were transduced with shRNA targeting ATX (shATX) or an irrelevant control (shControl).
  • shRNA-ATX cells were trans-complemented with wild-type ATX (shATX-wt) or a catalytically inactive mutant (ATX-T210A) and intracellular or extracellular HBV DNA levels measured.
  • Western blots depict extracellular ATX expression. The median and range of the data from three independent experiments are presented and were compared using Student's t-test (*** P O.001).
  • FIG. 9 LPA modulates hepatitis virus infection via stabilizing HIF-1 a.
  • Huh- 7 cells were transfected with HRE-Luc and cultured under serum-free conditions prior to incubating with LPA (10uM) in the presence or absence of LPAR antagonist KM6425 (10uM) and cultured under 20% oxygen for24h. HIF-1a expression was confirmed by Western blotting. Data are representative of three independent experiments and were compared using a Student's t-test (**** P ⁇ 0.0001).
  • B Differentiated HepG2-NTCP were transfected with siRNA targeting HIF-1 a or control for 48h, cultured without serum for 24h prior to infecting with HBV. HIF-1 a expression was confirmed by Western blotting.
  • HBV infection was quantified 7 days later by measuring cellular viral DNA and HBeAg. For all experiments infectivity is expressed relative to control and represents the mean of three replicate infections analysed by Student's t-test (*** P ⁇ 0.001 , **** P ⁇ 0.0001).
  • C HepG2-NTCP were cultured without serum, treated with LPA (10uM) and cell lysates separated by SDS-PAGE and probed for phospho-AKT (pT308 AKT) or total AKT (AKT).
  • FIG. 10 LPA receptor expression in SCID-upA chimeric mice. Expression of LPA receptors 1-6 mRNA in liver of HBV infected SCID-upA chimeric mice relative to naive mice. Mice were sacrificed at 37 days post-inoculation and HBV DNA levels were 5600-34200 lU/mL.
  • an autotaxin-lysophosphatidic acid signalling pathway modulator for use in the prophylaxis or treatment of hepatitis infection or a disease mediated by hepatitis infection.
  • Autotaxin (ATX) a member of the ectonucleotide pyrophosphatase/phosphodiesterase family of proteins, was originally identified as a motility-stimulating factor secreted from melanoma cells (Stracke (1992) J Biol Chem 267 (4):2524-2529).
  • ATX is over-expressed in various cancers and several studies have reported an association between ATX expression and rates of tumour progression, angiogenesis and metastasis (for review see Houben (201 1) Cancer metastasis reviews 30 (3-4), 557-565).
  • LPC membrane phospholipid lysophosphatidlycholine
  • LPA lysophosphatidic acid
  • LPAR G-protein coupled LPA receptors
  • ATX expression has been implicated in the progression of several inflammatory-associated pathologies namely, multiple sclerosis, rheumatoid arthritis, fibrosis and hepatitis (for review see Sevastou (2013) Biochimica et Biophysica Acta 1831 (1):42-60.
  • Hepatitis, or inflammation of the liver occurs as a result of virus infection, alcohol or autoimmune induced damage and chronic liver injury leads to scarring or fibrosis.
  • Enhanced ATX expression and concomitant LPA production has been associated with liver fibrosis (for review see Ikeda (2012) Journal of Clinical Chemistry 413(23-24), 1817-1821).
  • HBV hepatitis B
  • HCV hepatitis C
  • the data presented herein also confirms increased ATX mRNA and protein expression in HBV and HCV infected human hepatocytes transplanted in the uPA-SCID mouse, demonstrating a direct role for both viruses to regulate ATX expression.
  • an essential role for ATX has been identified in HCV replication, demonstrating a requirement for ATX enzymic activity to generate LPA that stimulates HCV RNA replication.
  • a role for ATX-LPA signaling has been highlighted in the assembly or transport of extracellular HBV particles, providing a new pathway to regulate HBV dissemination.
  • the data presented herein supports a model where HCV and HBV increase ATX expression and associated LPA genesis, providing an autocrine pathway to promote viral persistence and associated pathogenesis.
  • the ATX-LPA signaling axis plays an essential role in the lifecycle of two clinically important human pathogens HCV and HBV that are responsible for the majority of HCC cases across the world - highlighting the potential therapeutic value of targeting ATX-LPA to treat viral replication and associated HCC progression.
  • references herein to "autotaxin-lysophosphatidic acid signalling pathway modulator” refer to any agent, such as an inhibitor (i.e. competitive, non-competitive or un-competitive inhibitor) or antagonist (i.e. competitive, non-competitive or un-competitive antagonist), activator or agonist (i.e. full inverse agonist, partial inverse agonist, silent antagonist, partial agonist, full agonist or super agonist) capable of modulating the signaling effected by the ATX-LPA pathway.
  • an inhibitor i.e. competitive, non-competitive or un-competitive inhibitor
  • antagonist i.e. competitive, non-competitive or un-competitive antagonist
  • activator or agonist i.e. full inverse agonist, partial inverse agonist, silent antagonist, partial agonist, full agonist or super agonist
  • ATX-LPA refers to one or more biological components that participates in or is part of the conversion of lysophosphatidylcholine (LPC) to lysophosphatidyl acid (LPA) or any component upstream or downstream of said participating component.
  • LPC lysophosphatidylcholine
  • LPA lysophosphatidyl acid
  • the autotaxin- lysophosphatidic acid signalling pathway modulator is an inhibitor or antagonist of the autotaxin-lysophosphatidic acid signaling pathway.
  • the autotaxin-lysophosphatidic acid signalling pathway modulator i.e. inhibitor or antagonist of the autotaxin-lysophosphatidic acid signaling pathway
  • an autotaxin inhibitor any suitable ATX inhibitors may be used with the methods of the invention. Suitable examples include chemical compounds, antibodies which specifically bind to ATX or antibody fragments thereof, ATX substrates, ATX product analogs (such as Brp- LPA) or natural inhibitors (such as PF8380). Examples of suitable antibodies and antibody fragments include monoclonal antibodies, humanized antibodies, single chain antibodies, domain antibodies.
  • Suitable autotaxin inhibitors or antagonists include the autotaxin inhibitors disclosed in WO 2014/018881 , WO 2012/024620, WO 2012/166415, WO 2012/100018, WO 201 1/1 16867, WO 2011/006569, WO 2011/053597, WO 201 1/002918, US 201 1/01 10886, US 2010/222341 , WO 2010/1 12116, WO 2010/1 15491 , WO 2010/112124, WO 2010/063352, WO 2010/060532, US 2010/016258, WO 2009/151644 and US 2006/270634 (the contents of each of which are herein incorporated by reference) and the autotaxin inhibitors disclosed in US 2013/0202614 (the contents of which are herein incorporated by reference), such as: 6- (3-(piperazin-1-yl)propanoyl)benzo[d]oxazol-2(3H)-one (PF-8380), 3-[(4-
  • the autotaxin-lysophosphatidic acid signalling pathway modulator i.e. inhibitor or antagonist of the autotaxin-lysophosphatidic acid signaling pathway
  • the autotaxin-lysophosphatidic acid signalling pathway modulator is an LPC inhibitor or an antagonist of the LPC receptor.
  • the autotaxin-lysophosphatidic acid signalling pathway modulator i.e. inhibitor or antagonist of the autotaxin-lysophosphatidic acid signaling pathway
  • LPA lysophosphatidic acid
  • LPA inhibitors or antagonists examples include: the LPA inhibitors disclosed in US 2013/0202614 (the contents of which are herein incorporated by reference), such as: 3-(4-[4- ([1-(2-chlorophenyl)ethoxy]carbonylamino)-3-methyl-5-isoxazolyl]benzylsulfanyl)propanoic acid ( ⁇ 6425; LPA 1 ,3 antagonist); (4'- ⁇ 4-[(R)-1-(2-chlorophenyl)-ethoxycarbonylamino]-3- methyl-isoxazol-5-yl ⁇ -biphenyl-4-yl)-acetic acid (AM966; LPA3 antagonist); isoxazole and thiazole derivatives (such as [1-(2-chlorophenyl)ethyl N-(3-methyl-5-phenylisoxazol-4-yl)- carbamate (LPA2 antagonist)); 1-bromo-3(S)-hydroxy
  • the autotaxin-lysophosphatidic acid signalling pathway modulator i.e. inhibitor or antagonist of the autotaxin-lysophosphatidic acid signaling pathway
  • LPAR G-protein coupled lysophosphatidic acid receptor
  • the LPAR is an antagonist of an endothelial differentiated gene G- protein coupled receptor, such as LPAR 1 , 2, 3, 4, 5 or 6.
  • LPAR 1 , 2, 3, 4, 5 or 6 an endothelial differentiated gene G- protein coupled receptor
  • Ki 16425 a specific antagonist for the LPAR 1 and LPAR 3 receptors
  • the LPAR is an antagonist of LPAR 1 and/or 3, in particular LPAR 1.
  • infection of human hepatocytes transplanted into SCID-upA mice with HBV demonstrates up regulation of LPAR1.
  • the inhibition of HBV release by the LPAR antagonist KM6425 is thus mediated by LPAR1. Therefore, this data shows that LPAR1 receptor is involved in HBV infection and that inhibitors against the LPAR1 receptor in particular present useful anti-HBV drugs.
  • the autotaxin-lysophosphatidic acid signalling pathway modulator i.e. inhibitor or antagonist of the autotaxin-lysophosphatidic acid signaling pathway
  • the autotaxin-lysophosphatidic acid signalling pathway modulator is an inhibitor of hypoxia inducible factor 1 , alpha subunit (HIF-1 alpha).
  • Hypoxia- inducible factors are transcription factors that respond to changes in available oxygen in the cellular environment, to be specific, to decreases in oxygen, or hypoxia.
  • the inhibitor of hypoxia inducible factor 1 , alpha subunit comprises a phosphoinositide 3 kinase (PI3K) or mitogen activated protein kinase (MAPK) signaling modulator.
  • the phosphoinositide 3 kinase (PI3K) signaling modulator is an inhibitor of p1 10a class I phosphoinositide 3 kinase (PI3K), such as BYL-719.
  • the mitogen activated protein kinase (MAPK) signaling modulator is a MAPK/MEK inhibitor, such as U0126.
  • MAPK/MEK inhibitor such as U0126.
  • Data is provided herein which surprisingly found that the MAPK/MEK inhibitor U0126 inhibited LPA stabilization of HIF-1 alpha (see Fig.5A) therefore suggesting a role for MAPK signalling in LPA-dependent augmentation of HCV infection.
  • the hepatitis infection or disease mediated by hepatitis infection is a hepatitis B or C virus infection or a disease mediated by a hepatitis B or C virus infection.
  • the modulator of the invention may be of use in the prophylaxis or treatment of a hepatitis B infection or a disease mediated by a hepatitis B virus infection.
  • liver diseases mediated by hepatitis infection include liver diseases selected from: inflammation of the liver such as hepatitis (i.e. chronic hepatitis), liver fibrosis, cirrhosis or hepatocellular carcinoma, such as hepatocellular carcinoma.
  • liver diseases selected from: inflammation of the liver such as hepatitis (i.e. chronic hepatitis), liver fibrosis, cirrhosis or hepatocellular carcinoma, such as hepatocellular carcinoma.
  • hypoxia inducible factor 1 alpha subunit
  • HIF-1 alpha alpha subunit
  • ATX autotaxin
  • LPC lysophosphatidylcholine
  • LPA lysophosphatidic acid
  • biomarker means a distinctive biological or biologically derived indicator of a process, event, or condition. Biomarkers can be used in methods of diagnosis, e.g. clinical screening, and prognosis assessment and in monitoring the results of therapy, identifying patients most likely to respond to a particular therapeutic treatment, drug screening and development. Biomarkers and uses thereof are valuable for identification of new drug treatments and for discovery of new targets for drug treatment.
  • hypoxia inducible factor 1 alpha subunit (HIF-1 alpha), autotaxin (ATX), lysophosphatidylcholine (LPC) and lysophosphatidic acid (LPA) may be altered in a viral infected and inflamed liver.
  • HIF-1 alpha alpha subunit
  • ATX autotaxin
  • LPC lysophosphatidylcholine
  • LPA lysophosphatidic acid
  • the biomarker is selected from hypoxia inducible factor 1 , alpha subunit (HIF-1 alpha), autotaxin (ATX) or lysophosphatidic acid (LPA).
  • the biomarker is selected from hypoxia inducible factor 1 , alpha subunit (HIF-1 alpha) or lysophosphatidic acid (LPA).
  • the biomarker is selected from hypoxia inducible factor 1 , alpha subunit (HIF-1 alpha).
  • hypoxia inducible factor 1 alpha subunit (HIF- 1 alpha)
  • autotaxin lysophosphatidylcholine or lysophosphatidic acid in the biological sample is indicative of hepatitis infection or a disease mediated by hepatitis infection.
  • a method of monitoring efficacy of a therapy in a subject having, suspected of having, hepatitis infection or a disease mediated by hepatitis infection comprising:
  • hypoxia inducible factor 1 alpha subunit (HIF- 1 alpha)
  • autotaxin lysophosphatidylcholine or lysophosphatidic acid in the biological sample is indicative of an effect of said therapy.
  • references herein to "difference in the level” refer to either a higher or lower level of the biomarker(s) in the test biological sample compared with the control or reference sample(s).
  • the higher or lower level is a ⁇ 1 fold difference relative to the control or reference sample, such as a fold difference of 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1 , 0.05, 0.01 or any ranges therebetween.
  • the lower level is between a 0.1 and 0.9 fold difference, such as between a 0.2 and 0.5 fold difference, relative to the control or reference sample.
  • the higher or lower level is a > 1 fold difference relative to the control or reference sample, such as a fold difference of 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 10.5, 1 1 , 1 1.5, 12, 12.5, 15 or 20 or any ranges therebetween.
  • the higher level is between a 1 and 15 fold difference, such as between a 2 and 10 fold difference, relative to the control or reference sample.
  • HIF-1alpha, autotaxin and lysophosphatidic acid will be present in samples obtained from a subject having, suspected of having, hepatitis infection or a disease mediated by hepatitis infection compared with a normal control biological sample from a normal subject for the reasons as set out hereinbefore.
  • a lower level of lysophosphatidylcholine will be present in samples obtained from a subject having, suspected of having, hepatitis infection or a disease mediated by hepatitis infection compared with a normal control biological sample from a normal subject for the reasons as set out hereinbefore.
  • Monitoring methods of the invention can be used to monitor onset, progression, stabilisation, amelioration and/or remission.
  • detecting and/or quantifying the biomarker in a biological sample from a test subject may be performed on two or more occasions. Comparisons may be made between the level of biomarker in samples taken on two or more occasions. Assessment of any change in the level of the biomarker in samples taken on two or more occasions may be performed. Modulation of the biomarker level is useful as an indicator of the state of hepatitis infection or a disease mediated by hepatitis infection. An increase in the level of HIF-1 alpha, autotaxin and lysophosphatidic acid, over time is indicative of onset or progression, i.e.
  • a method of diagnosis or prognosis of or monitoring according to the invention may comprise quantifying the biomarker in a test biological sample from a test subject and comparing the level of the biomarker present in said test sample with one or more controls.
  • test samples may be taken on two or more occasions.
  • the method may further comprise comparing the level of the biomarker present in the test sample with one or more reference(s) and/or with one or more previous test sample(s) taken earlier from the same test subject, e.g. prior to commencement of therapy, and/or from the same test subject at an earlier stage of therapy.
  • the method may comprise detecting a change in the level of the biomarker in test samples taken on different occasions.
  • the method comprises comparing the amount of biomarker(s) in said test biological sample with the amount present in one or more samples taken from said individual prior to commencement of treatment, and/or one or more samples taken from said individual during treatment.
  • diagnosis encompasses identification, confirmation, and/or characterisation of hepatitis infection or a disease mediated by hepatitis infection.
  • prognosis encompasses the prediction of whether a patient it likely to develop hepatitis infection or a disease mediated by hepatitis infection.
  • Methods of monitoring and of diagnosis or prognosis according to the invention are useful to confirm the existence of a disorder; to monitor development of the disorder by assessing onset and progression, or to assess amelioration or regression of the disorder.
  • Methods of monitoring and of diagnosis or prognosis are also useful in methods for assessment of clinical screening, choice of therapy, evaluation of therapeutic benefit, i.e. for drug screening and drug development.
  • Efficient diagnosis, prognosis and monitoring methods provide very powerful "patient solutions" with the potential for improved prognosis, by establishing the correct diagnosis, allowing rapid identification of the most appropriate treatment (thus lessening unnecessary exposure to harmful drug side effects), reducing relapse rates.
  • Methods for monitoring efficacy of a therapy can be used to monitor the therapeutic effectiveness of existing therapies and new therapies in human subjects and in non-human animals (e.g. in animal models). These monitoring methods can be incorporated into screens for new drug substances and combinations of substances.
  • Any suitable animal may be used as a subject non-human animal, for example a non-human primate, horse, cow, pig, goat, sheep, dog, cat, fish, rodent, e.g. guinea pig, rat or mouse; insect (e.g. Drosophila), amphibian (e.g. Xenopus) or C. elegans.
  • the time elapsed between taking samples from a subject undergoing diagnosis or monitoring will be 3 days, 5 days, a week, two weeks, 1 month, 2 months, 3 months, 6 or 12 months.
  • Samples may be taken prior to and/or during and/or following therapy for hepatitis infection or a disease mediated by hepatitis infection. Samples can be taken at intervals over the remaining life, or a part thereof, of a subject.
  • detecting means confirming the presence of the biomarker present in the sample.
  • Quantifying the amount of the biomarker present in a sample may include determining the concentration of the biomarker present in the sample. Detecting and/or quantifying may be performed directly on the sample, or indirectly on an extract therefrom, or on a dilution thereof.
  • the presence of the biomarker is assessed by detecting and/or quantifying antibody or fragments thereof capable of specific binding to the biomarker that are generated by the subject's body in response to the biomarker and thus are present in a biological sample from a subject having hepatitis infection or a disease mediated by hepatitis infection.
  • Detecting and/or quantifying can be performed by any method suitable to identify the presence and/or amount of a specific protein in a biological sample from a patient or a purification or extract of a biological sample or a dilution thereof.
  • quantifying may be performed by measuring the concentration of the biomarker in the sample or samples.
  • Biological samples that may be tested in a method of the invention include whole blood, blood serum, plasma, cerebrospinal fluid (CSF), urine, saliva, or other bodily fluid (stool, tear fluid, synovial fluid, sputum), breath, e.g. as condensed breath, or an extract or purification therefrom, or dilution thereof.
  • Bio samples also include tissue homogenates, tissue sections and biopsy specimens from a live subject, or taken post-mortem.
  • the samples can be prepared, for example where appropriate diluted or concentrated, and stored in the usual manner. It will be understood that methods of the invention may be performed in vitro.
  • the biological sample is whole blood, blood serum or plasma, such as blood serum.
  • Detection and/or quantification of biomarkers may be performed by detection of the biomarker or of a fragment thereof, e.g. a fragment with C-terminal truncation, or with N-terminal truncation. Fragments are suitably greater than 4 amino acids in length, for example 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length.
  • the biomarker defined herein may be replaced by a molecule, or a measurable fragment of the molecule, found upstream or downstream of the biomarker in a biological pathway.
  • kits comprising a biosensor capable of detecting and/or quantifying hypoxia inducible factor 1 , alpha subunit (HIF-1 alpha), autotaxin, lysophosphatidylcholine or lysophosphatidic acid for monitoring, prognosing or diagnosing hepatitis infection or a disease mediated by hepatitis infection.
  • a biosensor capable of detecting and/or quantifying hypoxia inducible factor 1 , alpha subunit (HIF-1 alpha), autotaxin, lysophosphatidylcholine or lysophosphatidic acid for monitoring, prognosing or diagnosing hepatitis infection or a disease mediated by hepatitis infection.
  • biosensor means anything capable of detecting the presence of the biomarker. Examples of biosensors are described herein.
  • Biosensors according to the invention may comprise a ligand or ligands, as described herein, capable of specific binding to the biomarker. Such biosensors are useful in detecting and/or quantifying the biomarker of the invention.
  • the biomarker may be directly detected, e.g. by mass spectrometry.
  • the biomarker may be detected directly or indirectly via interaction with a ligand or ligands such as an antibody or a biomarker-binding fragment thereof, or other peptide, or ligand, e.g. aptamer, or oligonucleotide, capable of specifically binding the biomarker.
  • the ligand may possess a detectable label, such as a luminescent, fluorescent or radioactive label, and/or an affinity tag.
  • detecting and/or quantifying can be performed by one or more method(s) selected from the group consisting of: SELDI (-TOF), MALDI (-TOF), a 1-D gel-based analysis, a 2-D gel-based analysis, Mass spec (MS), reverse phase (RP) LC, size permeation (gel filtration), ion exchange, affinity, HPLC, UPLC and other LC or LC MS-based techniques.
  • Appropriate LC MS techniques include ICAT® (Applied Biosystems, CA, USA), or iTRAQ® (Applied Biosystems, CA, USA).
  • Liquid chromatography e.g. high pressure liquid chromatography (HPLC) or low pressure liquid chromatography (LPLC)
  • thin-layer chromatography e.g. high pressure liquid chromatography (HPLC) or low pressure liquid chromatography (LPLC)
  • NMR nuclear magnetic resonance
  • Methods according to the invention may comprise analysing a sample of blood serum by SELDI-TOF or MALDI-TOF to detect the presence or level of the biomarker. These methods are also suitable for clinical screening, prognosis, monitoring the results of therapy, identifying patients most likely to respond to a particular therapeutic treatment, for drug screening and development, and identification of new targets for drug treatment. Detecting and/or quantifying the biomarkers may be performed using an immunological method, involving an antibody, or a fragment thereof capable of specific binding to the biomarker.
  • Suitable immunological methods include sandwich immunoassays, such as sandwich ELISA, in which the detection of the biomarkers is performed using two antibodies which recognize different epitopes on a biomarker; radioimmunoassays (RIA), direct, indirect or competitive enzyme linked immunosorbent assays (ELISA), enzyme immunoassays (EIA), Fluorescence immunoassays (FIA), western blotting, immunoprecipitation and any particle- based immunoassay (e.g. using gold, silver, or latex particles, magnetic particles, or Q-dots).
  • Immunological methods may be performed, for example, in microtitre plate or strip format. Immunological methods in accordance with the invention may be based, for example, on any of the following methods.
  • Immunoprecipitation is the simplest immunoassay method; this measures the quantity of precipitate, which forms after the reagent antibody has incubated with the sample and reacted with the target antigen present therein to form an insoluble aggregate. Immunoprecipitation reactions may be qualitative or quantitative.
  • particle immunoassays In particle immunoassays, several antibodies are linked to the particle, and the particle is able to bind many antigen molecules simultaneously. This greatly accelerates the speed of the visible reaction. This allows rapid and sensitive detection of the biomarker.
  • Radioimmunoassay methods employ radioactive isotopes such as I 125 to label either the antigen or antibody.
  • the isotope used emits gamma rays, which are usually measured following removal of unbound (free) radiolabel.
  • EIA enzyme-linked immunosorbent assay
  • Fluorescent immunoassay refers to immunoassays which utilize a fluorescent label or an enzyme label which acts on the substrate to form a fluorescent product. Fluorescent measurements are inherently more sensitive than colorimetric (spectrophotometric) measurements. Therefore, FIA methods have greater analytical sensitivity than EIA methods, which employ absorbance (optical density) measurement.
  • Chemiluminescent immunoassays utilize a chemiluminescent label, which produces light when excited by chemical energy; the emissions are measured using a light detector.
  • Immunological methods according to the invention can thus be performed using well-known methods. Any direct (e.g., using a sensor chip) or indirect procedure may be used in the detection of the biomarker of the invention.
  • Biotin-Avidin or Biotin-Streptavidin systems are generic labelling systems that can be adapted for use in immunological methods of the invention.
  • One binding partner hapten, antigen, ligand, aptamer, antibody, enzyme etc
  • biotin is labelled with avidin or streptavidin.
  • avidin or streptavidin is conventional technology for immunoassays, gene probe assays and (bio)sensors, but is an indirect immobilisation route rather than a direct one.
  • a biotinylated ligand e.g.
  • an antibody or aptamer) specific for a biomarker of the invention may be immobilised on an avidin or streptavidin surface, the immobilised ligand may then be exposed to a sample containing or suspected of containing the biomarker in order to detect and/or quantify a biomarker of the invention. Detection and/or quantification of the immobilised antigen may then be performed by an immunological method as described herein.
  • antibody as used herein includes, but is not limited to: polyclonal, monoclonal, bispecific, humanised or chimeric antibodies, single chain antibodies, Fab fragments and F(ab')2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies and epitope-binding fragments of any of the above.
  • antibody as used herein also refers to immunoglobulin molecules and immunologically-active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen.
  • the immunoglobulin molecules of the invention can be of any class (e.g., IgG, IgE, IgM, IgD and IgA) or subclass of immunoglobulin molecule.
  • biosensors can be developed, accordingly, in methods and uses of the invention, detecting and quantifying can be performed using a biosensor, microanalytical system, microengineered system, microseparation system, immunochromatography system or other suitable analytical devices.
  • the biosensor may incorporate an immunological method for detection of the biomarker, electrical, thermal, magnetic, optical (e.g. hologram) or acoustic technologies. Using such biosensors, it is possible to detect the target biomarker at the anticipated concentrations found in biological samples.
  • an apparatus for monitoring hepatitis infection or a disease mediated by hepatitis infection which comprises a biosensor, microanalytical, microengineered, microseparation and/or immunochromatography system configured to detect and/or quantify one or more of the biomarkers defined herein.
  • the biomarker of the invention can be detected using a biosensor incorporating technologies based on "smart" holograms, or high frequency acoustic systems, such systems are particularly amenable to "bar code” or array configurations.
  • a biosensor incorporating technologies based on "smart" holograms, or high frequency acoustic systems, such systems are particularly amenable to "bar code” or array configurations.
  • smart hologram sensors Smart Holograms Ltd, Cambridge, UK
  • a holographic image is stored in a thin polymer film that is sensitised to react specifically with the biomarker.
  • the biomarker reacts with the polymer leading to an alteration in the image displayed by the hologram.
  • the test result read-out can be a change in the optical brightness, image, colour and/or position of the image.
  • a sensor hologram can be read by eye, thus removing the need for detection equipment.
  • a simple colour sensor can be used to read the signal when quantitative measurements are required. Opacity or colour of the sample does not interfere with operation of the sensor.
  • the format of the sensor allows multiplexing for simultaneous detection of several substances. Reversible and irreversible sensors can be designed to meet different requirements, and continuous monitoring of a particular biomarker of interest is feasible.
  • biosensors for detection of the biomarker of the invention combine biomolecular recognition with appropriate means to convert detection of the presence, or quantitation, of the biomarker in the sample into a signal.
  • Biosensors can be adapted for "alternate site” diagnostic testing, e.g. in the ward, outpatients' department, surgery, home, field and workplace.
  • Biosensors to detect the biomarker of the invention include acoustic, plasmon resonance, holographic and microengineered sensors. Imprinted recognition elements, thin film transistor technology, magnetic acoustic resonator devices and other novel acousto-electrical systems may be employed in biosensors for detection of the biomarker of the invention.
  • Biosensors can be performed on bench-top instruments, or can be incorporated onto disposable, diagnostic or monitoring platforms that can be used in a non-laboratory environment, e.g. in the physician's office or at the patient's bedside.
  • Suitable biosensors for performing methods of the invention include "credit" cards with optical or acoustic readers. Biosensors can be configured to allow the data collected to be electronically transmitted to the physician for interpretation and thus can form the basis for e-neuromedicine.
  • a method of identifying a substance capable of promoting or suppressing the generation of the biomarker in a subject comprising exposing a test cell to a test substance and monitoring the level of the biomarker within said test cell, or secreted by said test cell.
  • the test cell could be prokaryotic, however a eukaryotic cell will suitably be employed in cell- based testing methods.
  • the eukaryotic cell is a yeast cell, insect cell, Drosophila cell, amphibian cell (e.g. from Xenopus), C. elegans cell or is a cell of human, non-human primate, equine, bovine, porcine, caprine, ovine, canine, feline, piscine, rodent or murine origin.
  • the test substance can be a known chemical or pharmaceutical substance, such as, but not limited to, an anti-psychotic disorder therapeutic; or the test substance can be novel synthetic or natural chemical entity, or a combination of two or more of the aforesaid substances.
  • non-human animals or cells can be used that are capable of expressing the peptide.
  • Methods of screening for an autotaxin-lysophosphatidic acid signalling pathway modulator are also contemplated within the scope of the invention. Suitable methods of screening are described in the methods section herein and may typically involve the step of administering a test compound to Huh-7 and/or HepG2 hepatoma cells, which are model systems for supporting HCV and HBV replication, respectively.
  • the virally infected hepatoma cell comprises Huh-7 hepatoma cells. These cells have the advantage of supporting HCV replication.
  • the virally infected hepatoma cell comprises HepG2 hepatoma cells, these cells have the advantage of supporting HBV replication.
  • Screening methods also encompass a method of identifying a ligand capable of binding to the biomarker according to the invention, comprising incubating a test substance in the presence of the biomarker in conditions appropriate for binding, and detecting and/or quantifying binding of the biomarker to said test substance.
  • High-throughput screening technologies based on the biomarker, uses and methods of the invention e.g. configured in an array format, are suitable to monitor biomarker signatures for the identification of potentially useful therapeutic compounds, e.g. ligands such as natural compounds, synthetic chemical compounds (e.g. from combinatorial libraries), peptides, monoclonal or polyclonal antibodies or fragments thereof, which may be capable of binding the biomarker.
  • Methods of the invention can be performed in array format, e.g. on a chip, or as a multiwell array. Methods can be adapted into platforms for single tests, or multiple identical or multiple non-identical tests, and can be performed in high throughput format. Methods of the invention may comprise performing one or more additional, different tests to confirm or exclude diagnosis, and/or to further characterise a condition.
  • the invention further provides a substance, e.g. a ligand, identified or identifiable by an identification or screening method or use of the invention.
  • a substance e.g. a ligand, identified or identifiable by an identification or screening method or use of the invention.
  • Such substances may be capable of inhibiting, directly or indirectly, the activity of the biomarker, or of suppressing generation of the biomarker.
  • the term "substances” includes substances that do not directly bind the biomarker and directly modulate a function, but instead indirectly modulate a function of the biomarker.
  • Ligands are also included in the term substances; ligands of the invention (e.g. a natural or synthetic chemical compound, peptide, aptamer, oligonucleotide, antibody or antibody fragment) are capable of binding, suitably specific binding, to the biomarker.
  • Huh-7 Luc2a-JFH cells (provided by Robert Thimme, UNI Freiburg; Jo (2009) Gastroenterology 136 (4): 1391-1401) were propagated in the same growth media supplemented with G418. All cells were grown in a humidified atmosphere at 37°C in 5% C0 2 .
  • the primary antibodies used were: anti-NS5A 9E10 (C. Rice, Rockefeller University, USA); anti-ATX 4FAB (Baumforth (2005) Blood 106 (6), 2138-2146).
  • RNA was prepared (RNAeasy mini kit, Qiagen) from: human liver tissue of patients with a range of disease aetiologies, who underwent resection or transplantation at the Queen Elizabeth Hospital Birmingham, with patient consent and regional ethical committee approval obtained. Liver disease was diagnosed according to standard clinical, histological and radiological criteria. Normal liver tissue was obtained from surplus donor tissue used for reduced size transplantation; uPA-SCID mouse liver tissue transplanted with human hepatocytes or infected with HCV or HBV. Untransplanted mice were used as a negative control; Huh-7.5 cells naive or infected for 72h with HCV; HepG2 or HepG2.2.15 cells.
  • Pseudoviruses expressing luciferase reporters were generated following transfection of 293T cells with a 1 : 1 ratio of plasmids encoding HIV provirus expressing luciferase and HCV strain 1A38 or H77 E1 E2 envelope gps (HCVpp-1A38, HCVpp-H77), MLV gp (MLVpp) VSV gp (VSVpp) or empty vector (Env-pp), as previously described (Hsu (2003) PNAS(USA) 100(12), 7271-7276). 24 h post transfection cells were serum starved prior to harvesting at 48h post transfection in serum free media.
  • Target cells were seeded at 1.5x10 4 cells/cm 2 and serum starved for 16 h prior addition of HCVcc or pseudoparticles in serum free media for 24 h.
  • HCVcc infection was assessed following methanol fixation and staining for NS5A using the anti-NS5A 9E10 antibody; bound antibody was detected with an Alexa 488-conjugated anti-mouse IgG and quantified by enumerating NS5A positive cells.
  • Pseudoparticle infection was quantified by measuring cellular luciferase activity in a luminometer (Berthold Centro LB 960). Specific infectivity was calculated by subtracting the mean Env-pp signal from the HCVpp/MLVpp/VSVpp signals.
  • uPA-SCID mice infection uPA/SCID-bg mice were transplanted with primary human hepatocytes at 3 weeks of age by intrasplenic injection of 10 6 cells suspended in PBS as already described (Mercer (2001) Nat Med 7(8), 927-933). Successful engraftment was determined by measuring the human albumin concentration in the serum of transplanted mice by specific ELISA. HCV infected mice were inoculated with Jc1 recombinant virus from cell supernatant or mouse serum.
  • HBV infected mice were inoculated with an infectious mouse serum containing a genotype E patient-derived virus. Mice were sacrificed by spine dislocation after gaseous anesthesia and blood harvesting by intracardiac puncture 5 (HB) to 16 (HCV) weeks later. The liver was recovered, cut in small pieces and immediately frozen on dry ice for RNA extraction. Liver samples were also fixed in formalin for immunostaining purposes. Experiments were performed in the Inserm Unit 1 110 animal facility according to local laws and approved by the ethical committee of France (number AL/02/19/08/12 and AL/01/18/08/12). Autotaxin silencing and rescue.
  • Huh-7 Luc2a-JFH expressing cells or Huh-7 Luc2a-JFH transduced to express plKO.1 control or plKO.1 shATX silencing vectors were seeded at 1.5 x 10 4 /cm 2 and serum starved for 16 h prior to assay. Cells were untreated or incubated with HA130 for 24 h prior to lysis and quantification of luciferase performed as described.
  • Huh-7.5 cells were transfected with Jd GLuc for 24 h then serum starved for 16 h. Cells were washed extensively then incubated with HA130 for 24 h. Cell supernatants were harvested, heat inactivated and luciferase activity from secreted particles in the harvested supernatant determined.
  • Quantified protein lysates 50 ⁇ g were separated by SDS-PAGE and transferred to PVDF membranes (Sigma, UK) for incubation with anti-ATX.
  • HRP-conjugated anti-rat secondary antibody was detected by enhanced chemiluminescence (Geneflow, UK). Protein was visualised using the PXi imaging system (Syngene).
  • HIF-1alpha For detection of HIF-1alpha, cells were serum starved overnight prior to incubation with LPA (10 ⁇ ) diluted in serum free media containing 0.1 mg/ml fatty acid free in the presence or absence of KM 6425 (10 ⁇ ), U0126 (10 ⁇ ), wortmannin (200nM), BYL-779 (2 ⁇ ) or TGX- 221 (50nM) for 24 h at 37°C. Cells were lysed directly in Laemmli buffer (Laemmli (1970) Nature 227(5259), 680-685), lysates were separated by SDS-PAGE and transferred to PVDF membranes as described above.
  • Anti-HIF-1 alpha detection was performed and detected following incubation HRP-conjugated anti-mouse secondary antibody by enhanced chemiluminescence. Detection of LPA by mass spectrometry.
  • Huh-7.5 cells were serum starved for 16 h prior to harvesting of supernatants for 8 h in serum free medium. Following clarification supernatants were spiked with 30ng 17:0 LPA as an internal standard prior to LPA extraction. 8ml of supernatant was extracted with 16ml of n-butanol three times at room temperature, the combined extract was dried under vacuum (SpeedVac, Thermo) and re-dissolved in 100 ⁇ chloroform/methanol/water 2:5: 1.
  • LC- MS/MS Liquid-chromatography with tandem mass spectrometry (LC- MS/MS) analysis was performed on each sample using a Thermo Orbitrap Elite system (Thermo Fisher) hyphenated with a five-channel online degasser, four- pump, column oven, and autosampler with cooler Shimadzu Prominence HPLC system (Shimadzu) for lipids analysis. High resolution / accurate mass and tandem MS were used for molecular species identification and quantification. The identity of the lipid subspecies was further confirmed by reference to appropriate lipids standards. All the solvents used for lipid extraction and LC- MS/MS analysis were LC-MS grade from Fisher Scientific. The final amount of LPA (ng) is presented per 8 mL of conditioned media analysed.
  • hypoxia responsive element luciferase reporter assay To quantify HIF activity, Huh-7.5 cells were transfected to express hypoxia responsive element luciferase reporter (HRE-Luc) (provided by Margaret Ashcroft, University College London, UK; Chau (2005) Cancer Research 65(1 1), 4918-4928) using Fugene 6 (according to manufacturer's instructions). 24 h later, cells were seeded at 1.5 x 10 4 /cm 2 and serum starved for 16 h prior to assay. Cells were incubated for 24 h with LPA (10 ⁇ ), KM 6425 (10 ⁇ ), LPA plus KM6425, or NSC (25nM) diluted in serum free media containing 0.1 mg/ml fatty acid free BSA.
  • LPA 10 ⁇
  • KM 6425 10 ⁇
  • LPA plus KM6425 or NSC (25nM) diluted in serum free media containing 0.1 mg/ml fatty acid free BSA.
  • HRE-Luc activity was quantified by measuring cellular luciferase activity in a luminometer. Relative activity was calculated as a percentage of untreated cells and presented ⁇ S.E.M. where the mean activity value of replicate untreated cells wells was defined as 100%.
  • HepG2.2.15 cells or cells transduced to express plKO.1 control or plKO.1 shATX silencing vectors 72 h were seeded at 1.5 x 10 4 /cm 2 and serum starved for 16 h prior to assay. Parental cells were incubated with HA130 or DMSO control in serum free media. 24 h later cells or supernatants were harvested for quantification of HBV.
  • viral DNA was isolated from HepG2.2.15 supernatant using the QiaAMP MinElute Virus Spin Kit (Qiagen), while total cellular DNA was isolated using the QiaAMP DNA Mini Kit (Qiagen), both as per manufacturer's instructions.
  • HBV was then quantified using the Hepatitis B Virus qPCR Quantification Kit (PrimerDesign) as per manufacturer's description, using a Roche Lightcycler® (Roche).
  • HBsAg was quantified from viral supernatant using the HBsAg Antigen ELISA (Abnova) as per manufacturer's instructions.
  • the present experiment studied the impact of HCV and HBV infection on hepatic ATX expression in the absence of HCC.
  • a significant increase in ATX mRNA levels was confirmed in the liver biopsies from HCV and HBV non-cirrhotic patients (Fig.lA).
  • a comparable increase in ATX mRNA levels was noted in the liver tissue from patients with alcoholic liver disease (ALD), primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC) and autoimmune hepatitis (AIH), diseases characterised by persistent inflammation (Fig.lA).
  • ALD alcoholic liver disease
  • PBC primary biliary cirrhosis
  • PSC primary sclerosing cholangitis
  • AIH autoimmune hepatitis
  • HIF-1 alpha hypoxia inducible factor 1 a
  • Huh-7 hepatoma cells were transfected with plasmids encoding HIF-1 alpha and 36h later expression confirmed by western blotting (Fig.l B). It was confirmed that incubating Huh- 7 cells under physiological low oxygen levels found in the liver (3%) stabilized HIF-1alpha treatment and promoted ATX mRNA levels (data not shown).
  • HIF- 1 alpha dependent transcriptional responses using a HRE-luciferase reporter demonstrated a significant increase in ATX mRNA and control VEGF mRNA (Fig.l B), demonstrating that ATX is a HIF-target gene.
  • mice The human liver uPA-SCID mouse supports HCV and HBV infection (Mercer (2001 , supra); Meuleman (2006) Journal of Virology 80(6), 2797-2807, enabling us to directly investigate the impact of viral infection on ATX expression. All mice engrafted with human hepatocytes expressed human ATX to varying levels and infection with HCV or HBV promoted a significant increase in ATX mRNA levels (Fig.lC). In non-transplanted mice, ATX expression was not detected (data not shown) illustrating both the specificity of primers for human ATX and highlighting that viral-dependent modulation of ATX expression is of human hepatocyte origin.
  • ATX was clearly expressed in hepatocytes in both HCV and HBV infected livers, whereas no staining was observed in liver tissue from uninfected controls (Fig.lC).
  • Fig.lC liver tissue from uninfected controls
  • ATX expression was increased in HepG2.2.15 cells that support HBV replication (Sells (1987) PNAS(USA) 84(4), 1005-1009; Sun (2006) Journal of Hepatology 45(5), 636-645) compared to uninfected HepG2 cells, however, the present study failed to detect any significant change in ATX mRNA levels (Fig.l D), consistent with the lower ATX mRNA levels observed in the HBV infected SCID-uPA mice (Fig.lC).
  • ATX is a HIF-target gene and is up-regulated following HCV or HBV infection.
  • ATX is a secreted protein that binds LPC in its active site and catalyses its hydrolysis to LPA (Tokumura (2002) The Journal of Biological Chemistry 277(42), 39436- 39442; Umezu-Goto (2002) The Journal of Cell Biology 158(2), 227-233).
  • Inhibition of ATX can be achieved by ATX antagonists, that target the active site and block the enzymatic activity of ATX.
  • Huh-7.5 cells were infected with HCVcc strains SA13/JFH and J6/JFH in the presence of an increasing concentration of ATX inhibitor HA130.
  • Fig.2A A dose-dependent inhibition of HCV infection was observed following HA130 treatment (Fig.2A). To verify these observations, Huh-7.5 cells were transduced with an shRNA vector targeting ATX or an empty vector control. Quantitative RT-PCR (Fig.2B) and western blotting confirmed effective ATX silencing (Fig.2B). ATX silencing significantly inhibits HCV SA13/JFH and J6/JFH infectivity (Fig.2B), providing clear evidence for a role for ATX in HCV infection.
  • ATX-T210A enzymatically inactive ATX mutant T210A
  • Fig.2C control cells
  • ATX-LPA signalling axis in HCV infection control or ATX silenced cells were infected with HCV SA13/JFH or J6/JFH in the presence of HA130 or LPA.
  • HA130 inhibited HCV infection in control cells and had no effect on ATX silenced cells (Fig.2D), supportive of the lack of ATX secreted from these cells.
  • LPA enhanced HCV infection of control cells and restored infection in ATX silenced cells (Fig.2D), suggesting that LPA production via ATX activity is important in HCV infection.
  • LPA the product of LPC hydrolysis by ATX is an effector molecule.
  • the effect of exogenous LPA on hepatoma permissivity was investigated to support HCV infection.
  • LPA enhances HCV SA13/JFH and J6/JFH infection in a dose-dependent manner (Fig.3A).
  • LPA exerts its effects through a family of six currently identified LPA receptors (LPAR 1-6).
  • LPAR 1-6 A recent study reported that LPARs 1 , 3 and 6 were expressed in the liver and a comparable expression profile was noted in Huh-7 cells (Sokolov (2013) The Journal of Surgical Research 180(1), 104-1 13).
  • Quantitative RT-PCR confirmed expression of LPARs 1 , 3 and 6 in Huh-7.5 cells (data not shown).
  • HCVpp HCV pseudoparticle
  • Luc2a JFH expressing Huh-7 subgenomic replicon cell line (Luc2a-JFH) was incubated with HA130 resulting in a significant reduction in HCV replication (Fig.4B).
  • Silencing ATX in the Luc2a-JFH cells as confirmed by Western blot (data not shown), substantiated these observations (Fig.4B).
  • HA130 dose-dependently inhibited HCV replication as illustrated by the reduction in GLuc signal (Fig.4C).
  • Example 4 LPA promotes HCV infection via stabilization of HIF-1 alpha
  • HIF-1 alpha inhibition abrogates HCV replication (Wilson (2012) Journal of Hepatology 56(4), 803-809) the role of HIF-1alpha in ATX-LPA-dependent HCV infection was investigated.
  • Huh-7.5 cells were incubated with LPA in the presence or absence of LPAR inhibitor KM6425 for 24h and lysates probed for HIF-1alpha expression.
  • HIF- 1 alpha binds to and elicits its transcriptional effects through hypoxia responsive elements (HRE) (for review see Wenger (2005) Science's STKE : signal transduction knowledge environment 2005 (306):re12.). Consistent with our observation that LPA stabilizes HIF-1 alpha an LPA- dependent increase in HRE transcriptional activity was demonstrated (Fig.5B). As a positive control HRE-Luc expressing cells were incubated under hypoxic conditions at 3% oxygen for 24h, resulting in a 7-fold stimulation of HRE-Luc activity that was not further potentiated by LPA (Fig.5B).
  • NSC is a specific inhibitor of the HIF pathway (Chau (2005) Cancer Research 65(11), 4918- 4928), incubating Huh-7.5 cells with LPA in the presence of an increasing concentration of NSC reveals a dose-dependent reduction in LPA-promoted HCV infection (Fig.5C). Importantly this was observed at a concentration (25nM) that, in the absence of LPA, had no significant effect on HCV infection (Fig.5C). The HRE-Luc reporter assay confirmed the efficacy of 25nM NSC on inhibition of HIF (Fig.5D). These data demonstrate a role for HIF-1 alpha stabilization in LPA- dependent promotion of HCV infection.
  • HIF-1 alpha expression was studied in LPA treated Huh-7.5 cells pre-incubated with inhibitors of PI3K (wortmannin, BYL-719, TGX-221) or MAPK (U0126).
  • Western blotting revealed stabilization of HIF-1alpha was inhibited in the presence of the pan PI3K inhibitor wortmannin (Fig.5A).
  • BYL-719 an inhibitor of p110a class I PI3Ks, inhibited HIF-1alpha stabilization in response to LPA treatment whereas TG-221 an inhibitor of p110b class I PI3Ks did not (Fig.5A) illustrating a role for p110a class I PI3Ks.
  • Incubation with the MAPK/MEK inhibitor U0126 also inhibited LPA stabilization of HIF-1 alpha (Fig.5A).
  • HepG2.2.15 cells that support HBV replication and secrete particles, allowed the role of ATX in the viral lifecycle to be investigated. It was confirmed that HepG2.2.15 cells express ATX and incubating with HA130 significantly reduced the levels of HBV DNA in the extracellular media and yet had no effect on the levels of intracellular HBV DNA (Fig.6A), consistent with a role for ATX in the assembly or secretion of HBV particles.
  • HepG2.2.15 cells were transduced with an shRNA vector targeting ATX or empty vector control and rescue experiments performed by transfecting silenced cells with wild type ATX (ATX-wt) or the enzymatically inactive ATX mutant T210A (ATX-T210A).
  • Example 6 ATX plays a role in HBV infection of naive target cells
  • Example 7 A role for ATX in HBV persistently infected cells
  • Differentiated HBV infected HepG2.2.15 producer cells were cultured without serum for 24h and the effect of HA130 (100nM) on intracellular or extracellular HBV DNA assessed over a 24h period (Fig.8A).
  • differentiated HepG2.2.15 cells were transduced with shRNA targeting ATX (shATX) or an irrelevant control (shControl).
  • shRNA-ATX cells were trans-complemented with wild-type ATX (shATX-wt) or a catalytically inactive mutant (ATX-T210A) and intracellular or extracellular HBV DNA levels measured (Fig.8B).
  • Western blots depict extracellular ATX expression.
  • Example 8 LPA modulates hepatitis virus infection via stabilizing HIF-1 a
  • Huh-7 cells were transfected with HRE-Luc and cultured under serum-free conditions prior to incubating with LPA (10 ⁇ ) in the presence or absence of LPAR antagonist KM6425 (10 ⁇ ) and cultured under 20% oxygen for 24h (Fig.9A).
  • HIF-1 a expression was confirmed by Western blotting. Data are representative of three independent experiments and were compared using a Student's t-test (**** P ⁇ 0.0001).
  • Differentiated HepG2-NTCP were transfected with siRNA targeting HIF-1 a or control for 48h, cultured without serum for 24h prior to infecting with HBV.
  • HIF-1 a expression was confirmed by Western blotting.
  • HBV infection was quantified 7 days later by measuring cellular viral DNA and HBeAg (Fig.9B).
  • HepG2-NTCP were cultured without serum, treated with LPA (10 ⁇ ) and cell lysates separated by SDS- PAGE and probed for phospho-AKT (pT308 AKT) or total AKT (AKT) (Fig.9C).
  • Serum-free Huh-7 cells were treated with wortmannin (WM - 200nM), BYL-719 (2 ⁇ ) or TGX-221 (50nM) prior to a 15 min incubation with LPA (10 ⁇ ) before lysis and Western blotting for HIF-1 a (Fig.9D).
  • Fig.9E Differentiated HepG2-NTCP cells were cultured without serum for 24h, treated with wortmannin (200nM), BYL-779 (2 ⁇ ) or TGX-221 (50nM) for 15 mins prior to infecting with HBV and infection quantified 7 days later by measuring HBeAg and HCV NS5A expressing cells enumerated 24h post inoculation (Fig.9E). Infectivity is expressed relative to control and represents the mean of three replicate infections analysed by Student's t-test (**** P ⁇ 0.0001). The results in Fig.9 show that LPA elevates HIF-1 alpha expression and stabilisation in liver cells in a PI3kinase dependent manner, it also shows that infection of such cells with HBV requires HIF-1 alpha.
  • LPA receptors 1-6 mRNA in liver of HBV infected SCID-upA chimeric mice were sacrificed at 37 days post-inoculation and HBV DNA levels were 5600- 34200 lU/mL.
  • the results in Fig.10 show that infection of human hepatocytes transplanted into SCID-uPA mouse liver with HBV induces an increase in mRNA for LPAR1 with limited effect upon the expression of the other LPA receptors.
  • ATX exerts its effects via generation of LPA and downstream activation of signalling events via LPARs.
  • Studying the modulation of ATX activity for effect(s) on HCV infection it has been identified for the first time that ATX-LPA signalling has an important role in HCV replication.
  • LPARs 1 , 3 and 6 are expressed in the liver and (Sokolov (2013, supra)) hepatoma cells. The results herein demonstrate that ATX-LPA-dependent promotion of HCV infection is inhibited by KM6425 which suggests a role for LPAR1 or 3 in this process.
  • HCV replication Many aspects of HCV replication have yet to be understood, it was previously reported that inhibiting HIF-1 alpha reduced HCV replication (Wilson (2012, supra) demonstrating an essential role for HIF-1 alpha in HCV replication (Vassilaki (2013) Journal of Virology 87(5), 2935-2948). Under normal oxygen levels HIF-1 alpha, expression is tightly controlled by prolyl hydroxylases (PHDs) (Bruick (2001) Science (New York, NY) 294(5545), 1337-1340) targeting HIF-1 alpha for degradation via the proteasomal pathway.
  • PDHs prolyl hydroxylases
  • HIF-1 alpha overexpression in Huh-7.5 cells under normoxic conditions up-regulates ATX mRNA levels, demonstrating for the first time that ATX is a HIF-regulated gene. This observation is consistent with elevated ATX expression reported in tumors that frequently express HIFs.
  • LPA stabilizes HIF-1 alpha protein expression and HRE-transcriptional activity that was inhibited by KM6425, illustrating a role for LPARsl and/or 3.
  • LPA had no further effect on HRE-transcriptional activity.
  • LPA increases VEGF expression through induction of HIF-1alpha, which in turn stimulates ATX expression, providing a positive feedback that promotes cancer progression
  • HIF-1alpha increases VEGF expression through induction of HIF-1alpha, which in turn stimulates ATX expression, providing a positive feedback that promotes cancer progression
  • HBV infection stabilizes HIF-1 alpha (Liu (2014) British Journal of Cancer 1 10(4), 1066-1073; Yoo (2008) Oncogene 27(24), 3405-3413), signifying this pathway to be a common target for both viruses.
  • the study herein reveals that inhibition of the ATX-LPA signalling axis diminishes the secretion of HBV particles.
  • the viral protein HBx plays an important role in replication and stabilizes HIF-1 alpha (Yoo (2003) The Journal of Biological Chemistry 278(40), 39076-39084; Yoo (2004) FEBS letters 577(1-2), 121-126; Yoo (2008) supra), if a similar feedback loop exists its modulation could provide a mechanism for the reduced secretion of HBV observed following inhibition of ATX-LPA signalling.
  • the lack of effect of LPA on HBV production may be due to HIF-1alpha already being stabilized in these cells a theory supported by the inability of LPA to promote HRE-Luc under hypoxia.
  • Epstein Barr virus mediated generation of LPA via enhancement of ATX expression augments the proliferation and survival of EBV infected Hodgkin lymphoma (HL) cells and contributes to the pathogenesis of HL (Baumforth (2005) supra).
  • HCV and HBV infection promotes ATX expression and LPA has been shown to promote invasion of hepatoma cells (Wu (2010, supra)).
  • LPA transactivates receptor tyrosine kinase such as EGFR (Miyamoto (2004) Cancer Research 64(16), 5720-5727).
  • HCV has been shown to phosphorylate EGFR (Diao (2012) Journal of Virology 86(20), 10935-10949) and EGF has been identified as an important host cell factor in HCV infection (Lupberger (2011) Nature Medicine 17(5), 589-595).
  • HBx increases EGFR gene expression (Menzo (1993) Virology 196(2), 878-882) and serum EGF is increased in HBV infected patients (Barreiros (2009) International Journal of Cancer 124(1), 120-129). Increased EGF receptor signalling is associated with several cancers (for review see (Normanno (2006) Gene 366(1), 2-16).
  • the implication of EGFR signalling in HCV and HBV infection may have far reaching consequences in terms of ATX/LPA signalling and the provision of tumour progression.
  • LPARs are G-protein coupled receptors that fall into two distinct groups, endothelial differentiated gene (Edg) including LPARs 1 , 2 and 3 and non-Edg (puringenic) including LPARs 4, 5 and 6 receptors of the EDG. Consequently, ligand binding results in the activation of a myriad downstream signalling pathways including, amongst others, mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K). It has been shown herein that KM6325 inhibits LPA-stimulated HIF-1alpha stabilization and suggests a role for LPARs 1 and 3.
  • Edg endothelial differentiated gene
  • PI3K phosphatidylinositol-3-kinase
  • HCV non-structural protein NS5A which plays a key role in replication of the virus (Fridell (201 1) Journal of Virology 85(14), 7312-7320), enhances the phosphotransferase activity of PI3K p1 10 in HCV replicating cells (Street (2004) The Journal of Biological Chemistry 279(13), 12232-12241). Activation of PI3K signalling contributes to the maintenance of HCV replication and survival of host cells (Mannova (2005) Journal of Virology 79(14), 8742-8749).
  • HCV activation of the PI3K/Akt signalling pathway augments inflammation and fibrosis in the liver (Huang (201 1) Hepatology Research : the official journal of the Japan Society of Hepatology 41 (5), 430-436).
  • PI3K has been implicated in hepatic stellate cell driven fibrosis (Son (2009) Hepatology 50(5), 1512-1523). As a consequence of the liver attempting to heal itself virus infection leads to fibrosis.
  • LPA signaling drives chronic wound healing leading to fibrosis, indeed antagonists against LPAR1 and 3 are shown to have anti-fibrotic capabilities and LPA modulators are being developed as therapies for fibrosis (Rancoule (201 1) Expert Opinion on Investigational Drugs 20(5), 657-667; Rancoule (2014) Biochimica et Biophysica Acta 1841 (1):88-96; Budd (2013) Future Medicinal Chemistry 5(16), 1935-1952).
  • HPB the official journal of the International Hepato Pancreato Biliary Association 16(6), 534-542.
  • Such an increase during viral infection could exacerbate viral infection by further enhancement of ATX-LPA signalling.
  • HCV and HBV hepatocellular carcinoma
  • the data presented herein suggests modulation of ATX by viral-hepatitis may provide a mechanism for the increased incidence of HCC in virus-infected individuals.
  • a role for ATX in the lifecycle of HCV and HBV has been identified herein, highlighting potential new targets for therapy and the prospect of stratifying therapies for the treatment of viral-associated and non-associated HCC.

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Abstract

The invention relates to a method of diagnosing and treating hepatitis virus infection, such as hepatitis B or C virus infection, or a disease mediated by hepatitis infection, such as hepatitis B or C virus infection, by using the autotaxin-lysophosphatidic acid signalling pathway. The invention also relates to biomarkers for hepatitis infection, such as hepatitis B or C virus infection, or a disease mediated by hepatitis infection, such as hepatitis B or C virus infection.

Description

DIAGNOSTIC AND THERAPEUTIC METHOD INVOLVING THE AUTOTAXIN-LYSOPHOSPHATIDIC ACID SIGNALLING PATHWAY
FIELD OF THE INVENTION
The invention relates to a method of diagnosing and treating hepatitis virus infection, such as hepatitis B or C virus infection, or a disease mediated by hepatitis infection, such as hepatitis B or C virus infection. The invention also relates to biomarkers for hepatitis infection, such as hepatitis B or C virus infection, or a disease mediated by hepatitis infection, such as hepatitis B or C virus infection.
BACKGROUND OF THE INVENTION
Hepatitis B virus (HBV) is the major cause of hepatocellular carcinoma. HBV associated liver disease is a major health problem of global impact. An estimated 14 million European patients suffer with chronic hepatitis B with an associated mortality rate of 36,000 deaths/year. Hepatitis B associated liver disease includes cirrhosis and hepatocellular carcinoma (HCC), and is the leading cause of death among cirrhotic patients (El-Serag (2012) Gastroenterology 142(6), 1264-1273). In 2008, almost 750,000 new HCC cases were reported worldwide and given the growing incidence of HCC (Villanueva (2011) Gastroenterology 140, 1410), the economic burden will increase in Western populations during the next decades. Although early-stage tumors can be curatively treated with surgical approaches, efficient treatment options for advanced HCC are limited or absent. Thus, HBV-induced liver disease is a major challenge for public health.
Efficient treatment strategies to cure chronic hepatitis B are lacking. The absence of curative treatments for chronic hepatitis B is a key challenge in patient management (Kwon (2011) Nature Reviews Gastro & Hepatol 8, 275). The current standard of care consists of nucleos(t)ide analogues (NUCs), including lamivudine and tenofovir that suppress HBV replication and limit disease progression. Although these drugs limit disease progression, they rarely clear the virus. The unique epigenomic replication strategy of HBV potentiates viral persistence. As a consequence, viral relapse is commonly observed after cessation of treatment. Although NUCs with a high barrier of resistance have been introduced, drug resistant variants evolve during long-term treatment and cross-resistance to other agents represents a significant limitation to current therapies. Furthermore, the long-term safety of sustained therapy is unknown. It is important to note that 90% of acutely infected adults clear HBV via strong and multispecific T cell responses that eliminate infected cells by cytolytic and non-cytolytic means (Reherman (2005) Nature Rev Immunol 5, 215). In contrast, persistent infection is common amongst immune compromised patients or those who acquired their infection via perinatal transmission, highlighting the important role of host immune responses in resolving HBV. Attempts to stimulate host anti-viral immune responses with interferon alpha have been disappointing, with low efficacy and adverse effects preventing wide-spread use. Thus, new anti-viral agents with a different mechanism of action, such as immune-based therapies, are urgently needed.
Hepatitis C is an infectious disease affecting primarily the liver, caused by the hepatitis C virus (HCV). The infection is often asymptomatic, but chronic infection can lead to scarring of the liver and ultimately to cirrhosis, which is generally apparent after many years. In some cases, those with cirrhosis will go on to develop liver failure, liver cancer, or life-threatening esophageal and gastric varices. HCV is spread primarily by blood-to-blood contact associated with intravenous drug use, poorly sterilized medical equipment, and transfusions. An estimated 150-200 million people worldwide are infected with hepatitis C. The existence of hepatitis C (originally identifiable only as a type of non-A non-B hepatitis) was suggested in the 1970s and proven in 1989. Hepatitis C infects only humans and chimpanzees.
The virus persists in the liver in about 85% of those infected. This chronic infection can be treated with medication: the standard therapy is a combination of peginterferon and ribavirin, with either boceprevir or telaprevir added in some cases. Overall, 50-80% of people treated are cured. Those who develop cirrhosis or liver cancer may require a liver transplant. Hepatitis C is the leading reason for liver transplantation, though the virus usually recurs after transplantation. No vaccine against hepatitis C is available.
There is therefore an urgent need for diagnostic and therapeutic methods for the effective treatment of hepatitis infection such as hepatitis B or C virus infection or a disease mediated by hepatitis infection, such as hepatitis B or C virus infection.
SUMMARY OF THE INVENTION
According to a first aspect of the invention, there is provided an autotaxin-lysophosphatidic acid signalling pathway modulator for use in the prophylaxis or treatment of hepatitis infection or a disease mediated by hepatitis infection.
According to a further aspect of the invention, there is provided the use of hypoxia inducible factor 1 , alpha subunit (HIF-1 alpha), autotaxin (ATX), lysophosphatidylcholine (LPC) or lysophosphatidic acid (LPA) as a biomarker for hepatitis infection or a disease mediated by hepatitis infection. According to a further aspect of the invention, there is provided a method of diagnosing hepatitis infection or a disease mediated by hepatitis infection in an individual thereto, comprising:
(a) quantifying the amounts of hypoxia inducible factor 1 , alpha subunit (HIF-
1 alpha), autotaxin, lysophosphatidylcholine or lysophosphatidic acid in a biological sample obtained from an individual; and
(b) comparing the amounts of hypoxia inducible factor 1 , alpha subunit (HIF- 1 alpha), autotaxin, lysophosphatidylcholine or lysophosphatidic acid in the biological sample with the amounts present in a normal control biological sample from a normal subject,
wherein a difference in the level of hypoxia inducible factor 1 , alpha subunit (HIF- 1 alpha), autotaxin, lysophosphatidylcholine or lysophosphatidic acid in the biological sample is indicative of hepatitis infection or a disease mediated by hepatitis infection. According to a further aspect of the invention, there is provided a method of monitoring efficacy of a therapy in a subject having, suspected of having, hepatitis infection or a disease mediated by hepatitis infection, comprising:
(a) quantifying the amounts of hypoxia inducible factor 1 , alpha subunit (HIF- 1 alpha), autotaxin, lysophosphatidylcholine or lysophosphatidic acid in a biological sample obtained from an individual prior to and/or during and/or following therapy for hepatitis infection or a disease mediated by hepatitis infection; and
(b) comparing the amount of hypoxia inducible factor 1 , alpha subunit (HIF- 1 alpha), autotaxin, lysophosphatidylcholine or lysophosphatidic acid in said sample obtained prior to therapy with the amount present in one or more samples taken from said subject during and/or following therapy,
wherein a difference in the level of hypoxia inducible factor 1 , alpha subunit (HIF- 1 alpha), autotaxin, lysophosphatidylcholine or lysophosphatidic acid in the biological sample is indicative of an effect of said therapy. According to a further aspect of the invention, there is provided the use of a kit comprising a biosensor capable of detecting and/or quantifying hypoxia inducible factor 1 , alpha subunit (HIF-1 alpha), autotaxin, lysophosphatidylcholine or lysophosphatidic acid for monitoring, prognosing or diagnosing hepatitis infection or a disease mediated by hepatitis infection. According to a further aspect of the invention, there is provided a method of screening for an autotaxin-lysophosphatidic acid signalling pathway modulator, which comprises the steps of: (a) contacting a virally infected hepatoma cell with a test compound; and
(b) comparing the effect of said test compound upon the autotaxin- lysophosphatidic acid signalling pathway within said cell. According to a further aspect of the invention, there is provided an autotaxin-lysophosphatidic acid signalling pathway modulator obtainable by a method as defined herein.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 : Hepatic ATX expression in viral and non-viral associated liver disease. (A) ATX mRNA levels in liver tissue from normal (n=4), HBV (n=6), HCV (n=4), alcoholic liver disease (ALD) (n=4), primary biliary cirrhosis (PBC) (n=4), primary sclerosing cholangitis (PSC) (n=4), and autoimmune hepatitis (AIH) (n=2). (B) Representative immmunohistochemical stains of ATX in normal, HBV and HCV infected liver tissue. (C) ATX mRNA expression in human liver slices (n=5 donors) or Huh-7 cells cultured under normoxic (20% 02,) or hypoxic (3% 02) conditions for 16h. (D) Huh-7 cells were transfected to express human HIF-1a and a luciferase reporter under the control of a hypoxic responsive element (HRE) or empty pCDNA3.1 (Control) and cultured under normoxic conditions for 24h. HIF-1a expression was confirmed by Western blotting and an increase was noted in HRE- transcriptional activity expressed as relative light units (RLU) and ATX mRNA levels. uPA- SCID mice transplanted with human hepatocytes were infected with HBV (genotype E) or HCV (genotype 2) and sacrificed at 5 and 16 weeks post infection, respectively. Serum viral loads at the time of sacrifice ranged between 26,600 - 248,400 and 9,300 - 34,200 lU/mL for HBV and HCV respectively. Virus infection induced a significant increase in ATX mRNA (E) and hepatocellular protein expression (F). Primers were designed to detect human ATX and no PCR products were obtained from the tissue of untransplanted mice (data not shown). (G) HBV or HCV strain SA13/JFH infection increases ATX mRNA (shown as graph) and protein secreted from HepG2-NTCP or Huh-7 cells (upper panel), respectively. Infection was confirmed by measuring HBeAg and intracellular HBV DNA (6.7 x 105 HBV DNA copies/ng total DNA) or HCV RNA (8.4 x 103 HCV RNA copies/105 cells). Gene expression is expressed relative to control where all samples have been normalized to GAPDH. The median and range of each data set is presented and is representative of three independent PCR amplifications. Data was compared using a non-parametric Kruskall-Wallis ANOVA with Dunn's Multiple comparison test (A, C, D (right panel), E, G) [* P <0.05, ** P <0.01 , *** P <0.001 , **** P <0.0001]) or Student's t-test (D, left panel) [** P<0.01]).
Figure 2: Autotaxin plays an essential role in HCV infection. (A) Huh-7.5 cells were serum starved and incubated with increasing concentrations of HA130 for 60 min prior to 24 h infecting with HCVcc strains SA13/JFH or J6/JFH. The effect of increasing doses of HA130 on HCV infection was analysed by one-way ANOVA (trend analysis [[* P <0.05, *** P <0.001]) (B) ATX mRNA and protein expression in Huh-7.5 cells transduced with plKO.1 control (shControl) or plKO.1 shATX (shATX). For PCR quantification gene expression is expressed relative to control where samples have been normalized to GAPDH expression. The median and range of each data set is presented and is representative of three independent PCR amplifications. Data was compared using students T-test ([**** P <0.0001]). Control (shControl) and ATX- silenced (shATX) Huh-7.5 cells were serum starved prior to infection with HCVcc strains SA13/JFH□ or J6/JFH■ for 24h. Data was compared using a one-way ANOVA (Holm-Sidak [**** P <0.0001]) (C) Control (shControl) and ATX-silenced (shATX) Huh-7.5 cells were transfected to express ATX-wt or ATX-T210A and ATX expression determined by western blot. Serum starved Huh-7.5 cells expressing shControl minus/plus ATX-wt or ATX-T210A □, shATX minus/plus ATX-wt or ATX-T210A■ were infected with HCVcc SA13/JFH or J6/JFH for 24h. Data was compared using a one-way ANOVA (Holm-Sidak [* P <0.05, ** P <0.01]) (D) Serum starved Huh-7.5 cells expressing shControl minus/plus ATX-wt or ATX-T210A □, shATX minus/plus ATX-wt or ATX-T210A■ were incubated with HA130 (100nM, 60min)) or LPA (10μΜ, 15min) prior to infection with HCVcc SA13/JFH or J6/JFH for 24h. Data was compared using a one-way ANOVA (Holm-Sidak [* P <0.05, ** P <0.01 , *** P <0.001 , **** P <0.0001]).
Figure 3: LPA plays an essential role in HCV infection (A) Serum starved Huh-7.5 cells pre-incubated for 15 min with LPA and were infected with HCVcc strains SA13/JFH□ or J6/JFH■ for 24h. The effect of increasing doses of LPA on HCV infection was analysed by one-way ANOVA (trend analysis [[* P <0.05, *** P <0.001]). (B) Huh-7.5 cells were serum starved and incubated in the presence/absence of Ki 16425 (10μΜ) for 30min prior to 15min incubation in the presence/absence of LPA. Cells were subsequently infected with HCVcc SA13/JFH□ or J6/JFH■ for 24h. Data was compared using a one-way ANOVA (Holm-Sidak [* P <0.05, ** P <0.01 , *** P <0.001 , **** P <0.0001]). For all experiments infectivity is expressed relative to control and represents the mean of three replicate infections. (C) Supernatants harvested from serum starved Huh-7.5 cells were subjected to LC-MS/MS analysis and both amount and species of LPA quantified (ng).
Figure 4: ATX-LPA axis has an essential role in HCV RNA replication. (A) Huh-7.5 cells were serum starved and incubated with HA130 (100nM) for 60 min prior to 24 h infection with HCVpp-H77 □ or HCVpp-1A38 ■. Infectivity is expressed relative to control and represents the mean of three replicate infections. (B) Serum starved Huh-7 cells expressing the Luc2A-JFH replicon were incubated with HA130 (100nM) for 24h or transfected to express shControl or shATX (24h). Cells were lysed and luciferase detection performed. Infectivity is expressed relative to control and represents the mean of three replicate infections. Data was compared using students T-test ([*** P <0.001]). (C) Huh-7.5 cells were transfected to express GL.UC-SA13. After 24h cells were serum starved and incubated for a further 24h with increasing concentrations of HA130. Cells were lysed and luciferase detection performed. Infectivity is expressed relative to control and represents the mean of three replicate infections. The effect of increasing doses of HA130 on HCV infection was analysed by one-way ANOVA (trend analysis [*** P <0.001]). (D) Supernatants harvested from serum starved HCV infected (SA13/JFH□ or J6/JFH■) Huh-7.5 cells incubated with HA130 (100nM) for 24h were used to infect serum starve naive Huh-7.5 cells. Infectivity was quantified by NS5A detection 24h post infection. Infectivity is expressed relative to control and represents the mean of three replicate infections. Data was compared using a one-way ANOVA (Holm-Sidak [** P <0.01]). (E) Supernatants harvested from serum starved Huh-7.5 cells were subjected to LC-MS/MS analysis and both amount and species of LPA quantified (ng).
Figure 5: LPA promotes HCV infection via stabilization of HIF-1 alpha. (A) Serum starved Huh-7.5 cells were incubated with LPA (10μΜ) in the absence or presence of Ki16425 (10μΜ), U0126 (10μΜ), wortmannin (200nM), BYL-779 (2μΜ) or TGX-221 (50nM) for 24h. Lysates were separated by SDS-PAGE and detection of HIF-1alpha performed. (B) Huh-7.5 cells were serum starved and following pre-incubation with or without Ki16425 (10μΜ), U0126 (10μΜ), wortmannin (200nM), BYL-779 (2μΜ) or TGX-221 (50nM) (15min) incubated in the absence□ or presence■ of LPA (10μΜ) for 15min prior to infection with HCV (J6/JFH) for 24h. Infectivity is expressed relative to control and represents the mean of three replicate infections. Data is presented from a single experiment representative of three independent experiments and compared using students T-test ([* P <0.05], [**** P <0.0001]). (C) Huh-7.5 cells were transected with HRE-Luc and following serum starvation incubated with LPA (10μΜ) in the absence or presence of Ki16425 (10μΜ) under normoxic (20% oxygen□) or hypoxic (3% oxygen■) conditions for 24h. Cells were lysed and luciferase activity measured. Data is presented from a single experiment representative of three independent experiments and compared compared using a one-way ANOVA (Holm-Sidak [**** P <0.0001]). (D) Huh-7.5 cells were serum starved and incubated with NSC (25μΜ) in the absence□ or presence■ of LPA (10μΜ) for 15min prior to infection with HCV (SA13/JFH) for 24h. Infectivity is expressed relative to control and represents the mean of three replicate infections. Data is presented from a single experiment representative of three independent experiments and compared using students T-test ([** P <0.01]). (E) Huh-7.5 transfected to express HRE-Luc were serum starved and incubated NSC (25μΜ) under normoxic (20% oxygen □) or hypoxic (3% oxygen ■) conditions for 24h. Cells were lysed and luciferase detection performed. HRE-Luc activity is expressed relative to control and represents the mean of three replicates. Data is presented from a single experiment representative of three independent experiments and compared using students T-test ([**** P <0.0001]). Figure 6: ATX plays an essential role in the genesis of extracellular HBV. (A)
HepG2.2.15 cells were serum starved and incubated with HA130 (100nM) for 24h. Supernatants (Extracellular) were harvested and cells lysed (Intracellular) for detection of total HBV DNA. Data is presented as copies of HBV/ug DNA. Data is presented from a single experiment representative of two independent experiments and compared using a one-way ANOVA (Sidak [* P <0.05]). (B) ATX expression in HepG2.2.15 cells transduced with plKO.1 control (shControl) or plKO.1 shATX (shATX) minus/plus ATX-wt or ATX-T210A determined by western blot. Supernatants were harvested and the levels of HBV DNA and HBsAg secreted from serum starved HepG2.2.15 cells transduced with shControl orr shATX minus/plus ATX- wt or ATX-T210A measured. Data is presented as copies of HBV/ug DNA. Data is presented from a single experiment representative of two independent experiments and compared using a one-way ANOVA (Sidak [* P <0.005, ** P <0.01]).
Figure 7: ATX plays a role in HBV infection of naive target cells. Differentiated HepG2-NTCP cells were treated with HA130 (100nM) for 1 h or transduced with shRNA targeting ATX or an irrelevant control prior to infecting with HBV. ATX expression was confirmed by Western blotting. HBV infection was quantified 7 days later by measuring intracellular and extracellular viral DNA and HBV encoded pre-core or e antigen (HBeAg). For all experiments infectivity is expressed relative to control and represents the mean of three replicate infections analysed by Student's t-test (* P <0.05, *** P <0.001).
Figure 8: A role for ATX in HBV persistently infected cells. (A) Differentiated HBV infected HepG2.2.15 producer cells were cultured without serum for 24h and the effect of HA130 (100nM) on intracellular or extracellular HBV DNA assessed over a 24h period. (B) To confirm the inhibitory effect of HA130 on HBV particle secretion, differentiated HepG2.2.15 cells were transduced with shRNA targeting ATX (shATX) or an irrelevant control (shControl). shRNA-ATX cells were trans-complemented with wild-type ATX (shATX-wt) or a catalytically inactive mutant (ATX-T210A) and intracellular or extracellular HBV DNA levels measured. Western blots depict extracellular ATX expression. The median and range of the data from three independent experiments are presented and were compared using Student's t-test (*** P O.001).
Figure 9: LPA modulates hepatitis virus infection via stabilizing HIF-1 a. (A) Huh- 7 cells were transfected with HRE-Luc and cultured under serum-free conditions prior to incubating with LPA (10uM) in the presence or absence of LPAR antagonist KM6425 (10uM) and cultured under 20% oxygen for24h. HIF-1a expression was confirmed by Western blotting. Data are representative of three independent experiments and were compared using a Student's t-test (**** P <0.0001). (B) Differentiated HepG2-NTCP were transfected with siRNA targeting HIF-1 a or control for 48h, cultured without serum for 24h prior to infecting with HBV. HIF-1 a expression was confirmed by Western blotting. HBV infection was quantified 7 days later by measuring cellular viral DNA and HBeAg. For all experiments infectivity is expressed relative to control and represents the mean of three replicate infections analysed by Student's t-test (*** P <0.001 , **** P <0.0001). (C) HepG2-NTCP were cultured without serum, treated with LPA (10uM) and cell lysates separated by SDS-PAGE and probed for phospho-AKT (pT308 AKT) or total AKT (AKT). (D) Serum-free Huh-7 cells were treated with wortmannin (WM - 200nM), BYL-719 (2uM) or TGX-221 (50nM) prior to a 15 min incubation with LPA (10uM) before lysis and Western blotting for HIF-1 a. (E) Differentiated HepG2-NTCP cells were cultured without serum for 24h, treated with wortmannin (200nM), BYL-779 (2uM) or TGX-221 (50nM) for 15 mins prior to infecting with HBV and infection quantified 7 days later by measuring HBeAg and HCV NS5A expressing cells enumerated 24h post inoculation. Infectivity is expressed relative to control and represents the mean of three replicate infections analysed by Student's t-test (**** P <0.0001).
Figure 10: LPA receptor expression in SCID-upA chimeric mice. Expression of LPA receptors 1-6 mRNA in liver of HBV infected SCID-upA chimeric mice relative to naive mice. Mice were sacrificed at 37 days post-inoculation and HBV DNA levels were 5600-34200 lU/mL.
DETAILED DESCRIPTION OF THE INVENTION
According to a first aspect of the invention, there is provided an autotaxin-lysophosphatidic acid signalling pathway modulator for use in the prophylaxis or treatment of hepatitis infection or a disease mediated by hepatitis infection. Autotaxin (ATX), a member of the ectonucleotide pyrophosphatase/phosphodiesterase family of proteins, was originally identified as a motility-stimulating factor secreted from melanoma cells (Stracke (1992) J Biol Chem 267 (4):2524-2529). Indeed ATX is over-expressed in various cancers and several studies have reported an association between ATX expression and rates of tumour progression, angiogenesis and metastasis (for review see Houben (201 1) Cancer metastasis reviews 30 (3-4), 557-565). A 100kDa secreted glycoprotein, ATX hydrolyzes the membrane phospholipid lysophosphatidlycholine (LPC) to generate lysophosphatidic acid (LPA) that activates and signals via a family of six G-protein coupled LPA receptors (LPAR). More recently increased ATX expression has been implicated in the progression of several inflammatory-associated pathologies namely, multiple sclerosis, rheumatoid arthritis, fibrosis and hepatitis (for review see Sevastou (2013) Biochimica et Biophysica Acta 1831 (1):42-60. However, our understanding of the pathways regulating ATX expression in health and disease are limited. Hepatitis, or inflammation of the liver, occurs as a result of virus infection, alcohol or autoimmune induced damage and chronic liver injury leads to scarring or fibrosis. Enhanced ATX expression and concomitant LPA production has been associated with liver fibrosis (for review see Ikeda (2012) Journal of Clinical Chemistry 413(23-24), 1817-1821). In the healthy liver, ATX is rapidly removed from the circulation by sinusoidal endothelial cells (Jansen (2009) Cancer Letters 284(2), 216-221). However, during fibrosis phenotypic changes in the sinusoidal endothelium (Muro (1993) The American Journal of Pathology 143(1), 105-120) and associated stellate cells (Ikeda (1998) Biochemical and Biophysical Research Communications 248(2), 436-440), impair ATX uptake and degradation. Hepatitis B (HBV) and hepatitis C (HCV) viruses are the major human pathogens that infect the liver and cause liver injury that can progress to cirrhosis and hepatocellular carcinoma (HCC) (El-Serag (2012, supra)). Several studies have reported increased ATX expression in the liver and sera of HCV infected subjects (Watanabe (2007) Journal of Clinical Gastroenterology 41 (6), 616-623; Kremer (2010) Gastroenterology 139 (3): 1008-1018; Wu (2010) Molecular Cancer 9, 71 ; Cooper (2007) Journal of Gastrointestinal Surgery : Official Journal of the Society for Surgery of the Alimentary Tract 11 (12), 1628-1634; Kondo (2014) Clinica Chimica Acta; International Journal of Clinical Chemistry 433, 128-134), however, the mechanism underlying increased ATX expression was not investigated nor the role of ATX in the viral lifecycle. In contrast, there have been no reports to date on ATX expression in chronic hepatitis B. The data presented herein surprisingly demonstrate that ATX is transcriptionally regulated by hypoxia inducible factor-1 , alpha subunit (HIF-1 alpha), providing an explanation for increased expression in viral infected and inflamed liver. The data presented herein also confirms increased ATX mRNA and protein expression in HBV and HCV infected human hepatocytes transplanted in the uPA-SCID mouse, demonstrating a direct role for both viruses to regulate ATX expression. Importantly, an essential role for ATX has been identified in HCV replication, demonstrating a requirement for ATX enzymic activity to generate LPA that stimulates HCV RNA replication. Treating Huh-7 hepatoma cells with LPA stabilized HIF-1 alpha and associated transcriptional activity in a PI3K and MAPK dependent manner, providing a mechanism for LPA to promote HCV replication. A role for ATX-LPA signaling has been highlighted in the assembly or transport of extracellular HBV particles, providing a new pathway to regulate HBV dissemination. The data presented herein supports a model where HCV and HBV increase ATX expression and associated LPA genesis, providing an autocrine pathway to promote viral persistence and associated pathogenesis. In summary, the ATX-LPA signaling axis plays an essential role in the lifecycle of two clinically important human pathogens HCV and HBV that are responsible for the majority of HCC cases across the world - highlighting the potential therapeutic value of targeting ATX-LPA to treat viral replication and associated HCC progression.
References herein to "autotaxin-lysophosphatidic acid signalling pathway modulator" refer to any agent, such as an inhibitor (i.e. competitive, non-competitive or un-competitive inhibitor) or antagonist (i.e. competitive, non-competitive or un-competitive antagonist), activator or agonist (i.e. full inverse agonist, partial inverse agonist, silent antagonist, partial agonist, full agonist or super agonist) capable of modulating the signaling effected by the ATX-LPA pathway. The term "ATX-LPA" pathway refers to one or more biological components that participates in or is part of the conversion of lysophosphatidylcholine (LPC) to lysophosphatidyl acid (LPA) or any component upstream or downstream of said participating component.
The data shown herein confirms that increased expression of ATX has been linked with virally infected and inflamed liver, therefore, an inhibitor or antagonist of the ATX-LPA signaling pathway finds great utility in the invention. Thus, in one embodiment, the autotaxin- lysophosphatidic acid signalling pathway modulator is an inhibitor or antagonist of the autotaxin-lysophosphatidic acid signaling pathway.
In one embodiment, the autotaxin-lysophosphatidic acid signalling pathway modulator (i.e. inhibitor or antagonist of the autotaxin-lysophosphatidic acid signaling pathway) is an autotaxin inhibitor. It will be appreciated that any suitable ATX inhibitors may be used with the methods of the invention. Suitable examples include chemical compounds, antibodies which specifically bind to ATX or antibody fragments thereof, ATX substrates, ATX product analogs (such as Brp- LPA) or natural inhibitors (such as PF8380). Examples of suitable antibodies and antibody fragments include monoclonal antibodies, humanized antibodies, single chain antibodies, domain antibodies.
Examples of suitable autotaxin inhibitors or antagonists include the autotaxin inhibitors disclosed in WO 2014/018881 , WO 2012/024620, WO 2012/166415, WO 2012/100018, WO 201 1/1 16867, WO 2011/006569, WO 2011/053597, WO 201 1/002918, US 201 1/01 10886, US 2010/222341 , WO 2010/1 12116, WO 2010/1 15491 , WO 2010/112124, WO 2010/063352, WO 2010/060532, US 2010/016258, WO 2009/151644 and US 2006/270634 (the contents of each of which are herein incorporated by reference) and the autotaxin inhibitors disclosed in US 2013/0202614 (the contents of which are herein incorporated by reference), such as: 6- (3-(piperazin-1-yl)propanoyl)benzo[d]oxazol-2(3H)-one (PF-8380), 3-[(4-{[3-(4-fluorobenzyl)- 2,4-dioxo-1 ,3-thiazolan-5-yliden]methyl}phenoxy)methyl]benzene boronic acid (HA130), 4- tetradecanoylamino)benzyl]phosphonic acid (S32826), hexachlorophene, 2,2- methylenebis(4-chlorophenol), merbromin, eosin Y, bithionol, RJC 03297, NSC48300, NSC10881 , NSC86629, NSC13792, NSC50016, NSC78785, NSC9616, NSC75913, NSC75779, NSC12859, NSC8680, H2L 5210574, H2L 5761473, H2L 5564949, H2L 7839888, H2L 7921385, H2L 7905958 and analogs thereof, vinpocetine, hypericin, phenanthroline, damnacanthal, calmidazolium, flavonols, phenolic acids, free fatty acids, N- acyltyrosines (such as N-acetyl tyrosine), 2-amino-2-[2-(4-octylphenyl)ethyl]propane-1 ,3-diol (FTY720, fingolimod), 1-bromo-3(S)-hydroxy-4-(palmitoyloxy)butyl]phosphonate (Brp-LPA), phosphonates (such as b-hydroxy phosphonate derivatives of LPA and b-keto phosphonate derivatives of LPA), carba analogs of cPA (1-acyl-sn-glycero-2,3-cyclic phosphate), fatty alkyl phosphonates, fatty alkyl thiophosphates, Darmstoff analogs, aminooxy-LPA analogues, alpha-substituted phosphonate analogues of LPA including methylene phosphonate analogues (such as [3(S)-hydroxy-4-(oleoyloxy)butyl]phosphonate and [(3S)-hydroxy-4- palmitoyloxy)butyl]phosphonate), alpha-hydroxymethylene phosphonate analogues (such as dimethyl-[1 ,3(S)-dihydroxy-4-(oleoyloxy)butyl]phosphonate and dimethyl-[1 ,3(S)-dihydroxy-4- (palmitoyloxy)butyl]phosphonate), alpha-chloromethylene phosphonate analogues (such as [1 -chloro-3(S)-hydroxy-4-(oleoyloxy)butyl]phosphonate and [1 -chloro-3(S)-hydroxy-4- (palmitoyloxy)butyl]phosphonate), alpha-bromomethylene phosphonate analogues (such as [1-bromo-3(S)-hydroxy-4-(oleoyloxy)butyl]phosophonate and [1-bromo-3(S)-hydroxy-4- (palmitoyloxy)butyl]phosophonate) and alpha-hydroxymethylene phosphonate analogues (such as [(1S,3S)-dihydroxy-4-(palmitoyloxy)butyl]phosphonate); and LPC analogues including alkylphosphocholines (such as tetradecylphosphocholine, hexadecylphosphocholine, octadecylphosphocholine and oleylphosphocholine), alkylglycero- phosphonocholines (such as (R)-1-decyl-2-hydroxy-sn-glycero-3-phosphonocholine, (R)-1- dodecyl-2-hydroxy-sn-glycero-3-phosphonocholine, (R)-1-tetradecyl-2-hydroxy-sn-glycero-3- phosphonocholine, (R)-1-hexadecyl-2-hydroxy-sn-glycero-3-phosphonocholine, (R)-1- octadecyl-2-hydroxy-sn-glycero-3-phosphonocholine, (S)-1-decyl-2-hydroxy-sn-glycero-3- phosphonocholine, (S)-1-dodecyl-2-hydroxy-sn-glycero-3-phosphonocholine, (S)-1- tetradecyl-2-hydroxy-sn-glycero-3-phosphonocholine, (S)-1-hexadecyl-2-hydroxy-sn-glycero- 3-phosphonocholine and (S)-1-octadecyl-2-hydroxy-sn-glycero-3-phosphonocholine), alkylphosphonocholine (such as tetradecylphosphonocholine, hexadecylphosphonocholine, octadecylphosphonocholine and oleylphosphonocholine) and phosphoramidates (such as N- (2-trimethylaminoethyl)-0-tetradecyl phosphoramidate, N-(2-trimethylaminoethyl)-0- hexadecyl phosphoramidate, N-(2-trimethylaminoethyl)-0-octadecyl phosphoramidate and N- (2-trimethylaminoethyl)-0-oleyl phosphoramidate.
In an alternative embodiment, the autotaxin-lysophosphatidic acid signalling pathway modulator (i.e. inhibitor or antagonist of the autotaxin-lysophosphatidic acid signaling pathway) is an LPC inhibitor or an antagonist of the LPC receptor.
In an alternative embodiment, the autotaxin-lysophosphatidic acid signalling pathway modulator (i.e. inhibitor or antagonist of the autotaxin-lysophosphatidic acid signaling pathway) is a lysophosphatidic acid (LPA) inhibitor.
Examples of suitable LPA inhibitors or antagonists include: the LPA inhibitors disclosed in US 2013/0202614 (the contents of which are herein incorporated by reference), such as: 3-(4-[4- ([1-(2-chlorophenyl)ethoxy]carbonylamino)-3-methyl-5-isoxazolyl]benzylsulfanyl)propanoic acid (ΚΪΙ6425; LPA 1 ,3 antagonist); (4'-{4-[(R)-1-(2-chlorophenyl)-ethoxycarbonylamino]-3- methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (AM966; LPA3 antagonist); isoxazole and thiazole derivatives (such as [1-(2-chlorophenyl)ethyl N-(3-methyl-5-phenylisoxazol-4-yl)- carbamate (LPA2 antagonist)); 1-bromo-3(S)-hydroxy-4-(palmitoyloxy)butyl]phosphonate (Brp-LPA); carba analogs of cPA (1-acyl-sn-glycero-2,3-cyclic phosphate); fatty alkyl phosphonates; fatty alkyl thiophosphates; Darmstoff analogs; aminooxy-LPA analogues; alpha-substituted phosphonate analogues of LPA including methylene phosphonate analogues (such as [3(S)-hydroxy-4-(oleoyloxy)butyl]phosphonate and [(3S)-hydroxy-4- palmitoyloxy)butyl]phosphonate), alpha-hydroxymethylene phosphonate analogues (such as dimethyl-[1 ,3(S)-dihydroxy-4-(oleoyloxy)butyl]phosphonate and dimethyl-[1 ,3(S)-dihydroxy-4- (palmitoyloxy)butyl]phosphonate), alpha-chloromethylene phosphonate analogues (such as [1 -chloro-3(S)-hydroxy-4-(oleoyloxy)butyl]phosphonate and [1 -chloro-3(S)-hydroxy-4- (palmitoyloxy)butyl]phosphonate), alpha-bromomethylene phosphonate analogues (such as [1-bromo-3(S)-hydroxy-4-(oleoyloxy)butyl]phosophonate and [1-bromo-3(S)-hydroxy-4- (palmitoyloxy)butyl]phosophonate) and alpha-hydroxymethylene phosphonate analogues (such as [(1 S,3S)-dihydroxy-4-(palmitoyloxy)butyl]phosphonate); LPA analogues based on a glycerol backbone (such as Ether-LPA an LPA1 antagonist and DGPP an LPA1/3 antagonist); and LPA analogues with non-glycerol backbones (such as NOHPP an LPA1/2 antagonist and VPC-12249 an LPA1/3 antagonist).
In an alternative embodiment, the autotaxin-lysophosphatidic acid signalling pathway modulator (i.e. inhibitor or antagonist of the autotaxin-lysophosphatidic acid signaling pathway) is an antagonist of a G-protein coupled lysophosphatidic acid receptor (LPAR).
In a further embodiment, the LPAR is an antagonist of an endothelial differentiated gene G- protein coupled receptor, such as LPAR 1 , 2, 3, 4, 5 or 6. Data is provided herein which surprisingly demonstrated that Ki 16425 (a specific antagonist for the LPAR 1 and LPAR 3 receptors) inhibited ATX-LPA dependent promotion of HCV infection, thereby suggesting a key role for LPAR 1 and 3 in the pathway. Thus, in a yet further embodiment, the LPAR is an antagonist of LPAR 1 and/or 3, in particular LPAR 1. As presented by the data shown herein, infection of human hepatocytes transplanted into SCID-upA mice with HBV demonstrates up regulation of LPAR1. The inhibition of HBV release by the LPAR antagonist KM6425 is thus mediated by LPAR1. Therefore, this data shows that LPAR1 receptor is involved in HBV infection and that inhibitors against the LPAR1 receptor in particular present useful anti-HBV drugs.
In an alternative embodiment, the autotaxin-lysophosphatidic acid signalling pathway modulator (i.e. inhibitor or antagonist of the autotaxin-lysophosphatidic acid signaling pathway) is an inhibitor of hypoxia inducible factor 1 , alpha subunit (HIF-1 alpha). Hypoxia- inducible factors (HIFs) are transcription factors that respond to changes in available oxygen in the cellular environment, to be specific, to decreases in oxygen, or hypoxia. Data is provided herein which surprisingly demonstrated that ATX is transcriptionally regulated by HIF-1 alpha, therefore, an agent capable of inhibiting the function of HIF-1 alpha will also inhibit autotaxin expression and consequently inhibit the autotaxin-lysophosphatidic acid signalling pathway, resulting in minimizing of the effects of a hepatitis B or C virus infection or a disease mediated by a hepatitis B or C virus infection.
Data is also provided herein which surprisingly demonstrates that treating hepatoma cells with LPA stabilized HIF-1 alpha and associated transcriptional activity in a PI3K and MAPK dependent manner. Thus, in a further embodiment, the inhibitor of hypoxia inducible factor 1 , alpha subunit (HIF-1 alpha) comprises a phosphoinositide 3 kinase (PI3K) or mitogen activated protein kinase (MAPK) signaling modulator. In a yet further embodiment, the phosphoinositide 3 kinase (PI3K) signaling modulator is an inhibitor of p1 10a class I phosphoinositide 3 kinase (PI3K), such as BYL-719. Data is provided herein which surprisingly found that BYL-719, an inhibitor of p110a class I PI3Ks, inhibited HIF-1 alpha stabilization in response to LPA treatment whereas TG-221 , an inhibitor of p1 10b class I PI3Ks, did not (see Fig.5A) therefore illustrating an important role for p1 10a class I PI3Ks in LPA-dependent augmentation of HCV infection.
In an alternative embodiment, the mitogen activated protein kinase (MAPK) signaling modulator is a MAPK/MEK inhibitor, such as U0126. Data is provided herein which surprisingly found that the MAPK/MEK inhibitor U0126 inhibited LPA stabilization of HIF-1 alpha (see Fig.5A) therefore suggesting a role for MAPK signalling in LPA-dependent augmentation of HCV infection.
In one embodiment, the hepatitis infection or disease mediated by hepatitis infection is a hepatitis B or C virus infection or a disease mediated by a hepatitis B or C virus infection.
As discussed hereinbefore, there have been no reports to date on ATX expression in chronic hepatitis B. Thus, in one embodiment, the modulator of the invention may be of use in the prophylaxis or treatment of a hepatitis B infection or a disease mediated by a hepatitis B virus infection.
Examples of diseases mediated by hepatitis infection, such as a hepatitis B or C virus infection, include liver diseases selected from: inflammation of the liver such as hepatitis (i.e. chronic hepatitis), liver fibrosis, cirrhosis or hepatocellular carcinoma, such as hepatocellular carcinoma. According to a further aspect of the invention, there is provided the use of hypoxia inducible factor 1 , alpha subunit (HIF-1 alpha), autotaxin (ATX), lysophosphatidylcholine (LPC) or lysophosphatidic acid (LPA) as a biomarker for hepatitis infection or a disease mediated by hepatitis infection.
The term "biomarker" means a distinctive biological or biologically derived indicator of a process, event, or condition. Biomarkers can be used in methods of diagnosis, e.g. clinical screening, and prognosis assessment and in monitoring the results of therapy, identifying patients most likely to respond to a particular therapeutic treatment, drug screening and development. Biomarkers and uses thereof are valuable for identification of new drug treatments and for discovery of new targets for drug treatment.
Data is provided herein which clearly indicates that the levels of hypoxia inducible factor 1 , alpha subunit (HIF-1 alpha), autotaxin (ATX), lysophosphatidylcholine (LPC) and lysophosphatidic acid (LPA) may be altered in a viral infected and inflamed liver. For example, increased expression of ATX has been observed herein in viral infected and inflamed liver. ATX has been demonstrated to be transcriptionally regulated by HIF-1 alpha, therefore, levels of both ATX and HIF-1alpha are increased in a viral infected and inflamed liver compared with healthy controls. Consequently, increased levels of ATX and HIF-1 alpha will result in increased levels of LPA and decreased levels of LPC because ATX is known to hydrolyse LPC to generate LPA. Therefore, the levels of each of these components provides a direct marker indicative of hepatitis B and C infection compared with healthy controls.
In one embodiment, the biomarker is selected from hypoxia inducible factor 1 , alpha subunit (HIF-1 alpha), autotaxin (ATX) or lysophosphatidic acid (LPA). In a further embodiment, the biomarker is selected from hypoxia inducible factor 1 , alpha subunit (HIF-1 alpha) or lysophosphatidic acid (LPA). In an yet further embodiment, the biomarker is selected from hypoxia inducible factor 1 , alpha subunit (HIF-1 alpha). According to a further aspect of the invention, there is provided a method of diagnosing hepatitis infection or a disease mediated by hepatitis infection in an individual thereto, comprising:
(a) quantifying the amounts of hypoxia inducible factor 1 , alpha subunit (HIF- 1 alpha), autotaxin, lysophosphatidylcholine or lysophosphatidic acid in a biological sample obtained from an individual; and (b) comparing the amounts of hypoxia inducible factor 1 , alpha subunit (HIF- 1 alpha), autotaxin, lysophosphatidylcholine or lysophosphatidic acid in the biological sample with the amounts present in a normal control biological sample from a normal subject,
wherein a difference in the level of hypoxia inducible factor 1 , alpha subunit (HIF- 1 alpha), autotaxin, lysophosphatidylcholine or lysophosphatidic acid in the biological sample is indicative of hepatitis infection or a disease mediated by hepatitis infection.
According to a further aspect of the invention, there is provided a method of monitoring efficacy of a therapy in a subject having, suspected of having, hepatitis infection or a disease mediated by hepatitis infection, comprising:
(a) quantifying the amounts of hypoxia inducible factor 1 , alpha subunit (HIF- 1 alpha), autotaxin, lysophosphatidylcholine or lysophosphatidic acid in a biological sample obtained from an individual prior to and/or during and/or following therapy for hepatitis infection or a disease mediated by hepatitis infection; and
(b) comparing the amount of hypoxia inducible factor 1 , alpha subunit (HIF-
1 alpha), autotaxin, lysophosphatidylcholine or lysophosphatidic acid in said sample obtained prior to therapy with the amount present in one or more samples taken from said subject during and/or following therapy,
wherein a difference in the level of hypoxia inducible factor 1 , alpha subunit (HIF- 1 alpha), autotaxin, lysophosphatidylcholine or lysophosphatidic acid in the biological sample is indicative of an effect of said therapy.
It will be appreciated that references herein to "difference in the level" refer to either a higher or lower level of the biomarker(s) in the test biological sample compared with the control or reference sample(s).
In one embodiment, the higher or lower level is a < 1 fold difference relative to the control or reference sample, such as a fold difference of 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1 , 0.05, 0.01 or any ranges therebetween. In one embodiment, the lower level is between a 0.1 and 0.9 fold difference, such as between a 0.2 and 0.5 fold difference, relative to the control or reference sample.
In one embodiment, the higher or lower level is a > 1 fold difference relative to the control or reference sample, such as a fold difference of 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 10.5, 1 1 , 1 1.5, 12, 12.5, 15 or 20 or any ranges therebetween. In one embodiment, the higher level is between a 1 and 15 fold difference, such as between a 2 and 10 fold difference, relative to the control or reference sample.
It will be appreciated that a higher level of HIF-1alpha, autotaxin and lysophosphatidic acid will be present in samples obtained from a subject having, suspected of having, hepatitis infection or a disease mediated by hepatitis infection compared with a normal control biological sample from a normal subject for the reasons as set out hereinbefore. Conversely, it will be appreciated that a lower level of lysophosphatidylcholine will be present in samples obtained from a subject having, suspected of having, hepatitis infection or a disease mediated by hepatitis infection compared with a normal control biological sample from a normal subject for the reasons as set out hereinbefore.
Monitoring methods of the invention can be used to monitor onset, progression, stabilisation, amelioration and/or remission.
In methods of diagnosing, prognosing or monitoring according to the invention, detecting and/or quantifying the biomarker in a biological sample from a test subject may be performed on two or more occasions. Comparisons may be made between the level of biomarker in samples taken on two or more occasions. Assessment of any change in the level of the biomarker in samples taken on two or more occasions may be performed. Modulation of the biomarker level is useful as an indicator of the state of hepatitis infection or a disease mediated by hepatitis infection. An increase in the level of HIF-1 alpha, autotaxin and lysophosphatidic acid, over time is indicative of onset or progression, i.e. worsening of this disorder, whereas a decrease in the level of HIF-1alpha, autotaxin and lysophosphatidic acid indicates amelioration or remission of the disorder, or vice versa. Conversely, a decrease in the level of lysophosphatidylcholine, over time is indicative of onset or progression, i.e. worsening of this disorder, whereas an increase in the level of lysophosphatidylcholine indicates amelioration or remission of the disorder, or vice versa. A method of diagnosis or prognosis of or monitoring according to the invention may comprise quantifying the biomarker in a test biological sample from a test subject and comparing the level of the biomarker present in said test sample with one or more controls.
Also provided is a method of monitoring efficacy of a therapy for hepatitis infection or a disease mediated by hepatitis infection in a subject having such a disorder or suspected of having such a disorder, comprising detecting and/or quantifying the biomarker present in a biological sample from said subject. In monitoring methods, test samples may be taken on two or more occasions. The method may further comprise comparing the level of the biomarker present in the test sample with one or more reference(s) and/or with one or more previous test sample(s) taken earlier from the same test subject, e.g. prior to commencement of therapy, and/or from the same test subject at an earlier stage of therapy. The method may comprise detecting a change in the level of the biomarker in test samples taken on different occasions.
In one embodiment, the method comprises comparing the amount of biomarker(s) in said test biological sample with the amount present in one or more samples taken from said individual prior to commencement of treatment, and/or one or more samples taken from said individual during treatment.
The term "diagnosis" as used herein encompasses identification, confirmation, and/or characterisation of hepatitis infection or a disease mediated by hepatitis infection. The term "prognosis" as used herein encompasses the prediction of whether a patient it likely to develop hepatitis infection or a disease mediated by hepatitis infection.
Methods of monitoring and of diagnosis or prognosis according to the invention are useful to confirm the existence of a disorder; to monitor development of the disorder by assessing onset and progression, or to assess amelioration or regression of the disorder. Methods of monitoring and of diagnosis or prognosis are also useful in methods for assessment of clinical screening, choice of therapy, evaluation of therapeutic benefit, i.e. for drug screening and drug development. Efficient diagnosis, prognosis and monitoring methods provide very powerful "patient solutions" with the potential for improved prognosis, by establishing the correct diagnosis, allowing rapid identification of the most appropriate treatment (thus lessening unnecessary exposure to harmful drug side effects), reducing relapse rates. Methods for monitoring efficacy of a therapy can be used to monitor the therapeutic effectiveness of existing therapies and new therapies in human subjects and in non-human animals (e.g. in animal models). These monitoring methods can be incorporated into screens for new drug substances and combinations of substances. Any suitable animal may be used as a subject non-human animal, for example a non-human primate, horse, cow, pig, goat, sheep, dog, cat, fish, rodent, e.g. guinea pig, rat or mouse; insect (e.g. Drosophila), amphibian (e.g. Xenopus) or C. elegans. Suitably, the time elapsed between taking samples from a subject undergoing diagnosis or monitoring will be 3 days, 5 days, a week, two weeks, 1 month, 2 months, 3 months, 6 or 12 months. Samples may be taken prior to and/or during and/or following therapy for hepatitis infection or a disease mediated by hepatitis infection. Samples can be taken at intervals over the remaining life, or a part thereof, of a subject.
The term "detecting" as used herein means confirming the presence of the biomarker present in the sample. Quantifying the amount of the biomarker present in a sample may include determining the concentration of the biomarker present in the sample. Detecting and/or quantifying may be performed directly on the sample, or indirectly on an extract therefrom, or on a dilution thereof.
In alternative aspects of the invention, the presence of the biomarker is assessed by detecting and/or quantifying antibody or fragments thereof capable of specific binding to the biomarker that are generated by the subject's body in response to the biomarker and thus are present in a biological sample from a subject having hepatitis infection or a disease mediated by hepatitis infection.
Detecting and/or quantifying can be performed by any method suitable to identify the presence and/or amount of a specific protein in a biological sample from a patient or a purification or extract of a biological sample or a dilution thereof. In methods of the invention, quantifying may be performed by measuring the concentration of the biomarker in the sample or samples. Biological samples that may be tested in a method of the invention include whole blood, blood serum, plasma, cerebrospinal fluid (CSF), urine, saliva, or other bodily fluid (stool, tear fluid, synovial fluid, sputum), breath, e.g. as condensed breath, or an extract or purification therefrom, or dilution thereof. Biological samples also include tissue homogenates, tissue sections and biopsy specimens from a live subject, or taken post-mortem. The samples can be prepared, for example where appropriate diluted or concentrated, and stored in the usual manner. It will be understood that methods of the invention may be performed in vitro. In one embodiment, the biological sample is whole blood, blood serum or plasma, such as blood serum. Detection and/or quantification of biomarkers may be performed by detection of the biomarker or of a fragment thereof, e.g. a fragment with C-terminal truncation, or with N-terminal truncation. Fragments are suitably greater than 4 amino acids in length, for example 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length.
In one embodiment, the biomarker defined herein may be replaced by a molecule, or a measurable fragment of the molecule, found upstream or downstream of the biomarker in a biological pathway.
According to a further aspect of the invention, there is provided the use of a kit comprising a biosensor capable of detecting and/or quantifying hypoxia inducible factor 1 , alpha subunit (HIF-1 alpha), autotaxin, lysophosphatidylcholine or lysophosphatidic acid for monitoring, prognosing or diagnosing hepatitis infection or a disease mediated by hepatitis infection.
As used herein, the term "biosensor" means anything capable of detecting the presence of the biomarker. Examples of biosensors are described herein.
Biosensors according to the invention may comprise a ligand or ligands, as described herein, capable of specific binding to the biomarker. Such biosensors are useful in detecting and/or quantifying the biomarker of the invention.
The biomarker may be directly detected, e.g. by mass spectrometry. Alternatively, the biomarker may be detected directly or indirectly via interaction with a ligand or ligands such as an antibody or a biomarker-binding fragment thereof, or other peptide, or ligand, e.g. aptamer, or oligonucleotide, capable of specifically binding the biomarker. The ligand may possess a detectable label, such as a luminescent, fluorescent or radioactive label, and/or an affinity tag. For example, detecting and/or quantifying can be performed by one or more method(s) selected from the group consisting of: SELDI (-TOF), MALDI (-TOF), a 1-D gel-based analysis, a 2-D gel-based analysis, Mass spec (MS), reverse phase (RP) LC, size permeation (gel filtration), ion exchange, affinity, HPLC, UPLC and other LC or LC MS-based techniques. Appropriate LC MS techniques include ICAT® (Applied Biosystems, CA, USA), or iTRAQ® (Applied Biosystems, CA, USA). Liquid chromatography (e.g. high pressure liquid chromatography (HPLC) or low pressure liquid chromatography (LPLC)), thin-layer chromatography, NMR (nuclear magnetic resonance) spectroscopy could also be used.
Methods according to the invention may comprise analysing a sample of blood serum by SELDI-TOF or MALDI-TOF to detect the presence or level of the biomarker. These methods are also suitable for clinical screening, prognosis, monitoring the results of therapy, identifying patients most likely to respond to a particular therapeutic treatment, for drug screening and development, and identification of new targets for drug treatment. Detecting and/or quantifying the biomarkers may be performed using an immunological method, involving an antibody, or a fragment thereof capable of specific binding to the biomarker. Suitable immunological methods include sandwich immunoassays, such as sandwich ELISA, in which the detection of the biomarkers is performed using two antibodies which recognize different epitopes on a biomarker; radioimmunoassays (RIA), direct, indirect or competitive enzyme linked immunosorbent assays (ELISA), enzyme immunoassays (EIA), Fluorescence immunoassays (FIA), western blotting, immunoprecipitation and any particle- based immunoassay (e.g. using gold, silver, or latex particles, magnetic particles, or Q-dots). Immunological methods may be performed, for example, in microtitre plate or strip format. Immunological methods in accordance with the invention may be based, for example, on any of the following methods.
Immunoprecipitation is the simplest immunoassay method; this measures the quantity of precipitate, which forms after the reagent antibody has incubated with the sample and reacted with the target antigen present therein to form an insoluble aggregate. Immunoprecipitation reactions may be qualitative or quantitative.
In particle immunoassays, several antibodies are linked to the particle, and the particle is able to bind many antigen molecules simultaneously. This greatly accelerates the speed of the visible reaction. This allows rapid and sensitive detection of the biomarker.
In immunonephelometry, the interaction of an antibody and target antigen on the biomarker results in the formation of immune complexes that are too small to precipitate. However, these complexes will scatter incident light and this can be measured using a nephelometer. The antigen, i.e. biomarker, concentration can be determined within minutes of the reaction. Radioimmunoassay (RIA) methods employ radioactive isotopes such as I125 to label either the antigen or antibody. The isotope used emits gamma rays, which are usually measured following removal of unbound (free) radiolabel. The major advantages of RIA, compared with other immunoassays, are higher sensitivity, easy signal detection, and well-established, rapid assays. The major disadvantages are the health and safety risks posed by the use of radiation and the time and expense associated with maintaining a licensed radiation safety and disposal program. For this reason, RIA has been largely replaced in routine clinical laboratory practice by enzyme immunoassays. Enzyme (EIA) immunoassays were developed as an alternative to radioimmunoassays (RIA). These methods use an enzyme to label either the antibody or target antigen. The sensitivity of EIA approaches that of RIA, without the danger posed by radioactive isotopes. One of the most widely used EIA methods for detection is the enzyme-linked immunosorbent assay (ELISA). ELISA methods may use two antibodies one of which is specific for the target antigen and the other of which is coupled to an enzyme, addition of the substrate for the enzyme results in production of a chemiluminescent or fluorescent signal.
Fluorescent immunoassay (FIA) refers to immunoassays which utilize a fluorescent label or an enzyme label which acts on the substrate to form a fluorescent product. Fluorescent measurements are inherently more sensitive than colorimetric (spectrophotometric) measurements. Therefore, FIA methods have greater analytical sensitivity than EIA methods, which employ absorbance (optical density) measurement.
Chemiluminescent immunoassays utilize a chemiluminescent label, which produces light when excited by chemical energy; the emissions are measured using a light detector.
Immunological methods according to the invention can thus be performed using well-known methods. Any direct (e.g., using a sensor chip) or indirect procedure may be used in the detection of the biomarker of the invention.
The Biotin-Avidin or Biotin-Streptavidin systems are generic labelling systems that can be adapted for use in immunological methods of the invention. One binding partner (hapten, antigen, ligand, aptamer, antibody, enzyme etc) is labelled with biotin and the other partner (surface, e.g. well, bead, sensor etc) is labelled with avidin or streptavidin. This is conventional technology for immunoassays, gene probe assays and (bio)sensors, but is an indirect immobilisation route rather than a direct one. For example a biotinylated ligand (e.g. antibody or aptamer) specific for a biomarker of the invention may be immobilised on an avidin or streptavidin surface, the immobilised ligand may then be exposed to a sample containing or suspected of containing the biomarker in order to detect and/or quantify a biomarker of the invention. Detection and/or quantification of the immobilised antigen may then be performed by an immunological method as described herein.
The term "antibody" as used herein includes, but is not limited to: polyclonal, monoclonal, bispecific, humanised or chimeric antibodies, single chain antibodies, Fab fragments and F(ab')2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies and epitope-binding fragments of any of the above. The term "antibody" as used herein also refers to immunoglobulin molecules and immunologically-active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen. The immunoglobulin molecules of the invention can be of any class (e.g., IgG, IgE, IgM, IgD and IgA) or subclass of immunoglobulin molecule.
The identification of key biomarkers specific to a disease is central to integration of diagnostic procedures and therapeutic regimes. Using predictive biomarkers, appropriate diagnostic tools such as biosensors can be developed, accordingly, in methods and uses of the invention, detecting and quantifying can be performed using a biosensor, microanalytical system, microengineered system, microseparation system, immunochromatography system or other suitable analytical devices. The biosensor may incorporate an immunological method for detection of the biomarker, electrical, thermal, magnetic, optical (e.g. hologram) or acoustic technologies. Using such biosensors, it is possible to detect the target biomarker at the anticipated concentrations found in biological samples.
Thus, according to a further aspect of the invention there is provided an apparatus for monitoring hepatitis infection or a disease mediated by hepatitis infection, which comprises a biosensor, microanalytical, microengineered, microseparation and/or immunochromatography system configured to detect and/or quantify one or more of the biomarkers defined herein.
The biomarker of the invention can be detected using a biosensor incorporating technologies based on "smart" holograms, or high frequency acoustic systems, such systems are particularly amenable to "bar code" or array configurations. In smart hologram sensors (Smart Holograms Ltd, Cambridge, UK), a holographic image is stored in a thin polymer film that is sensitised to react specifically with the biomarker. On exposure, the biomarker reacts with the polymer leading to an alteration in the image displayed by the hologram. The test result read-out can be a change in the optical brightness, image, colour and/or position of the image. For qualitative and semi-quantitative applications, a sensor hologram can be read by eye, thus removing the need for detection equipment. A simple colour sensor can be used to read the signal when quantitative measurements are required. Opacity or colour of the sample does not interfere with operation of the sensor. The format of the sensor allows multiplexing for simultaneous detection of several substances. Reversible and irreversible sensors can be designed to meet different requirements, and continuous monitoring of a particular biomarker of interest is feasible.
Suitably, biosensors for detection of the biomarker of the invention combine biomolecular recognition with appropriate means to convert detection of the presence, or quantitation, of the biomarker in the sample into a signal. Biosensors can be adapted for "alternate site" diagnostic testing, e.g. in the ward, outpatients' department, surgery, home, field and workplace.
Biosensors to detect the biomarker of the invention include acoustic, plasmon resonance, holographic and microengineered sensors. Imprinted recognition elements, thin film transistor technology, magnetic acoustic resonator devices and other novel acousto-electrical systems may be employed in biosensors for detection of the biomarker of the invention.
Methods involving detection and/or quantification of the biomarker of the invention can be performed on bench-top instruments, or can be incorporated onto disposable, diagnostic or monitoring platforms that can be used in a non-laboratory environment, e.g. in the physician's office or at the patient's bedside. Suitable biosensors for performing methods of the invention include "credit" cards with optical or acoustic readers. Biosensors can be configured to allow the data collected to be electronically transmitted to the physician for interpretation and thus can form the basis for e-neuromedicine. There is provided a method of identifying a substance capable of promoting or suppressing the generation of the biomarker in a subject, comprising exposing a test cell to a test substance and monitoring the level of the biomarker within said test cell, or secreted by said test cell.
The test cell could be prokaryotic, however a eukaryotic cell will suitably be employed in cell- based testing methods. Suitably, the eukaryotic cell is a yeast cell, insect cell, Drosophila cell, amphibian cell (e.g. from Xenopus), C. elegans cell or is a cell of human, non-human primate, equine, bovine, porcine, caprine, ovine, canine, feline, piscine, rodent or murine origin.
The test substance can be a known chemical or pharmaceutical substance, such as, but not limited to, an anti-psychotic disorder therapeutic; or the test substance can be novel synthetic or natural chemical entity, or a combination of two or more of the aforesaid substances.
In methods for identifying substances of potential therapeutic use, non-human animals or cells can be used that are capable of expressing the peptide.
Methods of screening for an autotaxin-lysophosphatidic acid signalling pathway modulator are also contemplated within the scope of the invention. Suitable methods of screening are described in the methods section herein and may typically involve the step of administering a test compound to Huh-7 and/or HepG2 hepatoma cells, which are model systems for supporting HCV and HBV replication, respectively.
Thus, according to a further aspect of the invention, there is provided a method of screening for an autotaxin-lysophosphatidic acid signalling pathway modulator, which comprises the steps of:
(a) contacting a virally infected hepatoma cell with a test compound; and
(b) comparing the effect of said test compound upon the autotaxin- lysophosphatidic acid signalling pathway within said cell.
In one embodiment, the virally infected hepatoma cell comprises Huh-7 hepatoma cells. These cells have the advantage of supporting HCV replication.
In an alternative embodiment, the virally infected hepatoma cell comprises HepG2 hepatoma cells, these cells have the advantage of supporting HBV replication. Screening methods also encompass a method of identifying a ligand capable of binding to the biomarker according to the invention, comprising incubating a test substance in the presence of the biomarker in conditions appropriate for binding, and detecting and/or quantifying binding of the biomarker to said test substance. High-throughput screening technologies based on the biomarker, uses and methods of the invention, e.g. configured in an array format, are suitable to monitor biomarker signatures for the identification of potentially useful therapeutic compounds, e.g. ligands such as natural compounds, synthetic chemical compounds (e.g. from combinatorial libraries), peptides, monoclonal or polyclonal antibodies or fragments thereof, which may be capable of binding the biomarker.
Methods of the invention can be performed in array format, e.g. on a chip, or as a multiwell array. Methods can be adapted into platforms for single tests, or multiple identical or multiple non-identical tests, and can be performed in high throughput format. Methods of the invention may comprise performing one or more additional, different tests to confirm or exclude diagnosis, and/or to further characterise a condition.
The invention further provides a substance, e.g. a ligand, identified or identifiable by an identification or screening method or use of the invention. Such substances may be capable of inhibiting, directly or indirectly, the activity of the biomarker, or of suppressing generation of the biomarker. The term "substances" includes substances that do not directly bind the biomarker and directly modulate a function, but instead indirectly modulate a function of the biomarker. Ligands are also included in the term substances; ligands of the invention (e.g. a natural or synthetic chemical compound, peptide, aptamer, oligonucleotide, antibody or antibody fragment) are capable of binding, suitably specific binding, to the biomarker.
The following studies illustrate the invention: MATERIALS AND METHODS Cell lines, antibodies and reagents. Huh-7.5 and HepG2 (provided by Charles Rice, The Rockefeller University, NY, USA) (Blight (2002) Journal of Virology 76(24), 13001-13014; Tagawa (1995) Journal of Gastroenterology and Hepatology 10(5), 523-527), HepG2.2.15 cells (provided by Ulrike Protzer TUM Munich; Sureau (1986) Cell 47(1), 37-47) and 293T (purchased from the American Type Culture Collection) were propagated in Dulbecco's modified Eagle Medium (DM EM) supplemented with 10% fetal bovine serum (FBS) and 1 % non-essential amino acids (Invitrogen, CA). Huh-7 Luc2a-JFH cells (provided by Robert Thimme, UNI Freiburg; Jo (2009) Gastroenterology 136 (4): 1391-1401) were propagated in the same growth media supplemented with G418. All cells were grown in a humidified atmosphere at 37°C in 5% C02. The primary antibodies used were: anti-NS5A 9E10 (C. Rice, Rockefeller University, USA); anti-ATX 4FAB (Baumforth (2005) Blood 106 (6), 2138-2146). Secondary labelled antibodies were obtained from Invitrogen, CA: Alexa Fluor 488 goat anti-mouse IgG; Horseradish peroxidase (HRP) conjugated sheep anti-mouse and anti-rat secondary antibodies were obtained from GE Healthcare, UK and Jackson laboratories respectively. Agonists, inhibitors and antagonists were obtained from the following sources: HA130 (Echelon Biosciences), LPA and wortmannin (Sigma), KM6425 (Cayman Chemical), U0126 (Tocris Bioscience), BYL-719 (Active Biochem) and TGX-221 (Cayman Chemical). Quantification of ATX, HIF-1 alpha and HCV by RT-PCR. Following lysis and homogenisation utilising the gentleMACS system (Miltenyibiotec) RNA was prepared (RNAeasy mini kit, Qiagen) from: human liver tissue of patients with a range of disease aetiologies, who underwent resection or transplantation at the Queen Elizabeth Hospital Birmingham, with patient consent and regional ethical committee approval obtained. Liver disease was diagnosed according to standard clinical, histological and radiological criteria. Normal liver tissue was obtained from surplus donor tissue used for reduced size transplantation; uPA-SCID mouse liver tissue transplanted with human hepatocytes or infected with HCV or HBV. Untransplanted mice were used as a negative control; Huh-7.5 cells naive or infected for 72h with HCV; HepG2 or HepG2.2.15 cells.
Gene amplification was performed using a modification of the method of Cook and colleagues (Cook (2004) Journal of Clinical Microbiology 42(9), 4130-4136; Tang (2005) Journal of Virological Methods 126(1-2), 81-86) in a single tube RT-PCR in accordance with the manufacturer's guidelines (CellsDirect kit, Invitrogen, CA) and fluorescence monitored in a 7900 HT real time PCR machine (ABI, CA) (Mee (2008) Journal of Virology 82(1), 461-470). In all reactions the housekeeping gene GAPDH was included as an internal endogenous control for amplification efficiency and RNA quantification (primer-limited endogenous control, ABI). Genesis of virus and infections. Cell culture-derived virus particles were generated as previously described (Lindenbach (2005) Science (New York, NY) 309(5734), 623-626). Using the Megascript T7 kit (Ambion, Austin, TX), RNA was transcribed in vitro from full-length genomes and electroporated into Huh-7.5 cells. After 48h cells were serum starved for 16h prior to collection of supernatants in serum free media at 72 h post infection and storage at - 80°C. Pseudoviruses expressing luciferase reporters were generated following transfection of 293T cells with a 1 : 1 ratio of plasmids encoding HIV provirus expressing luciferase and HCV strain 1A38 or H77 E1 E2 envelope gps (HCVpp-1A38, HCVpp-H77), MLV gp (MLVpp) VSV gp (VSVpp) or empty vector (Env-pp), as previously described (Hsu (2003) PNAS(USA) 100(12), 7271-7276). 24 h post transfection cells were serum starved prior to harvesting at 48h post transfection in serum free media.
Target cells were seeded at 1.5x104 cells/cm2 and serum starved for 16 h prior addition of HCVcc or pseudoparticles in serum free media for 24 h. HCVcc infection was assessed following methanol fixation and staining for NS5A using the anti-NS5A 9E10 antibody; bound antibody was detected with an Alexa 488-conjugated anti-mouse IgG and quantified by enumerating NS5A positive cells. Pseudoparticle infection was quantified by measuring cellular luciferase activity in a luminometer (Berthold Centro LB 960). Specific infectivity was calculated by subtracting the mean Env-pp signal from the HCVpp/MLVpp/VSVpp signals. Relative infectivity was calculated as a percentage of untreated cells and presented ± S.E.M. where the mean infection value of replicate untreated cells wells was defined as 100%. uPA-SCID mice infection. uPA/SCID-bg mice were transplanted with primary human hepatocytes at 3 weeks of age by intrasplenic injection of 106 cells suspended in PBS as already described (Mercer (2001) Nat Med 7(8), 927-933). Successful engraftment was determined by measuring the human albumin concentration in the serum of transplanted mice by specific ELISA. HCV infected mice were inoculated with Jc1 recombinant virus from cell supernatant or mouse serum. HBV infected mice were inoculated with an infectious mouse serum containing a genotype E patient-derived virus. Mice were sacrificed by spine dislocation after gaseous anesthesia and blood harvesting by intracardiac puncture 5 (HB) to 16 (HCV) weeks later. The liver was recovered, cut in small pieces and immediately frozen on dry ice for RNA extraction. Liver samples were also fixed in formalin for immunostaining purposes. Experiments were performed in the Inserm Unit 1 110 animal facility according to local laws and approved by the ethical committee of Strasbourg (number AL/02/19/08/12 and AL/01/18/08/12). Autotaxin silencing and rescue. To generate lentiviral vectors 293T cells were transfected (Fugene 6 (Promega)) according to manufacturer's instructions) with plasmids encoding plKO.1 control (shControl) or plKO.1 shATX (shATX) (Open Biosystems) plus p8.2 gag pol and VSV-G (Flint (2006) Journal of Virology 80(22), 11331-1 1342). At 48 h and 72 h post transfected supernatants containing lentiviral particles expressing shControl or shATX were harvested. Huh-7.5 cells were incubated with the collected supernatants for 72 h and cells cultured in the presence of puromycin for selection. Knockdown of ATX expression was determined by western blot (see below). shControl and shATX expressing Huh-7.5 cells were transfected with His tagged wild type ATXB (ATX-wt) or T210A ATXB mutant (ATX-T210A) (Kanda (2008) Nature Immunology 9(4), 415-423) for 24 h prior to seeding for infection studies. Determining the effect of ATX inhibition on the HCV life cycle. Target cells were seeded at 1.5 x 104/cm2 and serum starved for 16 h prior to assay. Cells were incubated with HA130 (60min), LPA (in the presence of 0.1 mg/ml fatty acid free BSA)(15min), KM6425 or LPA plus Ki 16425 (15min) diluted in serum free media prior to addition of cell culture derived HCV or pseudoparticles for 24 h after which infection was assessed as described above.
Huh-7 Luc2a-JFH expressing cells or Huh-7 Luc2a-JFH transduced to express plKO.1 control or plKO.1 shATX silencing vectors were seeded at 1.5 x 104/cm2 and serum starved for 16 h prior to assay. Cells were untreated or incubated with HA130 for 24 h prior to lysis and quantification of luciferase performed as described.
Huh-7.5 cells were transfected with Jd GLuc for 24 h then serum starved for 16 h. Cells were washed extensively then incubated with HA130 for 24 h. Cell supernatants were harvested, heat inactivated and luciferase activity from secreted particles in the harvested supernatant determined.
Western blotting. Cells were serum starved overnight prior to harvesting in lysis buffer (PBS, 1 % Triton-X100, 0.1 % sodium deoxycholate, 0.1 % SDS) containing protease (Complete, Roche, UK) and phosphatase (PhoStop, Roche, UK) inhibitors. Cell lysates were clarified by centrifugation (20 000 x g, 30min) and the protein concentration determined using Protein Assay Reagent (Pierce, IL) according to manufacturer's instructions. Huh-7.5 cell supernatants were harvested over 24 h in serum free media prior to separation by SDS-PAGE. Quantified protein lysates (50 μg) were separated by SDS-PAGE and transferred to PVDF membranes (Sigma, UK) for incubation with anti-ATX. HRP-conjugated anti-rat secondary antibody was detected by enhanced chemiluminescence (Geneflow, UK). Protein was visualised using the PXi imaging system (Syngene).
For detection of HIF-1alpha, cells were serum starved overnight prior to incubation with LPA (10μΜ) diluted in serum free media containing 0.1 mg/ml fatty acid free in the presence or absence of KM 6425 (10μΜ), U0126 (10μΜ), wortmannin (200nM), BYL-779 (2μΜ) or TGX- 221 (50nM) for 24 h at 37°C. Cells were lysed directly in Laemmli buffer (Laemmli (1970) Nature 227(5259), 680-685), lysates were separated by SDS-PAGE and transferred to PVDF membranes as described above. Anti-HIF-1 alpha detection was performed and detected following incubation HRP-conjugated anti-mouse secondary antibody by enhanced chemiluminescence. Detection of LPA by mass spectrometry. Huh-7.5 cells were serum starved for 16 h prior to harvesting of supernatants for 8 h in serum free medium. Following clarification supernatants were spiked with 30ng 17:0 LPA as an internal standard prior to LPA extraction. 8ml of supernatant was extracted with 16ml of n-butanol three times at room temperature, the combined extract was dried under vacuum (SpeedVac, Thermo) and re-dissolved in 100μΙ chloroform/methanol/water 2:5: 1. Liquid-chromatography with tandem mass spectrometry (LC- MS/MS) analysis was performed on each sample using a Thermo Orbitrap Elite system (Thermo Fisher) hyphenated with a five-channel online degasser, four- pump, column oven, and autosampler with cooler Shimadzu Prominence HPLC system (Shimadzu) for lipids analysis. High resolution / accurate mass and tandem MS were used for molecular species identification and quantification. The identity of the lipid subspecies was further confirmed by reference to appropriate lipids standards. All the solvents used for lipid extraction and LC- MS/MS analysis were LC-MS grade from Fisher Scientific. The final amount of LPA (ng) is presented per 8 mL of conditioned media analysed. Hypoxia responsive element luciferase reporter assay. To quantify HIF activity, Huh-7.5 cells were transfected to express hypoxia responsive element luciferase reporter (HRE-Luc) (provided by Margaret Ashcroft, University College London, UK; Chau (2005) Cancer Research 65(1 1), 4918-4928) using Fugene 6 (according to manufacturer's instructions). 24 h later, cells were seeded at 1.5 x 104/cm2 and serum starved for 16 h prior to assay. Cells were incubated for 24 h with LPA (10μΜ), KM 6425 (10μΜ), LPA plus KM6425, or NSC (25nM) diluted in serum free media containing 0.1 mg/ml fatty acid free BSA. Cells were incubated under normal 20% oxygen conditions (normoxia) or 3% oxygen (hypoxia by placing in a hypoxic chamber (Don Whitley Scientific Limited). Following cell lysis HRE-Luc activity was quantified by measuring cellular luciferase activity in a luminometer. Relative activity was calculated as a percentage of untreated cells and presented ± S.E.M. where the mean activity value of replicate untreated cells wells was defined as 100%.
Determining the effect of ATX inhibition on the HBV life cycle. HepG2.2.15 cells or cells transduced to express plKO.1 control or plKO.1 shATX silencing vectors (72 h) were seeded at 1.5 x 104/cm2 and serum starved for 16 h prior to assay. Parental cells were incubated with HA130 or DMSO control in serum free media. 24 h later cells or supernatants were harvested for quantification of HBV. To quantify HBV DNA, viral DNA was isolated from HepG2.2.15 supernatant using the QiaAMP MinElute Virus Spin Kit (Qiagen), while total cellular DNA was isolated using the QiaAMP DNA Mini Kit (Qiagen), both as per manufacturer's instructions. DNA was then quantified using Nanodrop™. HBV was then quantified using the Hepatitis B Virus qPCR Quantification Kit (PrimerDesign) as per manufacturer's description, using a Roche Lightcycler® (Roche). HBsAg was quantified from viral supernatant using the HBsAg Antigen ELISA (Abnova) as per manufacturer's instructions.
RESULTS
Example 1 : Hepatic expression of ATX
The present experiment studied the impact of HCV and HBV infection on hepatic ATX expression in the absence of HCC. A significant increase in ATX mRNA levels was confirmed in the liver biopsies from HCV and HBV non-cirrhotic patients (Fig.lA). However, a comparable increase in ATX mRNA levels was noted in the liver tissue from patients with alcoholic liver disease (ALD), primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC) and autoimmune hepatitis (AIH), diseases characterised by persistent inflammation (Fig.lA). Tissue inflammation is frequently associated with increased hypoxia inducible factor 1 a (HIF- 1 alpha) expression, a major transcriptional activator that enables a cell to respond to low oxygen environment. To ascertain whether ATX is a target for HIF-1 alpha transcriptional activation, Huh-7 hepatoma cells were transfected with plasmids encoding HIF-1 alpha and 36h later expression confirmed by western blotting (Fig.l B). It was confirmed that incubating Huh- 7 cells under physiological low oxygen levels found in the liver (3%) stabilized HIF-1alpha treatment and promoted ATX mRNA levels (data not shown). It was also confirmed that HIF- 1 alpha dependent transcriptional responses using a HRE-luciferase reporter (data not shown) and demonstrated a significant increase in ATX mRNA and control VEGF mRNA (Fig.l B), demonstrating that ATX is a HIF-target gene.
The human liver uPA-SCID mouse supports HCV and HBV infection (Mercer (2001 , supra); Meuleman (2006) Journal of Virology 80(6), 2797-2807, enabling us to directly investigate the impact of viral infection on ATX expression. All mice engrafted with human hepatocytes expressed human ATX to varying levels and infection with HCV or HBV promoted a significant increase in ATX mRNA levels (Fig.lC). In non-transplanted mice, ATX expression was not detected (data not shown) illustrating both the specificity of primers for human ATX and highlighting that viral-dependent modulation of ATX expression is of human hepatocyte origin. ATX was clearly expressed in hepatocytes in both HCV and HBV infected livers, whereas no staining was observed in liver tissue from uninfected controls (Fig.lC). To investigate whether these observations could be recapitulated in vitro Huh-7 and HepG2 hepatoma cells were used, that respectively support HCV and HBV replication. A significant 18-fold increase in ATX mRNA levels was observed and associated increase in protein expression in HCV infected Huh-7 cells compared to uninfected cells (Fig.l D). ATX expression was increased in HepG2.2.15 cells that support HBV replication (Sells (1987) PNAS(USA) 84(4), 1005-1009; Sun (2006) Journal of Hepatology 45(5), 636-645) compared to uninfected HepG2 cells, however, the present study failed to detect any significant change in ATX mRNA levels (Fig.l D), consistent with the lower ATX mRNA levels observed in the HBV infected SCID-uPA mice (Fig.lC). In summary, ATX is a HIF-target gene and is up-regulated following HCV or HBV infection.
Example 2: Autotaxin-LPA signalling axis plays an essential role in HCV infection
The present data uncovering HCV specific up-regulation of ATX suggests a role for ATX in HCV infection. ATX is a secreted protein that binds LPC in its active site and catalyses its hydrolysis to LPA (Tokumura (2002) The Journal of Biological Chemistry 277(42), 39436- 39442; Umezu-Goto (2002) The Journal of Cell Biology 158(2), 227-233). Inhibition of ATX can be achieved by ATX antagonists, that target the active site and block the enzymatic activity of ATX. To study a role for ATX in HCV infection Huh-7.5 cells were infected with HCVcc strains SA13/JFH and J6/JFH in the presence of an increasing concentration of ATX inhibitor HA130. A dose-dependent inhibition of HCV infection was observed following HA130 treatment (Fig.2A). To verify these observations, Huh-7.5 cells were transduced with an shRNA vector targeting ATX or an empty vector control. Quantitative RT-PCR (Fig.2B) and western blotting confirmed effective ATX silencing (Fig.2B). ATX silencing significantly inhibits HCV SA13/JFH and J6/JFH infectivity (Fig.2B), providing clear evidence for a role for ATX in HCV infection.
Off-target effects are a common limitation of gene silencing, to substantiate the present findings rescue experiments were performed where silenced cells were transfected to express wild type ATX (ATX-wt). Expression of ATX-wt in ATX silenced cells, as confirmed by Western blotting (Fig.2C), restored HCV infection (Fig.2C) and boosted infection when overexpressed in control cells (Fig.2C) suggesting that ATX is limiting in Huh-7.5 cells. HA130 targets the active site of ATX preventing its enzymatic action on LPC and thus blocks the genesis of LPA. To confirm a role for ATX enzymatic activity in HCV infection, control and ATX-silenced cells were transfected to express an enzymatically inactive ATX mutant T210A (ATX-T210A). ATX- T210A failed to restore HCV infection of ATX silenced cells and had minimal effect on control cells (Fig.2C), supporting a role for ATX-dependent production of LPA in HCV infection. To further study the ATX-LPA signalling axis in HCV infection control or ATX silenced cells were infected with HCV SA13/JFH or J6/JFH in the presence of HA130 or LPA. HA130 inhibited HCV infection in control cells and had no effect on ATX silenced cells (Fig.2D), supportive of the lack of ATX secreted from these cells. Importantly, LPA enhanced HCV infection of control cells and restored infection in ATX silenced cells (Fig.2D), suggesting that LPA production via ATX activity is important in HCV infection.
LPA, the product of LPC hydrolysis by ATX is an effector molecule. The effect of exogenous LPA on hepatoma permissivity was investigated to support HCV infection. LPA enhances HCV SA13/JFH and J6/JFH infection in a dose-dependent manner (Fig.3A). LPA exerts its effects through a family of six currently identified LPA receptors (LPAR 1-6). A recent study reported that LPARs 1 , 3 and 6 were expressed in the liver and a comparable expression profile was noted in Huh-7 cells (Sokolov (2013) The Journal of Surgical Research 180(1), 104-1 13). Quantitative RT-PCR confirmed expression of LPARs 1 , 3 and 6 in Huh-7.5 cells (data not shown). To investigate the specificity of LPA-dependent augmentation of HCV infection, cells were incubated with LPA in the absence or presence of LPAR1/3 inhibitor KM6425 (Ohta (2003) Molecular Pharmacology 64(4), 994-1005). Pre-incubation with KM6425 abrogated LPA enhancement of HCV infection (Fig.3B) suggesting a role for LPAR1/3 in ATX-dependent HCV infection. Incubation with Ki 16425 alone reduced SA13/JFH infection (Fig.3B) suggesting endogenous production of LPA by Huh-7.5 cells. Indeed, lipid analysis of conditioned media from Huh-7.5 cells by mass spectrometry confirmed LPA expression (Fig.3C).
Example 3: Identifying a role for Autotaxin in the HCV lifecycle
To assess whether ATX has a specific role in HCV entry, the effect(s) of HA130 on the ability of Huh-7.5 cells were studied to support HCV pseudoparticle (HCVpp) infection. HA130 had no detectable effect on HCVpp (Fig.4A), MLVpp or VSVpp infectivity (data not shown), demonstrating no role for ATX in HCV entry. Unsurprisingly LPA treatment also had no effect (data not shown). Since HCVpp allows the study of gp-dependent entry independent of downstream replication and translation events, it can be concluded that ATX has no role in HCV entry.
Given the effects of ATX inhibition and silencing on HCV infection, the role of ATX in HCV genome replication was investigated. The Luc2a JFH expressing Huh-7 subgenomic replicon cell line (Luc2a-JFH) was incubated with HA130 resulting in a significant reduction in HCV replication (Fig.4B). Silencing ATX in the Luc2a-JFH cells, as confirmed by Western blot (data not shown), substantiated these observations (Fig.4B). In addition the effect of HA130 on full length HCV replication was investigated in cells transfected with HCV JCI GLuc reporter. HA130 dose-dependently inhibited HCV replication as illustrated by the reduction in GLuc signal (Fig.4C). Predictably, as measured by infection of naive target cells, when HCV infected Huh- 7.5 cells were incubated with HA130 they released less infectious virus (Fig. 4D). These data illustrate a role for the ATX-LPA signalling axis in HCV RNA replication.
Example 4: LPA promotes HCV infection via stabilization of HIF-1 alpha
LPA has been reported to induce HIF-1 alpha expression in ovarian and colon cancer cells (Kim (2006) Cancer Research 66(16), 7983-7990; Lin (2010) Gastrointestinal and Liver Physiology 299(5), G1128-1138). Since HIF-1alpha inhibition abrogates HCV replication (Wilson (2012) Journal of Hepatology 56(4), 803-809) the role of HIF-1alpha in ATX-LPA-dependent HCV infection was investigated. Huh-7.5 cells were incubated with LPA in the presence or absence of LPAR inhibitor KM6425 for 24h and lysates probed for HIF-1alpha expression. It was shown that LPA stabilizes HIF-1 alpha in Huh-7.5 cells (Fig.5A) and this is reversed by KM6425. HIF- 1 alpha binds to and elicits its transcriptional effects through hypoxia responsive elements (HRE) (for review see Wenger (2005) Science's STKE : signal transduction knowledge environment 2005 (306):re12.). Consistent with our observation that LPA stabilizes HIF-1 alpha an LPA- dependent increase in HRE transcriptional activity was demonstrated (Fig.5B). As a positive control HRE-Luc expressing cells were incubated under hypoxic conditions at 3% oxygen for 24h, resulting in a 7-fold stimulation of HRE-Luc activity that was not further potentiated by LPA (Fig.5B).
NSC is a specific inhibitor of the HIF pathway (Chau (2005) Cancer Research 65(11), 4918- 4928), incubating Huh-7.5 cells with LPA in the presence of an increasing concentration of NSC reveals a dose-dependent reduction in LPA-promoted HCV infection (Fig.5C). Importantly this was observed at a concentration (25nM) that, in the absence of LPA, had no significant effect on HCV infection (Fig.5C). The HRE-Luc reporter assay confirmed the efficacy of 25nM NSC on inhibition of HIF (Fig.5D). These data demonstrate a role for HIF-1 alpha stabilization in LPA- dependent promotion of HCV infection. Previous studies have shown that phosphoinositide 3 kinase (PI3K) and mitogen activated protein kinase (MAPK) signalling play a role in LPA-dependent HIF-1 alpha expression (Lee (2006) Clinical Cancer Research : an official journal of the American Association for Cancer Research 12(21), 6351-6358). HIF-1 alpha expression was studied in LPA treated Huh-7.5 cells pre-incubated with inhibitors of PI3K (wortmannin, BYL-719, TGX-221) or MAPK (U0126). Western blotting revealed stabilization of HIF-1alpha was inhibited in the presence of the pan PI3K inhibitor wortmannin (Fig.5A). More specifically, BYL-719, an inhibitor of p110a class I PI3Ks, inhibited HIF-1alpha stabilization in response to LPA treatment whereas TG-221 an inhibitor of p110b class I PI3Ks did not (Fig.5A) illustrating a role for p110a class I PI3Ks. Incubation with the MAPK/MEK inhibitor U0126 also inhibited LPA stabilization of HIF-1 alpha (Fig.5A). These data suggest a role for PI3K and MAPK signalling in LPA-dependent augmentation of HCV infection.
Example 5: A role for ATX in the HBV lifecycle
The availability of HepG2.2.15 cells that support HBV replication and secrete particles, allowed the role of ATX in the viral lifecycle to be investigated. It was confirmed that HepG2.2.15 cells express ATX and incubating with HA130 significantly reduced the levels of HBV DNA in the extracellular media and yet had no effect on the levels of intracellular HBV DNA (Fig.6A), consistent with a role for ATX in the assembly or secretion of HBV particles. To confirm these results HepG2.2.15 cells were transduced with an shRNA vector targeting ATX or empty vector control and rescue experiments performed by transfecting silenced cells with wild type ATX (ATX-wt) or the enzymatically inactive ATX mutant T210A (ATX-T210A). Western blotting confirmed effective silencing of ATX and recovery with ATX-wt and ATX-T21 OA. ATX silencing inhibits extracellular HBV DNA and HBS antigen levels and there was no significant effect on intracellular HBV DNA copy numbers (Fig.6B). Furthermore, expression of wt ATX but not the enzyme inactive mutant T210A restored the levels of extracellular HBV DNA (Fig.6B). These data illustrate a role for ATX-LPA signalling axis in the assembly and /or secretion HBV particles.
Example 6: ATX plays a role in HBV infection of naive target cells
Differentiated HepG2-NTCP cells were treated with HA130 (100nM) for 1 h or transduced with shRNA targeting ATX or an irrelevant control prior to infecting with HBV. ATX expression was confirmed by Western blotting. HBV infection was quantified 7 days later by measuring intracellular and extracellular viral DNA and HBV encoded pre-core or e antigen (HBeAg). For all experiments infectivity is expressed relative to control and represents the mean of three replicate infections analysed by Student's t-test (* P <0.05, *** P <0.001). The results in Fig.7 show that blocking ATX activity with HA130 or suppressing ATX expression with shRNA prevents infection of differentiated HepG2-NTCP liver cells in culture by HBV.
Example 7: A role for ATX in HBV persistently infected cells
Differentiated HBV infected HepG2.2.15 producer cells were cultured without serum for 24h and the effect of HA130 (100nM) on intracellular or extracellular HBV DNA assessed over a 24h period (Fig.8A). To confirm the inhibitory effect of HA130 on HBV particle secretion, differentiated HepG2.2.15 cells were transduced with shRNA targeting ATX (shATX) or an irrelevant control (shControl). shRNA-ATX cells were trans-complemented with wild-type ATX (shATX-wt) or a catalytically inactive mutant (ATX-T210A) and intracellular or extracellular HBV DNA levels measured (Fig.8B). Western blots depict extracellular ATX expression. The median and range of the data from three independent experiments are presented and were compared using Student's t-test (*** P <0.001). The results in Fig.8 show that inhibition of ATX activity with HA130 or suppression of ATX expression by shRNA specifically prevents HBV release from persistently HBV infected cells without affecting the cellular viral load.
Example 8: LPA modulates hepatitis virus infection via stabilizing HIF-1 a
Huh-7 cells were transfected with HRE-Luc and cultured under serum-free conditions prior to incubating with LPA (10μΜ) in the presence or absence of LPAR antagonist KM6425 (10μΜ) and cultured under 20% oxygen for 24h (Fig.9A). HIF-1 a expression was confirmed by Western blotting. Data are representative of three independent experiments and were compared using a Student's t-test (**** P <0.0001). Differentiated HepG2-NTCP were transfected with siRNA targeting HIF-1 a or control for 48h, cultured without serum for 24h prior to infecting with HBV. HIF-1 a expression was confirmed by Western blotting. HBV infection was quantified 7 days later by measuring cellular viral DNA and HBeAg (Fig.9B). For all experiments infectivity is expressed relative to control and represents the mean of three replicate infections analysed by Student's t-test (*** P <0.001 , **** P <0.0001). HepG2-NTCP were cultured without serum, treated with LPA (10μΜ) and cell lysates separated by SDS- PAGE and probed for phospho-AKT (pT308 AKT) or total AKT (AKT) (Fig.9C). Serum-free Huh-7 cells were treated with wortmannin (WM - 200nM), BYL-719 (2μΜ) or TGX-221 (50nM) prior to a 15 min incubation with LPA (10μΜ) before lysis and Western blotting for HIF-1 a (Fig.9D). Differentiated HepG2-NTCP cells were cultured without serum for 24h, treated with wortmannin (200nM), BYL-779 (2μΜ) or TGX-221 (50nM) for 15 mins prior to infecting with HBV and infection quantified 7 days later by measuring HBeAg and HCV NS5A expressing cells enumerated 24h post inoculation (Fig.9E). Infectivity is expressed relative to control and represents the mean of three replicate infections analysed by Student's t-test (**** P <0.0001). The results in Fig.9 show that LPA elevates HIF-1 alpha expression and stabilisation in liver cells in a PI3kinase dependent manner, it also shows that infection of such cells with HBV requires HIF-1 alpha.
Example 9: LPA receptor expression in SCID-upA chimeric mice
Expression of LPA receptors 1-6 mRNA in liver of HBV infected SCID-upA chimeric mice relative to naive mice. Mice were sacrificed at 37 days post-inoculation and HBV DNA levels were 5600- 34200 lU/mL. The results in Fig.10 show that infection of human hepatocytes transplanted into SCID-uPA mouse liver with HBV induces an increase in mRNA for LPAR1 with limited effect upon the expression of the other LPA receptors.
DISCUSSION
An essential role for ATX-LPA signalling has been demonstrated herein in HCV and HBV infection. The data has confirmed increased ATX in HCV infected patient liver tissue and shown that HBV also promotes ATX expression. The liver is a highly complex multicellular organ it is therefore difficult to interpret virus specific changes in ATX expression in whole tissue analysis. Increased expression of ATX in vitro has been identified following infection with HCV and HBV. Crucially, by both RT-PCR and immunohistochemistry, it has been possible to pinpoint HCV- and HBV-dependent promotion of ATX expression within human hepatocytes in the uPA-SCID mouse humanized liver model system.
ATX exerts its effects via generation of LPA and downstream activation of signalling events via LPARs. Studying the modulation of ATX activity for effect(s) on HCV infection, it has been identified for the first time that ATX-LPA signalling has an important role in HCV replication. LPARs 1 , 3 and 6 are expressed in the liver and (Sokolov (2013, supra)) hepatoma cells. The results herein demonstrate that ATX-LPA-dependent promotion of HCV infection is inhibited by KM6425 which suggests a role for LPAR1 or 3 in this process. Many aspects of HCV replication have yet to be understood, it was previously reported that inhibiting HIF-1 alpha reduced HCV replication (Wilson (2012, supra) demonstrating an essential role for HIF-1 alpha in HCV replication (Vassilaki (2013) Journal of Virology 87(5), 2935-2948). Under normal oxygen levels HIF-1 alpha, expression is tightly controlled by prolyl hydroxylases (PHDs) (Bruick (2001) Science (New York, NY) 294(5545), 1337-1340) targeting HIF-1 alpha for degradation via the proteasomal pathway. It has been shown herein that HIF-1 alpha overexpression in Huh-7.5 cells under normoxic conditions up-regulates ATX mRNA levels, demonstrating for the first time that ATX is a HIF-regulated gene. This observation is consistent with elevated ATX expression reported in tumors that frequently express HIFs. In addition it has been demonstrated herein that LPA stabilizes HIF-1 alpha protein expression and HRE-transcriptional activity that was inhibited by KM6425, illustrating a role for LPARsl and/or 3. Interestingly, when hepatoma cells were subjected to low oxygen (3%), LPA had no further effect on HRE-transcriptional activity. In ovarian cancer cells, LPA increases VEGF expression through induction of HIF-1alpha, which in turn stimulates ATX expression, providing a positive feedback that promotes cancer progression (Ptaszynska (2008) Molecular Cancer Research : MCR 6 (3):352-363; Kim (2006) Cancer Research 66(16), 7983-7990; Park (2007) Cancer Letters 258(1), 63-69). It is therefore proposed that the existence of a similar positive feedback loop concerning HIF-1 alpha and the ATX-LPA signalling pathway in hepatocytes that is hijacked by HCV to promote its replication.
Analogous to HCV, HBV infection stabilizes HIF-1 alpha (Liu (2014) British Journal of Cancer 1 10(4), 1066-1073; Yoo (2008) Oncogene 27(24), 3405-3413), signifying this pathway to be a common target for both viruses. The study herein reveals that inhibition of the ATX-LPA signalling axis diminishes the secretion of HBV particles. The viral protein HBx plays an important role in replication and stabilizes HIF-1 alpha (Yoo (2003) The Journal of Biological Chemistry 278(40), 39076-39084; Yoo (2004) FEBS letters 577(1-2), 121-126; Yoo (2008) supra), if a similar feedback loop exists its modulation could provide a mechanism for the reduced secretion of HBV observed following inhibition of ATX-LPA signalling. The lack of effect of LPA on HBV production may be due to HIF-1alpha already being stabilized in these cells a theory supported by the inability of LPA to promote HRE-Luc under hypoxia. Epstein Barr virus mediated generation of LPA via enhancement of ATX expression augments the proliferation and survival of EBV infected Hodgkin lymphoma (HL) cells and contributes to the pathogenesis of HL (Baumforth (2005) supra). We have shown that both HCV and HBV infection promotes ATX expression and LPA has been shown to promote invasion of hepatoma cells (Wu (2010, supra)). LPA transactivates receptor tyrosine kinase such as EGFR (Miyamoto (2004) Cancer Research 64(16), 5720-5727). HCV has been shown to phosphorylate EGFR (Diao (2012) Journal of Virology 86(20), 10935-10949) and EGF has been identified as an important host cell factor in HCV infection (Lupberger (2011) Nature Medicine 17(5), 589-595). HBx increases EGFR gene expression (Menzo (1993) Virology 196(2), 878-882) and serum EGF is increased in HBV infected patients (Barreiros (2009) International Journal of Cancer 124(1), 120-129). Increased EGF receptor signalling is associated with several cancers (for review see (Normanno (2006) Gene 366(1), 2-16). The implication of EGFR signalling in HCV and HBV infection may have far reaching consequences in terms of ATX/LPA signalling and the provision of tumour progression. LPARs are G-protein coupled receptors that fall into two distinct groups, endothelial differentiated gene (Edg) including LPARs 1 , 2 and 3 and non-Edg (puringenic) including LPARs 4, 5 and 6 receptors of the EDG. Consequently, ligand binding results in the activation of a myriad downstream signalling pathways including, amongst others, mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K). It has been shown herein that KM6325 inhibits LPA-stimulated HIF-1alpha stabilization and suggests a role for LPARs 1 and 3. In addition it has been revealed herein that LPA-dependent stabilization of HIF-1 alpha is PI3K and MAPK dependent. HCV non-structural protein NS5A, which plays a key role in replication of the virus (Fridell (201 1) Journal of Virology 85(14), 7312-7320), enhances the phosphotransferase activity of PI3K p1 10 in HCV replicating cells (Street (2004) The Journal of Biological Chemistry 279(13), 12232-12241). Activation of PI3K signalling contributes to the maintenance of HCV replication and survival of host cells (Mannova (2005) Journal of Virology 79(14), 8742-8749). Inhibition of HCV replication by curcumin and quercetin occurs via suppression of PI3K signalling (Chen (2012) International Journal of Molecular Medicine 30(5), 1021-1028; Pisonero-Vaquero (2014) Laboratory Investigation; a journal of technical methods and pathology 94(3), 262-274). Taken together these findings support ATX/LPA-dependent activation of HIF-1 alpha via PI3K signalling as playing an important role in HCV replication and survival of host cells. Indeed, based on the inhibitor BYL-779, the study herein specifically suggests a role for p1 10a class I PI3K. Host cell survival offers the potential for HCV infected patients to develop HCC. Studies have suggested a role for MAPK in phosphorylation of HCV NS5a (Reed (1999) The Journal of Biological Chemistry 274(39), 2801 1-28018), important in viral replication. Akin to ATX-LPA driven PI3K signalling in HIF-1 alpha stabilization and increased HCV replication MAPK signalling is another potential regulator.
HCV activation of the PI3K/Akt signalling pathway augments inflammation and fibrosis in the liver (Huang (201 1) Hepatology Research : the official journal of the Japan Society of Hepatology 41 (5), 430-436). PI3K has been implicated in hepatic stellate cell driven fibrosis (Son (2009) Hepatology 50(5), 1512-1523). As a consequence of the liver attempting to heal itself virus infection leads to fibrosis. LPA signaling drives chronic wound healing leading to fibrosis, indeed antagonists against LPAR1 and 3 are shown to have anti-fibrotic capabilities and LPA modulators are being developed as therapies for fibrosis (Rancoule (201 1) Expert Opinion on Investigational Drugs 20(5), 657-667; Rancoule (2014) Biochimica et Biophysica Acta 1841 (1):88-96; Budd (2013) Future Medicinal Chemistry 5(16), 1935-1952). A recent study has demonstrated that during hepatic regeneration in mice LPAR 1 , 3 and 6 expression is increased (Simo (2013) HPB : the official journal of the International Hepato Pancreato Biliary Association 16(6), 534-542). Such an increase during viral infection could exacerbate viral infection by further enhancement of ATX-LPA signalling.
Targeting the ATX-LPA signalling axis to disrupt the lifecycle of hepatotropic viruses HCV and HBV could provide an effective therapeutic strategy. The study herein has demonstrated the effectiveness of an antibody against LPA in inhibiting establishment of HCV infection. Unfortunately limitations in methodologies to study HBV have hampered the ability to test this antibody in relation to HBV infection. As already stated, HCV and HBV are major factors in the development of hepatocellular carcinoma (HCC), the most common liver malignancy in the world. With limited treatment options, the ATX-LPA signalling axis is an emerging target for cancer therapy. Recent studies support a role for LPA signalling in the resistance of tumours to chemotherapy and radiation-induced cell death (Brindley (2013) Biochimica et Biophysica Acta 1831 (1), 74-85) highlighting an important role for ATX and LPA in the development and progression of human cancer. Active research in this area has generated several ATX inhibitors {for review see Albers (2012) Chemical Reviews 112(5), 2593-2603; Barbayianni (2013) Expert opinion on therapeutic patents 23(9), 1123-1 132). Administration of dual ATX/LPA antagonists has unveiled reduced tumour growth and angiogenesis in engineered tumours in vivo (Xu (2009) Prostaglandins & other lipid mediators 89(3-4), 140-146). The data presented herein suggests modulation of ATX by viral-hepatitis may provide a mechanism for the increased incidence of HCC in virus-infected individuals. In the liver, increased ATX expression associated with inflammatory status and the data presented herein proposes ATX as an anti-viral target, this is attractive since there may be reduced off-target effects in healthy non-inflamed tissue. In summary, a role for ATX in the lifecycle of HCV and HBV has been identified herein, highlighting potential new targets for therapy and the prospect of stratifying therapies for the treatment of viral-associated and non-associated HCC.

Claims

1. An autotaxin-lysophosphatidic acid signalling pathway modulator for use in the prophylaxis or treatment of hepatitis B virus infection.
2. The modulator for use as defined in claim 1 , which is an autotaxin inhibitor.
3. The modulator for use as defined in claim 1 or claim 2, which is a lysophosphatidic acid (LPA) inhibitor.
4. The modulator for use as defined in claim 1 , which is an antagonist of a G-protein coupled lysophosphatidic acid receptor (LPAR).
5. The modulator for use as defined in claim 4, which is an LPAR 1 and/or 2 and/or 3 antagonist, such as an LPAR 1 and/or 3 antagonist.
6. The modulator for use as defined in claim 1 , which is an inhibitor of hypoxia inducible factor 1 , alpha subunit (HIF-1 alpha).
7. The modulator for use as defined in claim 6, wherein the inhibitor of hypoxia inducible factor 1 , alpha subunit (HIF-1 alpha) comprises a phosphoinositide 3 kinase (PI3K) or mitogen activated protein kinase (MAPK) signaling modulator.
8. The modulator for use as defined in claim 7, wherein the phosphoinositide 3 kinase (PI3K) signaling modulator is an inhibitor of p1 10a class I phosphoinositide 3 kinase (PI3K).
9. The modulator for use as defined in claim 7, wherein the mitogen activated protein kinase (MAPK) signaling modulator is a MAPK/MEK inhibitor.
10. Use of hypoxia inducible factor 1 , alpha subunit (HIF-1 alpha), autotaxin (ATX), lysophosphatidylcholine (LPC) or lysophosphatidic acid (LPA) as a biomarker for hepatitis B virus infection.
1 1. A method of diagnosing hepatitis B virus infection, comprising: (a) quantifying the amounts of hypoxia inducible factor 1 , alpha subunit (HIF- 1 alpha), autotaxin, lysophosphatidylcholine or lysophosphatidic acid in a biological sample obtained from an individual; and
(b) comparing the amounts of hypoxia inducible factor 1 , alpha subunit (HIF- 1 alpha), autotaxin, lysophosphatidylcholine or lysophosphatidic acid in the biological sample with the amounts present in a normal control biological sample from a normal subject,
wherein a difference in the level of hypoxia inducible factor 1 , alpha subunit (HIF- 1 alpha), autotaxin, lysophosphatidylcholine or lysophosphatidic acid in the biological sample is indicative of hepatitis B virus infection.
12. A method of monitoring efficacy of a therapy in a subject having, suspected of having, hepatitis B virus infection, comprising:
(a) quantifying the amounts of hypoxia inducible factor 1 , alpha subunit (HIF- 1 alpha), autotaxin, lysophosphatidylcholine or lysophosphatidic acid in a biological sample obtained from an individual prior to and/or during and/or following therapy for hepatitis B virus infection; and
(b) comparing the amount of hypoxia inducible factor 1 , alpha subunit (HIF- 1 alpha), autotaxin, lysophosphatidylcholine or lysophosphatidic acid in said sample obtained prior to therapy with the amount present in one or more samples taken from said subject during and/or following therapy,
wherein a difference in the level of hypoxia inducible factor 1 , alpha subunit (HIF- 1 alpha), autotaxin, lysophosphatidylcholine or lysophosphatidic acid in the biological sample is indicative of an effect of said therapy.
13. A method as defined in claim 1 1 or claim 12, wherein quantifying is performed by measuring the concentration of hypoxia inducible factor 1 , alpha subunit (HIF-1 alpha), autotaxin, lysophosphatidylcholine or lysophosphatidic acid in the or each sample.
14. A method as defined in any of claims 11 to 13, wherein detecting and/or quantifying is performed by one or more methods selected from SELDI (-TOF), MALDI (-TOF), a 1-D gel- based analysis, a 2-D gel-based analysis, Mass spec (MS), reverse phase (RP) LC, size permeation (gel filtration), ion exchange, affinity, HPLC, UPLC or other LC or LC-MS-based technique.
15. A method as defined in any of claims 11 to 14, wherein detecting and/or quantifying i performed using an immunological method.
16. A method as defined in any of claims 1 1 to 15, wherein the detecting and/or quantifying is performed using a biosensor or a microanalytical, microengineered, microseparation or immunochromatography system.
17. A method as defined in any of claims 1 1 to 16, wherein the biological sample is whole blood, blood serum, plasma, cerebrospinal fluid, urine, saliva, or other bodily fluid, or breath, condensed breath, or an extract or purification therefrom, or dilution thereof.
18. Use of a kit comprising a biosensor capable of detecting and/or quantifying hypoxia inducible factor 1 , alpha subunit (HIF-1 alpha), autotaxin, lysophosphatidylcholine or lysophosphatidic acid for monitoring, prognosing or diagnosing hepatitis B virus infection.
19. A method of screening for an autotaxin-lysophosphatidic acid signalling pathway modulator, which comprises the steps of:
(a) contacting a virally infected hepatoma cell with a test compound; and
(b) comparing the effect of said test compound upon the autotaxin- lysophosphatidic acid signalling pathway within said cell.
20. The method of screening as defined in claim 19, wherein the virally infected hepatoma cell comprises Huh-7 hepatoma cells
21. The method of screening as defined in claim 19, wherein the virally infected hepatoma cell comprises HepG2 hepatoma cells.
22. An autotaxin-lysophosphatidic acid signalling pathway modulator obtainable by a method as defined in any of claims 19 to 21.
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