WO2009115398A1 - Vitamin d compounds for the treatment of biliary diseases - Google Patents

Vitamin d compounds for the treatment of biliary diseases Download PDF

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Publication number
WO2009115398A1
WO2009115398A1 PCT/EP2009/052201 EP2009052201W WO2009115398A1 WO 2009115398 A1 WO2009115398 A1 WO 2009115398A1 EP 2009052201 W EP2009052201 W EP 2009052201W WO 2009115398 A1 WO2009115398 A1 WO 2009115398A1
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Prior art keywords
vitamin
biliary
vdr
compound
udca
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PCT/EP2009/052201
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French (fr)
Inventor
Chantal Housset
Nicolas Chignard
Raoul Poupon
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INSERM (Institut National de la Santé et de la Recherche Médicale)
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Publication of WO2009115398A1 publication Critical patent/WO2009115398A1/en

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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
    • A61K31/59—Compounds containing 9, 10- seco- cyclopenta[a]hydrophenanthrene 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
    • A61K31/59—Compounds containing 9, 10- seco- cyclopenta[a]hydrophenanthrene ring systems
    • A61K31/592—9,10-Secoergostane derivatives, e.g. ergocalciferol, i.e. vitamin D2
    • 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
    • A61K31/59—Compounds containing 9, 10- seco- cyclopenta[a]hydrophenanthrene ring systems
    • A61K31/593—9,10-Secocholestane derivatives, e.g. cholecalciferol, i.e. vitamin D3
    • 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

Definitions

  • the invention relates to a vitamin D compound for treating or preventing biliary diseases.
  • PBC Primary biliary cirrhosis
  • the disease is progressive in nature, with a significant proportion of affected patients going on to develop cirrhosis. It may affect up to 1 in 3-4,000 people and the sex ratio is at least 9 to 1 , women to men (reference: Medical care of the Liver Transplant Patients,
  • PSC Primary sclerosing cholangitis
  • UDCA ursodeoxycholic acid
  • the current therapy of PBC and PSC relies upon the administration of ursodeoxycholic acid (UDCA).
  • UDCA ursodeoxycholic acid
  • the efficacy of UDCA treatment has been demonstrated mainly in PBC.
  • the efficacy of UDCA is not constant in PBC; it is not complete in PSC or other biliary-type liver diseases, e.g. Cystic fibrosis liver disease (Colombo et al. J Pediatr Gastroenterol Nutr 2006, 43:S49-55), MDR3 defect-related liver diseases including the low-phospholipid associated intrahepatic cholelithiasis syndrome (Rosmorduc et al. Orphanet Journal of rare Diseases 2007, 2:1-6). Accordingly there is an existing need to identify further therapeutic agents for the treatment of PBC, PSC, particular forms of cholelithiasis and other liver diseases of biliary type.
  • the present invention relates to a vitamin D compound for treating or preventing biliary diseases.
  • the present invention also relates to pharmaceutical composition for treating or preventing biliary diseases which comprises a vitamin D compound.
  • the present invention relates to a method for treating and/or preventing biliary diseases comprising administering a subject in need thereof with a vitamin D compound of the invention in combination with ursodeoxycholic acid.
  • biliary disease or "biliary-type liver disease” refers to all liver diseases that target primarily or secondarily cholangiocytes and can also be called cholangiopathy.
  • biliary diseases encompass primary biliary cirrhosis (PBC), primary (and secondary) sclerosing cholangitis (PSC), cystic fibrosis liver disease, MDR3 defect-related liver diseases, intrahepatic cholelihiasis, biliary atresia and cholangiocarcinoma.
  • PBC primary biliary cirrhosis
  • PSC primary (and secondary) sclerosing cholangitis
  • cystic fibrosis liver disease MDR3 defect-related liver diseases
  • intrahepatic cholelihiasis biliary atresia
  • cholangiocarcinoma cholangiocarcinoma
  • vitamin D compound includes any compound being vitamin D or an analogue thereof that is capable of
  • a vitamin D compound includes vitamin D2 (or ergocalciferol) compounds, vitamin D3 (or cholecalciferol) compounds, isomers thereof, or derivatives/analogues thereof.
  • Preferred vitamin D compounds are vitamin D3 compounds which are ligands of (more preferably are agonists of) the vitamin D receptor.
  • the vitamin D compound e.g., the vitamin D3 compound
  • Vitamin D1 compounds, vitamin D2 compounds and vitamin D3 compounds include, respectively, vitamin D1 , D2, D3 and analogues thereof. Examples of specific vitamin D compounds suitable for use in the methods of the present invention are further described herein.
  • the agonistic activity of compounds towards the VDR may be determined using various methods. For example, cotransfection assays can be performed in HEK293 cells with a GAL4-responsive luciferase reporter plasmid and cytomegalovirus-based (CMX) expression plasmids for GAL4-SRC-1 and VP16 chimeras containing the full-length mammalian VDR. CMX-VP16 alone is used as control (no receptor). VDR activation is ascertained then by luciferase activity.
  • CMX-VP16 cytomegalovirus-based
  • Cells can also be cotransfected with the GAL4- responsive luciferase reporter and expression vectors containing the GAL4 DNA-binding domain fused to the ligand-binding domain of human VDR (residues 90-427). VDR activation is then ascertained by luciferase activity after normalization to an internal Beta-galactosidase control. Cells can also be transfected with a luciferase reporter gene plasmid driven by four copies in tandem of a consensus DR3 response element for VDR cloned upstream of the herpes virus simplex thymidine kinase gene promoter. VDR activation is ascertained by luciferase activity.
  • Ligand binding can also be performed to assay the ability of molecules to bind to VDR using lysates from COS-7 cells transfected with expression plasmids for VDR. Binding is performed overnight at 4°C in lysate buffer with 0.71 nM (18 Ci/mmol) [ 3 H]1 , 25(OH) 2 D3 and various potential competitor. Unbound [ 3 H]1 , 25(OH)2D3 is removed by adsorption to dextran-coated charcoal and the - A -
  • Ki values are calculated from a computer fit of competition curves.
  • treatment refers to inhibiting the disease or condition, i.e. arresting its development; relieving the disease or condition, i.e. causing regression of the condition; or relieving the conditions caused by the disease, i.e. symptoms of the disease.
  • prevention refers to preventing the disease or condition from occurring in a subject who has not yet been diagnosed as having it.
  • the invention relates to a vitamin D compound for treating or preventing biliary diseases.
  • the invention relates to a vitamin D compound for the treatment of primary biliary cirrhosis (PBC), primary (and secondary) sclerosing cholangitis, cystic fibrosis liver disease, MDR3 defect-related liver diseases, intrahepatic cholelihiasis, biliary atresia and cholangiocarcinoma.
  • PBC primary biliary cirrhosis
  • primary (and secondary) sclerosing cholangitis cystic fibrosis liver disease
  • MDR3 defect-related liver diseases intrahepatic cholelihiasis
  • biliary atresia cholangiocarcinoma
  • the invention relates to a vitamin D compound for the treatment of primary (and secondary) sclerosing cholangitis, cystic fibrosis liver disease, MDR3 defect-related liver diseases, intrahepatic cholelihiasis, biliary atresia and cholangiocarcinoma.
  • the invention relates to a vitamin D compound for the treatment of primary biliary cirrhosis (PBC)
  • PBC primary biliary cirrhosis
  • the invention relates to a vitamin D compound for the treatment of primary sclerosing cholangitis (PSC).
  • the vitamin D compound is vitamin D2 (ergocalciferol) which has the formula:
  • the vitamin D compound is vitamin D3 (cholecalciferol) which has the formula:
  • active vitamin D compounds of the present invention include, but are not limited to, the analogs, homologs and derivatives of vitamin D compounds described in the following patents, each of which is incorporated by reference: U. S. Patent Nos.
  • 4,391 ,802 (1a-hydroxy vitamin D derivatives); 4,717,721 (1a-hydroxy derivatives with a 17 side chain greater in length than the cholesterol or ergosterol side chains) ; 4,851 ,401 (cyclopentano-vitamin D analogs) ; 4,866,048 and 5,145,846 (vitamin D3 analogues with alkynyl, alkenyl, and alkanyl side chains) ; 5,120,722 (trihydroxycalciferol) ; 5,547,947 (fluoro-cholecalciferol compounds) ; 5,446,035 (methyl substituted vitamin D); 5,411 ,949 (23-oxa-derivatives) ; 5,237,110 (19-nor-vitamin D compounds ⁇ ; 4,857,518 (hydroxylated 24-homo-vitamin D derivatives).
  • vitamin D receptor agonists including paricalcitol (ZEMPLAR) (see US Patent 5,587,497), tacalcitol (BONALFA) (see US Patent 4,022,891 ), doxercalciferol (HECTOROL) (see Lam et al. (1974) Science 186, 1038), maxacalcitol (OXAROL(TM)) (see US Patent 4,891 ,364), calcipotriol (DAIVONEX(TM)) (see US Patent 4,866,048), falecalcitriol (FULSTAN).
  • ZEMPLAR paricalcitol
  • BONALFA see US Patent 4,022,891
  • HECTOROL doxercalciferol
  • maxacalcitol OXAROL(TM)
  • DAIVONEX(TM) see US Patent 4,866,048)
  • falecalcitriol FULSTAN
  • Other compounds include investigational drugs from Leo Pharmaceuticals including EB 1089 (24a,26a,27a-trihomo-22,24-diene-laa,25-(OH)2-D3, KH 1060 (20- epi-22- oxa-24a,26a,27a-trihomo-1a,25-(OH)2-D3), MC 1288 (1 ,25-(OH)2-20-epi-D3) and MC 903 (calcipotriol, 1a24s-(OH)2-22-ene-26,27-dehydro-D3); Roche Pharmaceutical drugs that include1 ,25-(OH)2-16-ene-D3, 1 ,25-(OH)2-16-ene- 23-yne- D3, and 25-(OH)2-16-ene-23-yne-D3; Chugai Pharmaceuticals 22- oxacalcitriol(22-oxa- 1a,25-(OH)2-D3; Ia-(OH)-Ds from the University of Illinois; and drugs from
  • Additional examples include 1a,25-(OH)2-26,27-d6-D3; 1 a,25-(OH)2-22-ene- D3;1a,25-(OH)2-D3; 1 a,25-(OH)2-D2;1 a,25-(OH)2-D4; 1 a,24,25-(OH)3-D3; 1a,24,25- (OH)3-D2;1a,24,25-(OH)3-D4; 1a-(OH)-25-FD3; 1a-(OH)-25-FD4; 1a-(OH)-25-FD2; 1a,24-(OH)2-D4; 1a,24-(OH)2-D3; 1a,24-(OH)2-D2; 1 a,24-(OH)2-25-FD4;1a,24-(OH)2- 25-FD3; 1 a,24-(OH)2; 1 a,24-(OH)2-25-FD4;1a,24-(OH)
  • Other example compounds that may be used by the invention include but are not limited to 1 ,25-di hydroxy- 16-ene-23-yne cholecalciferol, 1 ,25 -dihydroxy-21 -(3 - hydroxy- 3-methylbutyl)- 19-nor-cholecalciferol, 1 ,25-dihydroxy-21 -(2R,3- dihy[omega]Oxy-3-raethy]-butyl)-20R-cholecalciferol; 1 ,25-dihydroxy-21 -(2R53- dihydroxy-3-metbyl-butyl)-20S-cholecalciferol; 1 , 25-Dihydroxy-21 -(2Rs3-dibydroxy-3- metbyl-butyl)-20S- 19-nor-cholecalciferol; 1 ,25-Dihydroxy-20S-21 -(3-hydroxy-3-methyl- butyl)-24-keto-l 9-nor
  • Another object of the invention relates to a method for treating and/or preventing biliary diseases comprising administering a subject in need thereof with a vitamin D compound as above described.
  • the vitamin D compound may be administered in the form of a pharmaceutical composition, as defined below.
  • said compound is administered in a therapeutically effective amount.
  • a “therapeutically effective amount” is meant a sufficient amount of the vitamin D compound to treat and/or to prevent biliary diseases at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, gender and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.
  • a therapeutically effective amount of vitamin D compound may range from about 0.001 to 30 ug/kg body weight, preferably about 0.01 to 25 ug/kg body weight, more preferably about 0.1 to 20 ug/kg body weight, and even more preferably about 1 to 10 ug/kg, 2 to 9 ug/kg, 3 to 8 ug/kg, 4 to 7 ug/kg, or 5 to 6 ug/kg body weight.
  • an effective dosage may range from about 0.001 to 30 ug/kg body weight, preferably about 0.01 to 25 ug/kg body weight, more preferably about 0.1 to 20 ug/kg body weight, and even more preferably about 1 to 10 ug/kg, 2 to 9 ug/kg, 3 to 8 ug/kg, 4 to 7 ug/kg, or 5 to 6 ug/kg body weight.
  • the skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder,
  • the dose administered will also depend on the particular vitamin D compound used, the effective amount of each compound can be determined by titration methods known in the art.
  • treatment of a subject with a therapeutically effective amount of a vitamin D compound can include a single treatment or, preferably, can include a series of treatments.
  • a subject is treated with a vitamin D compound in the range of between about 0.1 to 20 ug/kg body weight, once per day for a duration of six months or longer, for example for life depending on management of the symptoms and the evolution of the condition.
  • an "on-off ' or intermittent treatment regimen can be considered.
  • the effective dosage of a vitamin D compound used for treatment may increase or decrease over the course of a particular treatment.
  • the vitamin D compound may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.
  • “Pharmaceutically” or “pharmaceutically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate.
  • a pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • the active principle alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings.
  • Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.
  • the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that easy syhngability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the Vitamin D compound of the invention can be formulated into a composition in a neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like.
  • Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, thmethylamine, histidine, procaine and the like.
  • inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, thmethylamine, histidine, procaine and the like.
  • the carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.
  • aqueous solutions For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage could be dissolved in 1 ml of isotonic NaCI solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • the Vitamin D compound of the invention may be formulated within a therapeutic mixture to comprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0 or even about 10 milligrams per dose or so. Multiple doses can also be administered.
  • parenteral administration such as intravenous or intramuscular injection
  • other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; liposomal formulations ; time release capsules ; and any other form currently used.
  • composition of the invention may further comprise a compound known to be suitable for treating or preventing biliary diseases.
  • Said compound may be ursodeoxycholic acid which has the formula:
  • composition of the invention may comprise other bile acid derivatives (e.g. nor-ursodeoxycholic acid) or other nuclear receptor agonists (e.g. farnesoid X receptor agonists).
  • other bile acid derivatives e.g. nor-ursodeoxycholic acid
  • other nuclear receptor agonists e.g. farnesoid X receptor agonists
  • Another aspect of the invention relates to method for treating and/or preventing biliary diseases comprising administering a subject in need thereof with a vitamin D compound of the invention in combination with ursodeoxycholic acid or other bile acid derivatives (e.g. nor-ursodeoxycholic acid) or other nuclear receptor agonists (e.g. farnesoid X receptor agonists).
  • ursodeoxycholic acid or other bile acid derivatives e.g. nor-ursodeoxycholic acid
  • other nuclear receptor agonists e.g. farnesoid X receptor agonists
  • FIGURES are a diagrammatic representation of FIGURES.
  • Figure 1 Cathelicidin expression in human biliary epithelial cells.
  • FIG. 1 Bile salts control VDR expression and activity in biliary epithelial cells.
  • A Proteins from biliary epithelial cells incubated with CDCA or UDCA for the indicated time were submitted to VDR and ⁇ -actin immunoblot-ECL analyses. Representative gels of five different experiments are shown.
  • B, C Proteins from biliary epithelial cells incubated for 2 h with CDCA or UDCA with or without PD 98059 were submitted to (B) p-ERK 1/2 and ERK 1/2 immunoblot-ECL analyses;
  • C VDR and ⁇ - actin immunoblot-ECL analyses. Representative gels of three different experiments are shown.
  • Biliary epithelial cells transfected with a VDRE-driven promoter were assayed for luciferase activity after incubation with CDCA or UDCA in the presence or absence of PD 98059. Data are expressed as fold over control and represent means ⁇ SEM of 3 experiments.
  • Biliary epithelial cells transfected with a VDRE-driven promoter were assayed for luciferase activity after incubation with the FXR agonist, GW4064 (1 ⁇ mol/L). Data represent means ⁇ SEM of 3 experiments. * P ⁇ 0.05 vs. control.
  • Bile salts induce cathelicidin expression in biliary epithelial cells.
  • A Total RNA from biliary epithelial cells treated with CDCA, UDCA, VD3 or the combination of bile salts with VD3 was subjected to real time RT-PCR with primers designated to amplify cathelicidin. Data represent means ⁇ SEM of 4 experiments performed in duplicate. * P ⁇ 0.05 vs. untreated cells. ** P ⁇ 0.05 vs. VD3 or bile salts alone.
  • Biliary epithelial cells transfected either with scramble or VDR siRNA were incubated with CDCA, UDCA or the combination of bile salts with VD3 and subjected to cathelicidin mRNA detection by real time RT-PCR. Data represent means ⁇ SEM of 3 experiments performed in duplicate. * P ⁇ 0.05 vs. scramble.
  • UDCA treatment causes increased expressions of VDR and cathelicidin in the human liver.
  • Total RNA from the liver of PBC patients before or after the onset of UDCA treatment was subjected to real time RT-PCR with primers designated to amplify either VDR or cathelicidin. Results are expressed as mRNA levels relative to the mean value of PBC patients before UDCA treatment.
  • B Comparison between PBC patients before and after the onset of UDCA treatment. * P ⁇ 0.05.
  • Figure 5 1 ⁇ , 25(OH) 2 D 3 induces cathelicidin expression through VDR in biliary epithelial cells.
  • Human biliary epithelial cells were transfected either with scramble (Sc) or VDR siRNA (Si) and subjected to (A) Detection of VDR mRNA by real time RT-PCR (Data represent means +/- SEM of 3 experiments performed in duplicate; * P ⁇ 0.005 vs scramble); (B) Detection of VDR and ⁇ -actin by immunoblot-ECL analyses (Representative gels of three different experiments are shown); (C) Incubation with VD3 for 24h before cathelicidin expression was analysed by real time RT-PCR (Data represent means +/- SEM of 3 experiments performed in duplicate. * P>0.05 vs. control. ** P ⁇ 0.005 vs. scramble).
  • DMEM and fetal bovine serum were purchased from Invitrogen (Cergy).
  • the human biliary epithelial cell line Mz-ChA-1 (Knuth A. et al. 1985) was cultured in DMEM, supplemented with 1 g/L glucose, 10 mmol/L Hepes and 10% fetal bovine serum, under 95% air and 5% CO2 at 37°C. The culture medium was renewed every 48 h.
  • Mz-ChA-1 cells were incubated with either CDCA (100 ⁇ mol/L) or UDCA (100 ⁇ mol/L) for 1 to 24h.
  • Mz- ChA-1 cells were preincubated with PD 98059 (50 ⁇ mol/L) for 1 h before either CDCA (100 ⁇ mol/L), UDCA (100 ⁇ mol/L) or 1 ⁇ ,25(OH) 2 D 3 (0.1 ⁇ mol/L) were added and maintained for the following two hours.
  • Electrophoretic mobility shift assay (EMSA) experiments were performed as recommended by the manufacturer (Panomics, Montigny-le-Bretonneux, France). Briefly, nuclear extracts (2 ⁇ g), Poly d(l-C) (0.1 ⁇ g/ ⁇ l) and VDRE biotin-labeled probe (1 ng/ ⁇ l) were incubated in binding buffer at room temperature for 30 min. Samples were then runned at 4°C through a 6% polyacrylamide gel at 120V. Gel was then transferred for 30-45 min at 300 mA to Biodyne B ® nylon membrane (Pall, Fontenay-sous-Bois, France).
  • the membranes were baked for 1 hour at 80°C in a dry oven and transferred to a UV crosslinker oven for 3 min. Membranes were then blocked by incubation at room temperature for 15 min, before streptavidin-HRP conjugate was added. The biotin-streptavidin complex was revealed by enhanced chemiluminescence using an ECL kit (Amersham, Les UNs, France).
  • Mz-ChA-1 cells were seeded into six-well plates (2 10 5 cells/well) and grown overnight in DMEM with 1 g/L glucose, 10 mmol/L Hepes and 10% fetal bovine serum, under 95% air and 5% CO 2 at 37°C.
  • Cells were transfected using Lipofectamine 2000 (Invitrogen, Cergy-Pontoise, France) with either a luciferase reporter gene plasmid driven by four copies in tandem of a consensus DR3 response element for VDR cloned upstream of the herpes virus simplex thymidine kinase gene promoter (Kahlen JP. et al.
  • CMX-hFXR-W469A human FXR
  • VDR siRNA Dharmacon Research Inc., Lafayette, CO
  • silencer negative control RNA Ambion, Cambridgeshire, England
  • Quantitative real time PCR was performed using the Sybr Green PCR Core Reagents Kit (Perkin Elmer Applied Biosystems, Courtaboeuf, France) on a LightCycler 1.5 (Roche Diagnostics, Mannheim, Germany).
  • the primers used in real time PCR were the following: 5' GCT AAC CTC TAC CGC CTC CT 3' (sense) (SEQ ID NO :1 ) and 5' GGT CAC TGT CCC CAT ACA CC 3' (antisense) (SEQ ID NO :2) for cathelicidin (Gombart AF. et al.
  • Quantitative real time PCR reactions were run with 200 nmol/L of cathelicidin sense and antisense primers and 50 nmol/L of each 18S sense and antisense primers. Data were collected and analyzed with Roche LightCycler Software 3.5.3 (Roche Diagnostics, Mannheim, Germany). Data were expressed as a relative amount (2 " ⁇ CT ) of the control value.
  • Statistical analysis Comparisons were made using the Student's t test, the paired Wilcoxon or unpaired Mann-Whitney rank sum test. P ⁇ 0.05 was considered as significant.
  • liver epithelial cells which are constantly exposed to bile salts, express cathelicidin.
  • cathelicidin expression was abundant both in biliary epithelial cells and within the lumen of the biliary tract ( Figure 1 B), indicating that this antibacterial peptide is secreted in bile in response to infection.
  • VDR activation is regulated by VDR in other cell types, we tested the potential of VDR activation to control cathelicidin expression in biliary epithelial cells. As shown in figure 1C and in Figure 5, VDR activation by 1 ⁇ ,25(OH) 2 D 3 (VD3) increased cathelicidin expression in biliary epithelial cells.
  • bile salts can stimulate the expression or the activity of nuclear receptors (e.g. FXR) by ligand-independent mechanisms (Lew JL. Et al. 2004).
  • FXR nuclear receptors
  • both bile salts induced a sustained increase in VDR protein levels. Because on the one hand, bile salts are able to activate ERK V 2 (Yoon JH. Et al.
  • VDR protein expression in nuclear fractions of biliary epithelial cells we analyzed VDR protein expression in nuclear fractions of biliary epithelial cells.
  • UDCA like VD3, induced VDR nuclear translocation, while CDCA had little or no effect (Figure 2D). Consistent with this finding, in electrophoretic mobility shift assay (EMSA) experiments, UDCA clearly induced the binding of VDR to VDRE sequences, while CDCA had only a minor effect on DNA-protein complex formation (Figure 2E).
  • ESA electrophoretic mobility shift assay
  • cathelicidin which is known to neutralize the deleterious effects of bacterial products (Larrick JW. Et al 1994), could account for the clearance of endotoxins previously reported in PBC patients under UDCA treatment (Sasatomi K. et al. 1998; Ballot E; et 2004).
  • the increase in cathelicidin expression in biliary epithelial cells was higher when UDCA was combined with vitamin D.
  • VPAC1 expression is regulated by FXR agonists in the human gallbladder epithelium. Hepatology 42, 549-557 (2005).
  • Bile acids induce cyclooxygenase-2 expression via the epidermal growth factor receptor in a human cholangiocarcinoma cell line. Gastroenterology 122, 985-993 (2002).

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Abstract

The present invention relates to uses, methods and compositions for treating or preventing biliary diseases such as primary sclerosing cholangitis (PSC). More specifically, the invention relates to a vitamin D compound for treating or preventing biliary diseases. The invention encompasses the use of said vitamin D compound in combination with ursodeoxycholic acid.

Description

VITAMIN D COMPOUNDS FOR THE TREATMENT OF BILIARY DISEASES
FIELD OF THE INVENTION:
The invention relates to a vitamin D compound for treating or preventing biliary diseases.
BACKGROUND OF THE INVENTION:
Under normal conditions, while being exposed to bacterial invasion from the intestine by ascending route or via the portal venous circulation, the biliary tract remains sterile. Mucous secretion, bile flow, IgA and bile salts are multiple defense mechanisms responsible for this pathogen-free environment (Sung, J.Y et al. 1992). In disease states such as primary biliary cirrhosis (PBC), sterility may be disrupted as evidenced by the presence of endotoxins in biliary epithelial cells (Hopf, U. et al. 1989).
Primary biliary cirrhosis (PBC), is an autoimmune liver disease leading to the destruction of intrahepatic bile ducts (Kaplan, MM, N Engl J Med, 335:1570-80 (1996)).
The disease is progressive in nature, with a significant proportion of affected patients going on to develop cirrhosis. It may affect up to 1 in 3-4,000 people and the sex ratio is at least 9 to 1 , women to men (reference: Medical care of the Liver Transplant Patients,
3rd Edition published 2006, edited by Paul G. Killenberg, page 155).
Primary sclerosing cholangitis (PSC) is another chronic liver disease characterized by fibrosing inflammation and obliteration of intra and extrahepatic bile ducts, resulting in biliary cirrhosis and is associated with an increased risk of cholangiocarcinoma. The majority of patients is young, male and have coexisting inflammatory bowel disease. PSC is found with a prevalence of 10/100,000 in Northern
European populations.
The current therapy of PBC and PSC relies upon the administration of ursodeoxycholic acid (UDCA). The efficicacy of UDCA treatment has been demonstrated mainly in PBC. However, the efficacy of UDCA is not constant in PBC; it is not complete in PSC or other biliary-type liver diseases, e.g. Cystic fibrosis liver disease (Colombo et al. J Pediatr Gastroenterol Nutr 2006, 43:S49-55), MDR3 defect-related liver diseases including the low-phospholipid associated intrahepatic cholelithiasis syndrome (Rosmorduc et al. Orphanet Journal of rare Diseases 2007, 2:1-6). Accordingly there is an existing need to identify further therapeutic agents for the treatment of PBC, PSC, particular forms of cholelithiasis and other liver diseases of biliary type.
SUMMARY OF THE INVENTION:
The present invention relates to a vitamin D compound for treating or preventing biliary diseases.
The present invention also relates to pharmaceutical composition for treating or preventing biliary diseases which comprises a vitamin D compound.
Finally, the present invention relates to a method for treating and/or preventing biliary diseases comprising administering a subject in need thereof with a vitamin D compound of the invention in combination with ursodeoxycholic acid.
DETAILED DESCRIPTION OF THE INVENTION:
Main Definitions:
The term "biliary disease" or "biliary-type liver disease" refers to all liver diseases that target primarily or secondarily cholangiocytes and can also be called cholangiopathy. Examples of biliary diseases encompass primary biliary cirrhosis (PBC), primary (and secondary) sclerosing cholangitis (PSC), cystic fibrosis liver disease, MDR3 defect-related liver diseases, intrahepatic cholelihiasis, biliary atresia and cholangiocarcinoma. As used herein, the term "vitamin D compound" includes any compound being vitamin D or an analogue thereof that is capable of treating biliary diseases. Generally, compounds which are ligands for the vitamin D receptor (VDR ligands) and which are capable of treating biliary disease are considered to be within the scope of the invention. Vitamin D compounds are preferably agonists of the vitamin D nuclear receptor.
A vitamin D compound includes vitamin D2 (or ergocalciferol) compounds, vitamin D3 (or cholecalciferol) compounds, isomers thereof, or derivatives/analogues thereof. Preferred vitamin D compounds are vitamin D3 compounds which are ligands of (more preferably are agonists of) the vitamin D receptor. Preferably the vitamin D compound (e.g., the vitamin D3 compound) is a more potent agonist of the vitamin D receptor than the native ligand (i.e. vitamin D3). Vitamin D1 compounds, vitamin D2 compounds and vitamin D3 compounds include, respectively, vitamin D1 , D2, D3 and analogues thereof. Examples of specific vitamin D compounds suitable for use in the methods of the present invention are further described herein. The agonistic activity of compounds towards the VDR may be determined using various methods. For example, cotransfection assays can be performed in HEK293 cells with a GAL4-responsive luciferase reporter plasmid and cytomegalovirus-based (CMX) expression plasmids for GAL4-SRC-1 and VP16 chimeras containing the full-length mammalian VDR. CMX-VP16 alone is used as control (no receptor). VDR activation is ascertained then by luciferase activity. Cells can also be cotransfected with the GAL4- responsive luciferase reporter and expression vectors containing the GAL4 DNA-binding domain fused to the ligand-binding domain of human VDR (residues 90-427). VDR activation is then ascertained by luciferase activity after normalization to an internal Beta-galactosidase control. Cells can also be transfected with a luciferase reporter gene plasmid driven by four copies in tandem of a consensus DR3 response element for VDR cloned upstream of the herpes virus simplex thymidine kinase gene promoter. VDR activation is ascertained by luciferase activity. Ligand binding can also be performed to assay the ability of molecules to bind to VDR using lysates from COS-7 cells transfected with expression plasmids for VDR. Binding is performed overnight at 4°C in lysate buffer with 0.71 nM (18 Ci/mmol) [3H]1 , 25(OH)2D3 and various potential competitor. Unbound [3H]1 , 25(OH)2D3 is removed by adsorption to dextran-coated charcoal and the - A -
supernatant removed for scintillation counting. Ki values are calculated from a computer fit of competition curves.
As used herein, the term "treatment" refers to inhibiting the disease or condition, i.e. arresting its development; relieving the disease or condition, i.e. causing regression of the condition; or relieving the conditions caused by the disease, i.e. symptoms of the disease.
As used herein, the term "prevention" refers to preventing the disease or condition from occurring in a subject who has not yet been diagnosed as having it.
Therapeutic methods and uses
The invention relates to a vitamin D compound for treating or preventing biliary diseases. In a particular embodiment, the invention relates to a vitamin D compound for the treatment of primary biliary cirrhosis (PBC), primary (and secondary) sclerosing cholangitis, cystic fibrosis liver disease, MDR3 defect-related liver diseases, intrahepatic cholelihiasis, biliary atresia and cholangiocarcinoma.
In a more particular embodiment, the invention relates to a vitamin D compound for the treatment of primary (and secondary) sclerosing cholangitis, cystic fibrosis liver disease, MDR3 defect-related liver diseases, intrahepatic cholelihiasis, biliary atresia and cholangiocarcinoma.
In another particular embodiment, the invention relates to a vitamin D compound for the treatment of primary biliary cirrhosis (PBC) In a preferred embodiment, the invention relates to a vitamin D compound for the treatment of primary sclerosing cholangitis (PSC).
In a particular embodiment, the vitamin D compound is vitamin D2 (ergocalciferol) which has the formula:
Figure imgf000006_0001
In a particular embodiment, the vitamin D compound is vitamin D3 (cholecalciferol) which has the formula:
Figure imgf000006_0002
A large number of other active vitamin D compounds are known and can be used in the practice of the invention. The active vitamin D compounds of the present invention include, but are not limited to, the analogs, homologs and derivatives of vitamin D compounds described in the following patents, each of which is incorporated by reference: U. S. Patent Nos. 4,391 ,802 (1a-hydroxy vitamin D derivatives); 4,717,721 (1a-hydroxy derivatives with a 17 side chain greater in length than the cholesterol or ergosterol side chains) ; 4,851 ,401 (cyclopentano-vitamin D analogs) ; 4,866,048 and 5,145,846 (vitamin D3 analogues with alkynyl, alkenyl, and alkanyl side chains) ; 5,120,722 (trihydroxycalciferol) ; 5,547,947 (fluoro-cholecalciferol compounds) ; 5,446,035 (methyl substituted vitamin D); 5,411 ,949 (23-oxa-derivatives) ; 5,237,110 (19-nor-vitamin D compounds}; 4,857,518 (hydroxylated 24-homo-vitamin D derivatives).
Particular examples include ROCALTROL (Roche Laboratories); and CALCIJEX injectable calcitriol. Other examples are vitamin D receptor agonists including paricalcitol (ZEMPLAR) (see US Patent 5,587,497), tacalcitol (BONALFA) (see US Patent 4,022,891 ), doxercalciferol (HECTOROL) (see Lam et al. (1974) Science 186, 1038), maxacalcitol (OXAROL(TM)) (see US Patent 4,891 ,364), calcipotriol (DAIVONEX(TM)) (see US Patent 4,866,048), falecalcitriol (FULSTAN). Other compounds include ecalcidene, calcithiazol and tisocalcitate. Other compounds include atocalcitol, lexacalcitol and seocalcitol. Another compound of possible interest is secalciferol ("OSTEO D"). Other compounds include investigational drugs from Leo Pharmaceuticals including EB 1089 (24a,26a,27a-trihomo-22,24-diene-laa,25-(OH)2-D3, KH 1060 (20- epi-22- oxa-24a,26a,27a-trihomo-1a,25-(OH)2-D3), MC 1288 (1 ,25-(OH)2-20-epi-D3) and MC 903 (calcipotriol, 1a24s-(OH)2-22-ene-26,27-dehydro-D3); Roche Pharmaceutical drugs that include1 ,25-(OH)2-16-ene-D3, 1 ,25-(OH)2-16-ene- 23-yne- D3, and 25-(OH)2-16-ene-23-yne-D3; Chugai Pharmaceuticals 22- oxacalcitriol(22-oxa- 1a,25-(OH)2-D3; Ia-(OH)-Ds from the University of Illinois; and drugs from the Institute of Medical Chemistry-Schering AG that include ZK 161422 (20-methyl-1 ,25-(OH)2-D3) and ZK 157202 (20-methyl- 23-ene-1 ,25-(OH)2-D3); 1a-(OH)-D2; 1a-(OH)-D3 and 1a- (OH)-D4. Additional examples include 1a,25-(OH)2-26,27-d6-D3; 1 a,25-(OH)2-22-ene- D3;1a,25-(OH)2-D3; 1 a,25-(OH)2-D2;1 a,25-(OH)2-D4; 1 a,24,25-(OH)3-D3; 1a,24,25- (OH)3-D2;1a,24,25-(OH)3-D4; 1a-(OH)-25-FD3; 1a-(OH)-25-FD4; 1a-(OH)-25-FD2; 1a,24-(OH)2-D4; 1a,24-(OH)2-D3; 1a,24-(OH)2-D2; 1 a,24-(OH)2-25-FD4;1a,24-(OH)2- 25-FD3; 1 a,24-(OH)2-25-FD2; 1a,25-(OH)2-26,27-F6-22-ene-D3; 1a,25-(OH)2-26,27- F6-D3; 1a,25S-(OH)2-26-F3-D3;1 a,25-(OH)2-24-F2-D3; 1 a,25S,26-(OH)2-22-ene- D3; 1 a,25R,26-(OH)2-22-ene-D3; 1 a,25-(OH)2-D2; 1 a,25(OH)2-24-epi-D3; 1 a,25-(OH)2- 23-yne-D3; 1a,25-(OH)2-24R-F-D3;1 a,25S,26-(OH)2-D3; 1a,24R-(OH)2-25F-D3; 1a,25- (OH)2-26,27-F6-23-yne-D3; 1 a,25R-(OH)2-26-F3-D3; 1 a,25,28-(OH)3-D2; 1 a,25-(OH)2- 16-ene-23-yne-D3; 1 a,24R,25-(OH)3-D3; 1 a,25-(OH)2-26,27-F6-23-ene-D3; 1 a,25R-
(OH)2-22-ene-26-F3-D3; 1 [alpha],25S-(OH)2-22-enc-26-F3-D3; 1 a,25R-(OH)2-D3-
26,26,26-d3;1a,25S-(OH)2-D3-26,26,26-d3; and 1a,25R-(OH)2-22-ene-D3-26,26,26-D3.
Other example compounds that may be used by the invention include but are not limited to 1 ,25-di hydroxy- 16-ene-23-yne cholecalciferol, 1 ,25 -dihydroxy-21 -(3 - hydroxy- 3-methylbutyl)- 19-nor-cholecalciferol, 1 ,25-dihydroxy-21 -(2R,3- dihy[omega]Oxy-3-raethy]-butyl)-20R-cholecalciferol; 1 ,25-dihydroxy-21 -(2R53- dihydroxy-3-metbyl-butyl)-20S-cholecalciferol; 1 , 25-Dihydroxy-21 -(2Rs3-dibydroxy-3- metbyl-butyl)-20S- 19-nor-cholecalciferol; 1 ,25-Dihydroxy-20S-21 -(3-hydroxy-3-methyl- butyl)-24-keto-l 9-nor-cholecalciferol; 1 ,25-Dihydroxy-20S-21 -(3-hydroxy-3-methyl-butyl)- 24-keto-cholecalciferol; 1 ,25-DiliydiOxy-21 (3-hydroxy-3-trifluoromethyl-4-trifluoro- butynyl)-26,27-hexadeutero-19-nor- 20S-cholecalciferol; 1 ,25-Dihydroxy-21 (3 -hydroxy- 3-trifluorome%l-4-thfluoro-butynyl)-26,27-hexadeutero-20S- cholecalciferol; 1 ,3-di-O- acetyl- 1 ,25~dihydroxy-23-yne-cholecalciferol; 1 ,3-di-O-acetyl- 1 ,25-di hydroxy- 16-ene- 23-yne-cholecalciferol; 1 ,3-di-O-acetyl- 1 ,25-dihydroxy- 16,23E-diene-cholecalciferol; l,3~di~O-acetyl-l,25-dihydroxy-16-ene-cholecalciferol; l,3525-Th-O-acctyl-l,25-dihydroxy- 16-ene-23-yne-26,27-hexafluoro-cholecalciferol; 1 ,3-di-O-acetyl- 1 ,25-diliydroxy- 16- ene-23->'ne-26,27-hexafluoro-cholecalciferol; 1 ,3-Di-O-acetyl- 1 ,25-dihydroxy- 16,23E- diene-25R-26-trifluoro-cholecalciferol; 1 ,3-Di-O-acetyl- 1 ,25-Dihydroxy-l6-ene-23-yne- 26,27-hexafluoro-l 9-[upsilon]or-cholecalciferol; 1 ,3,25-Th-O-acetyl- 1 ,25-Dihydroxy- 16-ene-23 -yne-26,27-hexafl[iota][iota]oro-l 9-nor-cholecalciferol; 1 ,3-di-O-acetyl- 1 ,25- dihydroxy- 16-ene- 19-nor-cholecalciferol; I 33-Di-O-acetyl-3,25-dihydroxy-l6-erje-23- yne-19-nor-cho3eca]ciferol: l^-Di-O-acetyl-l^δ-dihydroxy^O-cyclopropyl^S-yne-i 9-nor- cholecalcifcrol; 1 ,3-Di-O-acetyl- 1 ,25-dihydroxy- 16-ene-23-yne-26,27-bishomo- 19-nor- cholecalciferol; 1 ,3,25-tri-O-acetyl- l,25-dihydroxy-20-cyclopropyl-23-yne-26,27- hexafluoro-l 9-nor- cholecalciferol; l53-di-O-acetyl-l525-dihydroxy-20-cyclopropyl-23-yne- 26,27-hexafluoro-19-nor- cholecalciferol; l^-di-O-acetyl-I^S-dihydroxy^O-cyclopropyl- 23-yne-choleca]ciferol; l,3-di-O-acetyl-l,25-dihydiOxy-20-cyclopropyl-23E-ene-26,27- hexafiuoro-19-nor- cholecalciferol; 1 ,3-di-O-acetyl- 1 ,25-dihydroxy-20-cyclopropyl-23Z- ene-26,27-hexafluoro- 19-nor- cholecalciferol; 1 ,3-di-O-acetyl- 1 s25-dihydroxy-20- cyclopropyl-cholecalciferol; 1 ,3-di-O-acetyl- 1 ,25-dihydroxy- 1 [beta]-eneΛO-cyclopropyl- 1 θ-nor-cholecalciferol; and l,3-Dd-O-acetyl-l525-dihydrox.y-16-ene-20-cyclopropyl- cholecalciferol; 1 ,25-dihydroxy-21 (3-hydroxy-3- trifluoromethyl-4-trifluoro-butynyl)-26,27- hexadeutero-19-nor-20S-choleca]ciferol.
Other non-limiting examples of vitamin D compounds that may be of use in accordance with the invention include those described in published international applications: WO 01/40177, WO0010548, WO0061776, WO0064869, WO0064870, WO0066548, WO0104089, WO0116099, WO0130751 , WO0140177, WO0151464, WO0156982, WO0162723, WO0174765, WO0174766, WO0179166, WO0190061 , WO0192221 , WO0196293, WO02066424, WO0212J 82, WO0214268, WO03004036, WO03027065, WO03055854, WO03088977, WO04037781 , WO04067504, WO8000339, WO8500819, WO8505622, WO8602078, WO8604333, WO8700834, WO8910351 , WO9009991 , WO9009992, WO9010620, WO9100271 , WO9100855, WO9109841 , WO9112239, WO9112240, WO9115475, WO9203414, WO9309093, WO9319Q44, WO9401398, WO9407851 , WO9407852, WO9408958, WO9410139, WO9414766, WO9502577, WO9503273, WO9512575, WO9527697, WO9616035, WO9616036, WO9622973, WO9711053, WO972081 1 , WO9737972, WO9746522, WO98J 8759, WO9824762, WO9828266, WO9841500, WO9841501 , WO9849138, WO9851663, WO9851664, WO985J 678, WO9903829, WO9912894, WO9915499, WO9918070, WO9943645, WO9952863, WO2006/051106, those described in U.S. Patent Nos.: 6,503,893,6,482,812, 6,441 ,207, 6,410,523,6,399,797, 6,392,071 ,6,376,480, 6,372,926,6,372,731 , 6,359,152, 6,329,357,6,326,503, 6,310,226,6,288,249, 6,281 ,249,6,277,837, 6,218,430, 6,207,656,6,197,982, 6,127,559,6,103,709, 6,080,878,6,075,015, 6,072,062, 6,043,385,6,017,908, 6,017,907,6,013,814, 5,994,332,5,976,784, 5,972,917, 5,945,410,5,939,406, 5,936,105,5,932,565, 5,929,056,5,919,986, 5,905,074, 5,883,271 ,5,880,113, 5,877,168,5,872,140, 5,847,173,5,843,927, 5,840,938, 5,830,885,5,824,811 , 5,811 ,562,5,786,347, 5,767,111 ,5,756,733, 5,716,945, 5,710,142,5,700,791 , 5,665,716,5,663,157, 5,637,742,5,612,325, 5,589,471 , 5,585,368,5,583,125, 5,565,589,5,565,442, 5,554,599,5,545,633, 5,532,228, 5,508,392,5,508,274, 5,478,955,5,457,217, 5,447,924,5,446,034, 5,414,098, 5,403,940,5,384,313, 5,374,629,5,373,004, 5,371 ,249,5,430,196, 5,260,290, 5,393,749,5,395,830, 5,250,523,5,247,104, 5,397,775,5,194,431 , 5,281 ,731 , 5,254,538,5,232,836, 5,185,150,5,321 ,018, 5,086,191 ,5,036,061 , 5,030,772, 5,246,925,4,973,584, 5,354,744,4,927,815, 4,804,502,4,857,518, 4,851 ,401 , 4,851 ,400, 4,847,012,4,755,329, 4,940,700,4,619,920, 4,594,192,4,588,716, 4,564,474,4,552,698, 4,588,528,4,719,204, 4,719,205, 4,689,180, 4,505,906, 4,769,181 ,4,502,991 , 4,481 ,198,4,448,726, 4,448,721 ,4,428,946, 4,411 ,833, 4,367,177,4,336,193, 4,360,472,4,360,471 , 4,307,231 , 4,307,025,4,358,406, 4,305,880,4,279,826, and 4,248,791 , and those described in published US Patent Applications: US2001007907, US2003083319, US2003125309, US2003130241 , US2003171605, US2004167105.
Another object of the invention relates to a method for treating and/or preventing biliary diseases comprising administering a subject in need thereof with a vitamin D compound as above described.
The vitamin D compound may be administered in the form of a pharmaceutical composition, as defined below. Preferably, said compound is administered in a therapeutically effective amount.
By a "therapeutically effective amount" is meant a sufficient amount of the vitamin D compound to treat and/or to prevent biliary diseases at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, gender and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.
A therapeutically effective amount of vitamin D compound (i.e., an effective dosage) may range from about 0.001 to 30 ug/kg body weight, preferably about 0.01 to 25 ug/kg body weight, more preferably about 0.1 to 20 ug/kg body weight, and even more preferably about 1 to 10 ug/kg, 2 to 9 ug/kg, 3 to 8 ug/kg, 4 to 7 ug/kg, or 5 to 6 ug/kg body weight. The skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. In addition, the dose administered will also depend on the particular vitamin D compound used, the effective amount of each compound can be determined by titration methods known in the art. Moreover, treatment of a subject with a therapeutically effective amount of a vitamin D compound can include a single treatment or, preferably, can include a series of treatments. In one example, a subject is treated with a vitamin D compound in the range of between about 0.1 to 20 ug/kg body weight, once per day for a duration of six months or longer, for example for life depending on management of the symptoms and the evolution of the condition. Also, as with other chronic treatments an "on-off ' or intermittent treatment regimen can be considered. It will also be appreciated that the effective dosage of a vitamin D compound used for treatment may increase or decrease over the course of a particular treatment.
Pharmaceutical compositions:
The vitamin D compound may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.
"Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.
Preferably, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syhngability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The Vitamin D compound of the invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, thmethylamine, histidine, procaine and the like.
The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.
For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCI solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
The Vitamin D compound of the invention may be formulated within a therapeutic mixture to comprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0 or even about 10 milligrams per dose or so. Multiple doses can also be administered.
In addition to the compounds of the invention formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; liposomal formulations ; time release capsules ; and any other form currently used.
In a particular embodiment, pharmaceutical composition of the invention may further comprise a compound known to be suitable for treating or preventing biliary diseases. Said compound may be ursodeoxycholic acid which has the formula:
Figure imgf000014_0001
In another embodiment, pharmaceutical composition of the invention may comprise other bile acid derivatives (e.g. nor-ursodeoxycholic acid) or other nuclear receptor agonists (e.g. farnesoid X receptor agonists).
Another aspect of the invention relates to method for treating and/or preventing biliary diseases comprising administering a subject in need thereof with a vitamin D compound of the invention in combination with ursodeoxycholic acid or other bile acid derivatives (e.g. nor-ursodeoxycholic acid) or other nuclear receptor agonists (e.g. farnesoid X receptor agonists).
The invention will further be illustrated in view of the following figures and examples.
FIGURES:
Figure 1. Cathelicidin expression in human biliary epithelial cells.
Representative immunostaining of cathelicidin in the biliary epithelium (A) within normal human liver and (B) in the liver of patients with suppurative cholangitis. Immunostaining is localized in biliary epithelial cells (arrows and higher magnification within insets in the lower left corners) and in infiltrating inflammatory cells (arrowheads). Inset in the upper right corner of figure 1 B illustrates positive staining in the biliary tract lumen. (C) Biliary epithelial cells (Mz-ChA-1 ) were treated with VD3 for the indicated time and subjected to cathelicidin mRNA detection by real time RT-PCR. Data represent means ± SEM of 4 experiments performed in duplicate. Values at all time points were significantly different from basal value (P < 0.05).
Figure 2. Bile salts control VDR expression and activity in biliary epithelial cells. (A) Proteins from biliary epithelial cells incubated with CDCA or UDCA for the indicated time were submitted to VDR and β-actin immunoblot-ECL analyses. Representative gels of five different experiments are shown. (B, C) Proteins from biliary epithelial cells incubated for 2 h with CDCA or UDCA with or without PD 98059 were submitted to (B) p-ERK 1/2 and ERK 1/2 immunoblot-ECL analyses; (C) VDR and β- actin immunoblot-ECL analyses. Representative gels of three different experiments are shown. (D) Nuclear proteins from biliary epithelial cells incubated for 2 h with CDCA, UDCA or VD3 were submitted to VDR, Lamin A/C and β-actin immunoblot-ECL analyses. Representative gels of three different experiments are shown. (E) Nuclear proteins from biliary epithelial cells incubated with CDCA, UDCA or VD3 were subjected to electrophoretic mobility shift assay using a VDRE consensus sequence. Representative gel of three different experiments are shown. (F) Biliary epithelial cells transfected with a VDRE-driven promoter were assayed for luciferase activity after incubation with CDCA or UDCA. Data represent means ± SEM of 4 experiments. * P < 0.05 vs. untreated cells. ** P < 0.05 vs. VD3 or bile salts alone. (G) Biliary epithelial cells transfected with a VDRE-driven promoter were assayed for luciferase activity after incubation with CDCA or UDCA in the presence or absence of PD 98059. Data are expressed as fold over control and represent means ± SEM of 3 experiments. (H) Biliary epithelial cells transfected with a VDRE-driven promoter were assayed for luciferase activity after incubation with the FXR agonist, GW4064 (1 μmol/L). Data represent means ± SEM of 3 experiments. * P < 0.05 vs. control.
Figure 3. Bile salts induce cathelicidin expression in biliary epithelial cells. (A) Total RNA from biliary epithelial cells treated with CDCA, UDCA, VD3 or the combination of bile salts with VD3 was subjected to real time RT-PCR with primers designated to amplify cathelicidin. Data represent means ± SEM of 4 experiments performed in duplicate. * P < 0.05 vs. untreated cells. ** P < 0.05 vs. VD3 or bile salts alone. (B, C) Biliary epithelial cells transfected either with scramble or VDR siRNA were incubated with CDCA, UDCA or the combination of bile salts with VD3 and subjected to cathelicidin mRNA detection by real time RT-PCR. Data represent means ± SEM of 3 experiments performed in duplicate. * P < 0.05 vs. scramble. (D) Biliary epithelial cells were incubated with CDCA or GW4064 in the presence of either a control plasmid or a FXR dominant negative plasmid (DN hFXR) and subjected to real time RT-PCR with primers designated to amplify cathelicidin. Data represent means ± SEM of 3 experiments performed in duplicate. * P < 0.05 vs. respective control condition. Figure 4. UDCA treatment causes increased expressions of VDR and cathelicidin in the human liver. Total RNA from the liver of PBC patients before or after the onset of UDCA treatment was subjected to real time RT-PCR with primers designated to amplify either VDR or cathelicidin. Results are expressed as mRNA levels relative to the mean value of PBC patients before UDCA treatment. (A) Correlation between VDR expression and cathelicidin expressions in all human liver samples; R = 0.84401 ; P < 0.05. (B) Comparison between PBC patients before and after the onset of UDCA treatment. * P < 0.05.
Figure 5: 1α, 25(OH)2D3 induces cathelicidin expression through VDR in biliary epithelial cells. Human biliary epithelial cells were transfected either with scramble (Sc) or VDR siRNA (Si) and subjected to (A) Detection of VDR mRNA by real time RT-PCR (Data represent means +/- SEM of 3 experiments performed in duplicate; * P<0.005 vs scramble); (B) Detection of VDR and β-actin by immunoblot-ECL analyses (Representative gels of three different experiments are shown); (C) Incubation with VD3 for 24h before cathelicidin expression was analysed by real time RT-PCR (Data represent means +/- SEM of 3 experiments performed in duplicate. * P>0.05 vs. control. ** P<0.005 vs. scramble).
EXAMPLE:
Material & Methods:
Reagents: DMEM and fetal bovine serum were purchased from Invitrogen (Cergy
Pontoise, France). 1α,25(OH)2D3 was provided by Sigma (Saint-Quentin Fallavier, France). GW4064 was purchased from Tocris (Bristol, UK). Sodium chenodeoxycholate (CDCA) and sodium ursodeoxycholate (UDCA) (99 % pure), as well as PD98059 were obtained from Calbiochem (Meudon, France). Ribonuclease inhibitor RNazine was purchased from Promega (Charbonnieres, France), Moloney murine leukemia virus reverse transcriptase from Invitrogen (Cergy Pontoise, France), and Taq DNA polymerase from Perkin-Elmer (Les UNs, France).
lmmunohistochemical staining : Liver tissue sections (4 μm) were sequentially incubated with an anti-cathelicidin antibody (Hycult Biotechnology, Le Perray-en-
Yvelines, France) at a dilution of 1 : 150. After microwave antigen retrieval (750 W for 15 minutes followed by 150 W for 15 minutes in 1OmM citrate buffer; pH 6), immunolabelling was performed using a Supersensitive Link-Label lmmunohistochemistry detection system (Biogenex, San Ramon, CA) according to the manufacturer's protocol. Peroxidase activity was detected using 3-amino-9-ethyl carbazole as the substrate and Mayer's hematoxylin for counterstaining.
Cell culture: The human biliary epithelial cell line Mz-ChA-1 (Knuth A. et al. 1985) was cultured in DMEM, supplemented with 1 g/L glucose, 10 mmol/L Hepes and 10% fetal bovine serum, under 95% air and 5% CO2 at 37°C. The culture medium was renewed every 48 h.
Preparation of cell extracts: Whole cell extracts, nuclear extracts (NEs), and cytosolic extracts (CEs) were prepared as follows. For whole cell extracts, Mz-ChA-1 cells were lysed in a buffer composed of 50 mmol/L Tris (pH 7.4), 150 mmol/L NaCI, 0.5% Sodium deoxycholate, 0.1 % SDS, and 1 % NP-40. For preparation of cytoplasmic and nuclear extracts, we performed cell fractionation with the NE-PER kit (Pierce, Berbieres, France) as recommended by the manufacturer with minor modifications. Briefly, cells were incubated 10 min with accutase in order to detach cells from culture flasks. Cells were then collected by centrifugation at 1 ,500 * g for 5 min. The cell pellet was resuspended in adequate volume of CER buffers. Cells were then centrifuged at maximal speed for 5 min. The cytoplasmic extract (supernatant) was collected and stored at -80 °C. The pellet fraction was resuspended in NER buffer and centrifuged at maximal speed for 10 min. The supernatant containing the nuclear extract was collected and stored at -80 °C. Protein concentration was determined using the BCA Protein Assay (Pierce, Berbieres, France). lmmunoblot analyses: Mz-ChA-1 cells were incubated with either CDCA (100 μmol/L) or UDCA (100 μmol/L) for 1 to 24h. For the MAPK inhibition experiments, Mz- ChA-1 cells were preincubated with PD 98059 (50 μmol/L) for 1 h before either CDCA (100 μmol/L), UDCA (100 μmol/L) or 1α,25(OH)2D3 (0.1 μmol/L) were added and maintained for the following two hours. Whole cell protein extracts (20 μg) were subjected to electrophoresis through a 10% SDS polyacrylamide gel, and then transferred to nitrocellulose membranes, lmmunoblotting was performed using antibodies raised against human VDR, ERK, p-ERK (Santa Cruz Biotechnology, Le Perray en Yvelines, France) and β-actin (Sigma, Saint-Quentin Fallavier, France). Immunoreactivity was revealed by enhanced chemiluminescence (Amersham, Les UNs, France).
Electrophoretic mobility shift assay (EMSA): EMSA experiments were performed as recommended by the manufacturer (Panomics, Montigny-le-Bretonneux, France). Briefly, nuclear extracts (2 μg), Poly d(l-C) (0.1 μg/μl) and VDRE biotin-labeled probe (1 ng/μl) were incubated in binding buffer at room temperature for 30 min. Samples were then runned at 4°C through a 6% polyacrylamide gel at 120V. Gel was then transferred for 30-45 min at 300 mA to Biodyne B® nylon membrane (Pall, Fontenay-sous-Bois, France). After transfer, the membranes were baked for 1 hour at 80°C in a dry oven and transferred to a UV crosslinker oven for 3 min. Membranes were then blocked by incubation at room temperature for 15 min, before streptavidin-HRP conjugate was added. The biotin-streptavidin complex was revealed by enhanced chemiluminescence using an ECL kit (Amersham, Les UNs, France).
Transient transfection and reporter gene assay: Mz-ChA-1 cells were seeded into six-well plates (2 105 cells/well) and grown overnight in DMEM with 1 g/L glucose, 10 mmol/L Hepes and 10% fetal bovine serum, under 95% air and 5% CO2 at 37°C. Cells were transfected using Lipofectamine 2000 (Invitrogen, Cergy-Pontoise, France) with either a luciferase reporter gene plasmid driven by four copies in tandem of a consensus DR3 response element for VDR cloned upstream of the herpes virus simplex thymidine kinase gene promoter (Kahlen JP. et al. 1996), the dominant negative form of human FXR (CMX-hFXR-W469A) (Makishima M. et al. 1999), a mixture of four synthetic VDR siRNA (Dharmacon Research Inc., Lafayette, CO) or with a silencer negative control RNA (Ambion, Cambridgeshire, England). Six hours after transfection, cells were transferred overnight to serum free medium before either CDCA (100 μmol/L), UDCA (100 μmol/L) or 1α,25(OH)2D3 (0.1 μmol/L) were added. Luciferase activity was measured 24h after the onset of stimulation and normalized to protein content in order to calculate relative activities.
Real time RT-PCR: Total RNA was extracted from Mz-ChA-1 cells treated or not with CDCA (100 μmol/L), UDCA (100 μmol/L), 1α,25(OH)2D3 (1 μmol/L) or from liver tissue using RNA plus lysis solution (Quantum, Montreuil-sous-Bois, France). One microgram of total RNA was denatured by heating at 72°C for 10 min, and then incubated in 25 μl of a reaction buffer containing 10 mmol/L DTT, 0.5 mmol/L dNTP, 20 U of RNazine, 5 μmol/L random hexamers, and 200 U of Moloney murine leukemia virus reverse transcriptase. Reverse transcription was allowed to proceed for 1 h at 37°C. Quantitative real time PCR was performed using the Sybr Green PCR Core Reagents Kit (Perkin Elmer Applied Biosystems, Courtaboeuf, France) on a LightCycler 1.5 (Roche Diagnostics, Mannheim, Germany). The primers used in real time PCR were the following: 5' GCT AAC CTC TAC CGC CTC CT 3' (sense) (SEQ ID NO :1 ) and 5' GGT CAC TGT CCC CAT ACA CC 3' (antisense) (SEQ ID NO :2) for cathelicidin (Gombart AF. et al. 2005); 5' GCC ACT GGC TTT CAC TTC AA 3' (sense) (SEQ ID NO :3) and 5' TCC TTG GTG ATG CGG CAC T 3' (antisense) (SEQ ID NO :4) for VDR (Genebank NM_001017535); 5' GGC ATC GTT TAT GGT CGGA A 3'(sense) (SEQ ID NO :5) and 5' GGC ATC GTT TAT GGT CGG AA 3' (antisense) (SEQ ID NO :6) for 18S (Chignard N. et al. 2005). Quantitative real time PCR reactions were run with 200 nmol/L of cathelicidin sense and antisense primers and 50 nmol/L of each 18S sense and antisense primers. Data were collected and analyzed with Roche LightCycler Software 3.5.3 (Roche Diagnostics, Mannheim, Germany). Data were expressed as a relative amount (2"ΔΔCT) of the control value. Statistical analysis : Comparisons were made using the Student's t test, the paired Wilcoxon or unpaired Mann-Whitney rank sum test. P < 0.05 was considered as significant.
Results:
First, we investigated whether liver epithelial cells, which are constantly exposed to bile salts, express cathelicidin. In normal human liver, we detected cathelicidin expression by immunostaining in biliary epithelial cells (Figure 1A) and in hepatocytes. Furthermore, we observed that in liver specimens from subjects with suppurative cholangitis, cathelicidin was abundant both in biliary epithelial cells and within the lumen of the biliary tract (Figure 1 B), indicating that this antibacterial peptide is secreted in bile in response to infection. Because cathelicidin expression is regulated by VDR in other cell types, we tested the potential of VDR activation to control cathelicidin expression in biliary epithelial cells. As shown in figure 1C and in Figure 5, VDR activation by 1α,25(OH)2D3 (VD3) increased cathelicidin expression in biliary epithelial cells.
Neither the endogenous bile salt, chenodeoxycholic acid (CDCA), nor the therapeutic bile salt, UDCA, are ligands of VDR (Makishima M et al. 2002). However, bile salts can stimulate the expression or the activity of nuclear receptors (e.g. FXR) by ligand-independent mechanisms (Lew JL. Et al. 2004). Thus, we assayed the ability of CDCA and of UDCA to regulate VDR expression in biliary epithelial cells. As shown in figure 2A, both bile salts induced a sustained increase in VDR protein levels. Because on the one hand, bile salts are able to activate ERK V2 (Yoon JH. Et al. 2004), and on the other hand, ERK 1/2 activation results in increased VDR expression (Gilad LA. et al. 2005), we examined the effects of bile salts on VDR protein levels after inhibition of the ERK 1/2 pathway. In this condition, the increase in VDR protein expression elicited by CDCA or UDCA was blunted (Figure 2B,C), indicating that bile salts induce VDR protein expression through the MAPK pathway.
Because VDR is transcriptionally active when translocated to the nucleus, we analyzed VDR protein expression in nuclear fractions of biliary epithelial cells. UDCA, like VD3, induced VDR nuclear translocation, while CDCA had little or no effect (Figure 2D). Consistent with this finding, in electrophoretic mobility shift assay (EMSA) experiments, UDCA clearly induced the binding of VDR to VDRE sequences, while CDCA had only a minor effect on DNA-protein complex formation (Figure 2E). We next tested the effects of bile salts in biliary epithelial cells transfected with a luciferase reporter gene cloned under the control of four copies of VDRE (Kahlen JP. Et al. 1996). In this system, both UDCA and CDCA, either alone or combined with VD3 increased promoter activity (Figure 2F). However, in keeping with VDR dependent and independent actions of UDCA and CDCA, respectively, ERK 1/2 inhibition abrogated UDCA-induced promoter activity, but did not alter the effect of CDCA (Figure 2G). CDCA being the principal FXR activator present in bile (Wang H. et al. 1999), and in line with the possibility that FXR can bind to, and activate transcription through, a variety of nonconsensus response elements (Lafitte BA. Et al. 2000), we hypothesized that the effect of CDCA on VDRE-driven promoter was mediated by FXR. Supporting this possibility, the pharmacological activation of FXR caused a significant increase in VDRE-driven promoter activity (Figure 2H).
We next determined whether bile salts were able to control cathelicidin transcript levels in biliary epithelial cells. CDCA and UDCA alone elicited a significant rise in cathelicidin expression in biliary epithelial cells (Figure 3A). When combined with VD3, CDCA and UDCA caused increases cathelicidin expression, which were significantly higher compared to those induced by either bile salt or VD3 alone (Figure 3A). To determine the contribution of VDR in CDCA or UDCA-induced cathelicidin expression, we performed VDR knock-down experiments in biliary epithelial cells (Figure 5). In agreement with the fact that CDCA does not cause VDR activation, the inhibition of VDR expression was without significant consequence on the effect of this bile salt, alone or combined with VD3, on cathelicidin expression (Figure 3B). By contrast, the increase in cathelicidin expression triggered by UDCA, either alone or in combination with VD3, was blunted in cells with diminished VDR expression (Figure 3C). The role of FXR in the effect of CDCA was then assayed by using biliary epithelial cells that expressed a FXR dominant negative mutant. In these cells, the induction of cathelicidin expression induced either by CDCA or by FXR pharmacological activation was significantly diminished compared to control cells (Figure 3D). Altogether, these results indicate that CDCA controls cathelicidin expression through FXR activation, while UDCA has the unique ability to increase cathelicidin expression through VDR activation in human biliary epithelial cells.
To test the in vivo implications of these findings, we compared VDR and cathelicidin transcript levels in liver biopsies from PBC patients who were either naive of treatment or under UDCA therapy. A positive correlation was found between the transcript levels of VDR and cathelicidin in human liver (Figure 4A). Of particular interest with respect to UDCA action, when compared to naive PBC patients, the patients under UDCA treatment displayed a significant increase in VDR and cathelicidin expressions (Figure 4, B and C). These results provide evidence that the novel effect of UDCA identified in vitro, occurs in treated patients and could contribute to the beneficial therapeutic action of UDCA.
In summary, we herein show that bile salts control innate immunity in human biliary epithelial cells through nuclear receptors. Furthermore, our data suggest that the therapeutic benefit provided by UDCA (Poupon RE. et al. 1991 ; Corpechot C. et al. 2005) may result, at least in part, from the ability of this bile salt to increase cathelicidin expression in vivo. Consistent with a VDR-mediated effect, VDR and cathelicidin expressions are both increased by UDCA in patients with PBC. Abnormal accumulation of endotoxins in biliary epithelial cells is a feature of PBC (Sasatomi K. et al. 1998). Thus, cathelicidin, which is known to neutralize the deleterious effects of bacterial products (Larrick JW. Et al 1994), could account for the clearance of endotoxins previously reported in PBC patients under UDCA treatment (Sasatomi K. et al. 1998; Ballot E; et 2004). Importantly, the increase in cathelicidin expression in biliary epithelial cells was higher when UDCA was combined with vitamin D. Taken together, our results infer that in PBC and in other inflammatory biliary diseases, which involve bacterial factors, a significant benefit would arise from a strategy systematically combining UDCA with vitamin D therapy.
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Claims

CLAIMS:
1. A vitamin D compound for treating or preventing biliary diseases.
2. The vitamin D compound according to claim which is ergocalciferol, or cholecalciferol.
3. A pharmaceutical composition for treating or preventing biliary diseases which comprises a vitamin D compound.
4. The pharmaceutical composition according to claim 3 which further comprises ursodeoxycholic acid.
5. The vitamin D compound according to claim 1 or 2 or the pharmaceutical composition according to claim 3 or 4 for the treating or preventing of primary biliary cirrhosis (PBC), primary (and secondary) sclerosing cholangitis, cystic fibrosis liver disease, MDR3 defect-related liver diseases, intrahepatic cholelihiasis, biliary atresia and cholangiocarcinoma.
6. The vitamin D compound according to claim 1 or 2 or the pharmaceutical composition according to claim 3 or 4 for treating or preventing primary sclerosing cholangitis (PSC).
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