WO2011077245A2 - Compositions - Google Patents

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WO2011077245A2
WO2011077245A2 PCT/IB2010/003447 IB2010003447W WO2011077245A2 WO 2011077245 A2 WO2011077245 A2 WO 2011077245A2 IB 2010003447 W IB2010003447 W IB 2010003447W WO 2011077245 A2 WO2011077245 A2 WO 2011077245A2
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group
formula
lxr
substituent
cells
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PCT/IB2010/003447
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French (fr)
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WO2011077245A3 (en
Inventor
Vincenzo Russo
Catia Traversari
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Fondazione Centro San Raffaele Del Monte Tabor
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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
    • A61K31/33—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
    • A61K31/4433—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with oxygen as a ring hetero atom
    • 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
    • A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
    • 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
    • A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
    • A61K31/381—Heterocyclic compounds having sulfur as a ring hetero atom having five-membered 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
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • 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
    • A61K31/4164—1,3-Diazoles
    • A61K31/4178—1,3-Diazoles not condensed 1,3-diazoles and containing further heterocyclic rings, e.g. pilocarpine, nitrofurantoin
    • 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
    • A61K31/4196—1,2,4-Triazoles
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene
    • 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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to novel treatment of cancer patients with therapeutics interfering with oxysterol synthesis and metabolism to restore a successful antitumour response.
  • the present invention also relates to novel treatment of cancer patients with therapeutics to prevent migration of pro-angiogenic cells. More particularly, the invention relates to a novel combination of an oxysterol modulator and a CD25-depleting monoclonal antibody for the treatment of cancer.
  • the invention also relates to LXR ligand inactivators for the treatment of cancer.
  • the invention further relates to LXR inhibitors/antagonists for the treatment of cancer.
  • the present invention also relates to the use of an LXR ligand for promoting migration and/ or for isolating mouse CD11b+GR1 + cells or human CD1 + cells from a population.
  • Tumors evade the immune system through mechanisms conditioning their microenvironment. Recently, some immunoescape processes have been molecularly elucidated 1 . Among them, the activation of different metabolic pathways 2,3 leads to immunosuppressive effects.
  • LXR The nuclear Liver X Receptors (LXR) a and ⁇ are involved in lipid and cholesterol homeostasis 4,5,6 .
  • LXRp is expressed ubiquitously, while LXRa is expressed in liver, adipose tissue, adrenal glands, intestine, lungs and cells of myelomonocytic lineage 7 . Both receptors are triggered by oxidized cholesterol (oxysterols) 5
  • Oxsterols oxidized cholesterol
  • DC Dendritic cells initiate adaptive immune responses 9 , including antitumor activity following their CCR7-dependent migration to lymphoid organs. Indeed, DC ablation abrogates the induction of antigen-specific effector cells 10 .
  • the ability of DC to initiate an immune response depends on their migration to lymphoid organs where they present antigens to naive T and B cells 1 . This process requires the expression of the lymphoid-homing receptor CCR7 by maturing DC 12,13 .
  • the present invention provides novel therapeutics for use in the treatment of cancer and novel methods of treatment.
  • LXR ligands released by tumor cells could also be active on other cell types such as macrophages or lymphocytes.
  • mouse CD11 b+GR1 + bone-marrow derived cells migrate to LXR-releasing tumors in a CXCR2 dependent manner, and that mouse CD11b+GR1+ bone-marrow derived cells promote neoangiogenesis and tumor growth.
  • this new pathway mediated by LXR ligands occurring within the tumor microenvironment, is responsible for the migration of CD11b+GR1+ myeloid cells within the tumor.
  • CD11b+GR1+ myeloid cells within the tumor.
  • the present invention opens new avenues for the treatment of cancer patients with drugs interfering with sterol metabolism to restore a successful antitumour immune response.
  • the use of oxysterol inhibitors has been confirmed to prevent migration of CDl 1 b+GR1+ pro- angiogenic cells.
  • the present invention relates to the novel use of a combination of an oxysterol modulator and a CD25-depleting monoclonal antibody for the treatment of cancer.
  • the present invention further relates to the novel use of LXR ligand inactivators for l o the treatment of cancer.
  • the present invention further relates to the novel use of LXR inhibitors/antagonists for the treatment of cancer.
  • statins the most used cholesterol-lowering drugs
  • statins have been described to have an immune suppressive activity blocking T cell proliferation and dampening DC activation.
  • statins also inhibit the formation of molecules (i.e. farnesyl pyrophosphate and geranylgeranyl pyrophospate) involved in functional posttranslational modification (i.e. prenylation) of small GTPase proteins including Rho, Rac and CdC42, while ZA by acting downstream mevalonic acid synthesis, blocks only cholesterol/oxysterol5 formation.
  • squalene synthase inhibitors e.g. lanosterol 14-alpha demethylase inhibitor, farnesyl diphosphate synthase (FDPS) inhibitor
  • FDPS farnesyl diphosphate synthase
  • the present invention is directed towards novel compositions and methods of treating cancer.
  • the present invention is directed towards therapeutics interfering with oxysterol synthesis and metabolism to restore a successful antitumour response, for treating cancer.
  • a pharmaceutical composition comprising a combination of (i) an oxysterol modulator and (ii) a CD25- depleting monoclonal antibody for treating cancer.
  • the oxysterol modulator blocks cholesterol synthesis downstream of mevalonic acid.
  • the oxysterol modulator is selected from a squalene synthase inhibitor and a lanosterol 14-alpha demethylase inhibitor.
  • the lanosterol 14-alpha demethylase inhibitor is an azole.
  • azoles include, but are not limited to, fluconazole (Diflucan) and itraconazole (Sporanox).
  • the lanosterol 14-alpha demethylase inhibitor is SKF 104976.
  • the squalene synthase inhibitor is zaragozic acid or a derivative thereof.
  • the derivative is a conjugate, a salt, an ester, an amide or a carbamate of zaragozic acid, optionally substituted.
  • zaragozic acid derivatives include, but are not limited to,
  • X is H, a halogen (F, CI, Br, I), OH or CH 3 ; Y is halogen (F, CI, Br, I), OH or CH 3 , and wherein Z 1 t Z 2 and Z 3 are each independently H, C1-5 alkyl, C1-5 alkyl substituted with (i) phenyl, (ii) phenyl substituted with methyl, methoxy, halogen (F, CI, Br, I) or hydroxy, (iii) C1-5 alkylcarbonyloxy, (iv) C6-10 arylcarbonyloxy, (v) C1-5
  • R is selected from:
  • R 2 is selected from: H, and — ;
  • R 3 is C 1-5 alkyl;
  • Z is selected from (i) H, (ii) Ci. 5 alkyl; (iii) C h alky! substituted with (a) C ⁇ alkylcarbonyloxy, (b) arylcarbonyloxy, (c) Ci. 5 alkoxycarbonyloxy, (d) aryloxycarbonyloxy; (e) C1-5 alkyl , (f) , (g) or the groups (a) to (d) form a a 5 to 10 membered mono or bicyclic ring with C 1-5 alkyl, (iv) cycloalkyl; or a pharmaceutically acceptable salt thereof.
  • the squalene synthase inhibitor is zaragozic acid.
  • the squalene synthase inhibitor is selected from lapaquistat (TAK-475), terbinafine, ER-27856 (5- ⁇ A/-[2-butenyl-3-(2-methoxyphenyl)]-N- methylamino ⁇ -1 ,1-penthylidenebis(phosphonic acid) tri-sodium salt), RPR-107393 (3- hydroxy-3-[4-(quinolin-6-yl)phenyl]-1 -azabicyclo [2-2-2]octane dihydrochloride) and YM-53601 ((E)-2-[2-fluoro-2-quinuclidin-3-ylidene ethoxy]-9H-carbazole monohydrochloride).
  • the squalene synthase inhibitor is a phosphonic acid derivative.
  • An example of a phosphonic acid derivative is is represented by the following general
  • Rl OR3 wherein R, represents a hydrogen atom, a hydroxyl group, an acyloxyalkyi group, an alkyloxycarbonyl group, a lower alkyl group which may have a substituent or a lower alkoxy group which may have a substituent; R 2 and R 3 may be the same or different from each other and each represents a hydrogen atom, a lower alkyl group which may have a substituent, an alkali metal or a prodrug ester forming group; RA represents a group represented by the formula: 0 (wherein R4 represents a hydrogen atom, a lower alkyl group, an alkali metal or an acyloxyalkyi group which may have a substituent), a group represented by the
  • R6 alkali metal or a group represented by the formula: R6 wherein R 5 represents a hydrogen atom, a lower alkyl group, an alkali metal or a prodrug ester forming group; and R6 represents a lower alkyl group or a group represented by the formula: -OR7 (wherein R7 represents a hydrogen atom, a lower alkyl group, an alkali metal or a prodrug ester forming group)]; and
  • RB represents a group represented by the formula: S--T-- [wherein S represents an alkenyl roup which may have a substituent or a group represented by the formula:
  • R12 may be the same or different from one another and each represents (1 ) a hydrogen atom, (2) an alkyl group which may have a substituent, (3) an alkenyl group which may have a substituent, (4) a lower alkoxy group which may have a substituent, (5) a carbamoyl group which may have a substituent, (6) a carbamoyloxy group which may have a substituent, (7) a hydroxyl group, (8) an acyl group, (9) a halogen atom, (10) a group represented by the following formula: (wherein R13 and R14 may be the same or different from each other and each represents a lower alkyl group which may have a substituent, or alternatively R13 and R1 may form together with the nitrogen atom to which they are bonded, a ring which may further contain an oxygen atom, a sulfur atom or a nitrogen atom and which may have one or two, mono- or divalent substituent(s); p is 0 or 1 ; and
  • R15, R16, R17, R18 and R19 may be the same or different from one another and each represents a hydrogen atom, a hydroxyl group, a lower alkyl group or a lower alkoxy group which may have a substituent;
  • ring B represents an aromatic ring;
  • Y represents an alkylene chain which may have a substituent, an alkenylidene chain which may have a substituent, an alkynylidene chain which may have a substituent, a group represented by the formula: 0 , a group represented by the formula: -0-, or a single bond), or alternatively two adjacent groups of R8, R9, R10, R1 1 and R12 may together form a ring;
  • X represents a single bond, an alkylene chain which may have a substituent, an alkenylidene chain which may have a substituent or a group represented by the formula: -(CH2)u -Z--(CH2)v - (wherein Z is a
  • R20 represents a hydrogen atom, a lower alkyl group which may have a substituent or a lower alkenyl group which may have a substituent
  • R21 represents a hydrogen atom, a lower alkyl group which may have a substituent, a lower alkenyl group which
  • N represents a hydrogen atom, a lower alkyl group which may have a substituent or a lower alkenyl group which may have a substituent
  • u is an integer of 0 to 3
  • v is an integer of 0 to 6
  • T represents (1) a single bond
  • R23 represents a hydrogen atom, a cycioalkyi group, a cycloalkylalkyl group, a lower alkyl group which may have a substituent or a lower alkenyl group which may have a substituent
  • W represents a group represented by the formula: -0-, a group represented by the formula: 0 , a group represented by
  • R23, W, s and t are each as defined above; and R29 represents a hydrogen atom, a cycioalkyi group, a cycloalkylalkyl group, a lower alkyl group which may have a substituent or a lower alkenyl group which may have a substituent),
  • N represents a group represented by the formula: N (wherein R25 represents a hydrogen atom, a cycioalkyi group, a lower alkyl group which may have a substituent or a lower alkenyl group which may have a substituent), or
  • x and y are independent of each other and are each an integer of 0 to 3.
  • X is H or P0 3 H 2 and X 2 is H or P0 3 H 2 ; and wherein R1 and R2 are H- or - CH 3 ; and R3, R4, R5 and R6 are independently selected from H- or an alkyl group selected from the group consisting of methyl, ethyl, propyl, butyl or tert-butyl; or, a pharmaceutically acceptable salt thereof.
  • the CD25-depleting monoclonal antibody is selected from Basiliximab (an anti-mouse CD25 antibody), daclizumab (Zenapax), inolimomab (Leucotac), HuMax-TAC and PC61.
  • an LXR ligand inactivator for treating cancer.
  • the LXR ligand inactivator may be an LXRa ligand inactivator or an LXR ligand inactivator.
  • the LXR ligand inactivator is a sulfotransferase enzyme. In one embodiment the sulfotransferase enzyme is SULT2B1b.
  • the LXR ligand inactivator is administered using gene therapy.
  • an LXR inhibitor or antagonist for treating cancer is provided.
  • the LXR inhibitor or antagonist is an LXRa inhibitor or antagonist or an LXRp inhibitor or antagonist.
  • the LXR antagonist is a cholesterol oxide, an oxysterol or a sterol or derivative thereof.
  • the sterol is selected from a hydroxycholesterol and a sulfated oxysterol.
  • the cholesterol oxide is a functionalised cholesterol oxide selected from 7 -hydroxycholesterol, a-epoxycholesterol, ⁇ -epoxycholesterol, 7-keto- cholesterol, cholestane triol, 7a-hydroxycholesterol, 25-hydroxycholesterol, 22(R)- hydroxy-cholesterol, 24(S)-hydroxy-cholesterol, 27-hydroxy-cholesterol.
  • the sulfated oxysterol is selected from 24-OHChol-3-sulfate and 24-OHChol-3, 24-sulfate.
  • the LXR inhibitor or antagonist is selected from a polyunsaturated fatty acid, a geranyl geraniol or geranylgeranyl pyrophosphate, 5 ⁇ x,6a-epoxycholesterol sulphate (ECHS), 7-ketocholesterol-3-sulphate, and a
  • R1 represents a hydrogen atom, a lower alkyl group, a lower halogenated alkyl group, an unsubstituted or substituted phenyl group, or an unsubstituted or substituted benzyl group
  • R2 represents a I .I .I .S.S.S-hexafluoro ⁇ -hydroxypropan- -yl group or a carboxymethyl group
  • R3 represents a hydrogen atom, a lower alkyl group, a lower alkoxy group or a halogen atom
  • Y represents CO or S02.
  • LXR inhibitors or antagonists include, but are not limited to, Liver X Receptor antagonist BMS (Bristol-Myers Squibb Company (BMY)) and Liver X Receptor antagonist EXELIXIS (Exelixis Inc (EXEL)).
  • the pharmaceutical composition further comprises a pharmaceutically acceptable excipient, diluent or carrier.
  • composition comprising a LXR ligand inactivator and further comprising a pharmaceutically acceptable excipient, diluent or carrier.
  • a pharmaceutical composition comprising the LXR inhibitor or antagonist and further comprising a pharmaceutically acceptable excipient, diluent or carrier.
  • the pharmaceutical composition, ligand inactivator or LXR inhibitor or antagonist is combined with a chemotherapeutic agent for treating cancer.
  • chemotherapeutic agent examples include cytotoxic antibiotics such as aclarubicin, bleomycin, dactinomycin, daunorubicin, doxorubicin, epirubicin, idarubicin, mitomycin, and mitoxantrone (mitozantrone); alkylating agents such as busulfan, carmustine, chlorambucil, chlormethine hydrochloride, mustine hydrochloride, cyclophosphamide, estramustine phosphate, ifosfamide, lomustine, melphalan, thiotepa, and treosulfan; antimetabolites such as capecitabine, cladribine, cytarabine, fludarabine, fluorouracil, gemcitabine, mercaptopurine, methotrexate, raltitrexed, tegafur, and thioguanine; vinca alkaloids, such as etoposide, vinblastine
  • combination therapies comprising an oxysterol modulator.
  • a pharmaceutical composition comprising an oxysterol modulator in combination with a further cancer treatment.
  • the oxysterol modulator is administered in combination with a chemotherapeutic agent.
  • the oxysterol modulator is administered in combination with immunotherapy (active immunotherapy and vaccination strategy).
  • an LXR ligand for isolating CD11b+GR1 + cells from a population In a further aspect there is provided the use of an LXR ligand for isolating mouse CD1 1 b+GR1 + and/or human CD14+ cells from a population. According to a further aspect there is provided the use of an LXR ligand for promoting migration of mouse CD11 b+GR1+ and/or human CD14+ cells.
  • the use involves a migration assay.
  • the LXR ligand is 22R-HC.
  • FIG. 1 Tumors producing factors inhibiting CCR7 on DC.
  • Fig. 2 Tumor-CM trigger LXRa activation in DC.
  • RLA Relative Luciferase Activity.
  • Fig. 3 Blocking of LXRa signaling abrogates CCR7 inhibition
  • (c) showing CCR7 expression/inhibition (d) Luciferase assay for LXRa activation by the tumor-CM described in Fig. 3c * , P ⁇ 0.05; * * * , P ⁇ 0.01 (Student's f-test). Mean and s.d. of three experiments (results with MR255-CM in Fig.
  • Fig. 4 Dampening of DC migration and T cell priming by tumor-CM and LXR ligands and generation of antitumor responses by avoiding LXRa signaling,
  • (c,d) Tumor growth (c) and survival (d) of RMA-bearing mice treated with ZA or vehicle. Arrow indicates the onset of ZA treatment.
  • Fig. 5 Dissection of the role of DC, CCR7 and LXRa, and characterization of infiltrating cells,
  • Fig. 7 Zaragozic Acid strongly delays the growth of 7 days established Lewis Lung Carcinoma (LLC) and potentiates the antitumor activity of immunotherapy, (a) **, P ⁇ 0.003; ***, P ⁇ 0.0008 (/ test). Mean and s.d. of one out of three experiments (4-5 mice/group), (b) Mean tumor weight at sacrifice. The weight of tumors from untreated mice is higher than the weight of tumors collected from ZA-treated mice. ***, P ⁇ 0.0009 (/ test). Mean and s.d. of one experiment (4-5 mice/group), (c) Zaragozic Acid improves the antitumor activity of active immunotherapy.
  • LLC Lewis Lung Carcinoma
  • Fig. 8 Injection of therapeutic doses of Zaragozic Acid is not associated with general and liver-specific side effects, (a) The weight of LLC-bearing mice left untreated or treated with ZA, VAX, or ZA+VAX did not differ significantly before and after treatments, (b) Blood levels of GOT and GPT enzymes (liver function) were not altered by the treatments (LLC, LLC +ZA, LLC +VAX, LLC +ZA +VAX). Fig.
  • Bone marrow-derived CD11b * GR1 * cells migrate in vitro towards the LXR ligand 22R-HC. This migration is independent of LXRs and is inhibited by Pertussis toxin (PTX).
  • PTX Pertussis toxin
  • CD11b + GR1 + from LXRs KO mice migrate to 22R-HC, demonstrating that CD11b + GR1 + migration to LXR ligands is independent of LXR engagement.
  • One representative experiment out of two is shown, (d) The treatment of CD11b * GR1 * with PTX (100 or 500 ng) inhibits their migration to the LXR ligand 22R-HC, indicating that the receptor mediating LXR ligand migration is a G Protein Coupled Receptor (GPCR). As expected, PTX control does not affect migration.
  • GPCR G Protein Coupled Receptor
  • Fig. 11 Selective migration of the CD11 b high GR1 hi9h cells to the LXR ligand 22R-HC.
  • the cells isolated by functional sorting were characterized by qRT-PCR chemokine receptors expression, by flow cytometry for the expression of lineage-specific cell surface markers and functionally.
  • the CD11b hi9f, GR1 high cells that migrate to LXR ligands have higher levels of mRNAs and proteins for CCR1, CXCR4 and CXCR2 chemokine receptors than non migrating cells.
  • One representative experiment out of two is shown.
  • Fig. 12 The migration of bone marrow-derived CD11 b + GR1 + cells towards the LXR ligand 22R-HC is mediated by the CXCR2 chemokine receptor, (a) The pre-treatment of CD11b + GR1 + cells with the LXR ligand 22R-HC blocks their migration to 22R-HC and to the CXCR2 ligand CXCL5, but not to SDF1a and MIP-1a that are ligands of CXCR4 and CCR1 receptors, respectively. One representative experiment out of two is shown, (b) The pre-treatment of CD11 b + GR1 + cells with the CXCR2 ligand CXCL5 blocks their migration to both 22R-HC and CXCL5.
  • Fig. 14 In vivo analysis of the pro-angiogenic ability of CD1 1 b hi9h GR1 high cells migrating to LXR ligands.
  • RMA tumor cells co-injected with LXR ligands migrating CD1 1 b high GR1 hi9h cells display an higher percentage of endothelial CD31 + CD45 " cells than RMA tumor cells co-injected with non migrating cells. **, P ⁇ 0.001 ; ***, P ⁇ 0.0001 (Anova). Mean and s.d. of two experiments with 4-5 mice/group is shown.
  • Fig. 15 Migration of human CD14 + monocytes to LXR ligands.
  • CD14* monocytes isolated from peripheral blood mononuclear cells of a healthy donor or from a melanoma patient, specifically migrate to the LXR ligand 22R-HC but not to 22S-HC.
  • Figure 16 Role of LXRa/LXRa ligands in antitumor immune responses. Schematic representation of the effects of tumor-released LXRa ligands on the generation of antitumor immune responses. Tumor cells release LXRa ligands that affect CCR7 expression on maturing DC. As a consequence, DC do not migrate to draining lymph node and fail to elicit antitumor CD8+ T cells.
  • FIG. 17 Phenotypic and mRNA analysis of human DC treated with MSR3-CM.
  • One representative experiment out of three is shown
  • CCR7 inhibition is independent of the activation stimulus used.
  • DC co-cultured with MSR3 cells black bars
  • DC co-cultured with NIH-3T3 grey bars
  • DC co-cultured with NIH-3T3 grey bars
  • DC co-cultured with NIH-3T3 grey bars
  • DC co-cultured with 3T3- CD40L in the absence or presence of MSR3-CM release similar amounts of IL-12.
  • DC co-cultured with MSR3- CD40L do not up regulate CXCR4.
  • One representative experiment out of ten is shown.
  • FIG. 18 Phenotypic and functional analysis of DC activated in the presence of CM from the tumors LOVO, RMA, CALU-1 , G43 and Det.
  • One representative experiment out of three is shown,
  • One representative experiment out of two is shown.
  • FIG 19 Effect of natural and synthetic LXR ligands on DC.
  • One representative experiment out of three is shown,
  • FIG. 20 (a) HEK293 cells expressing the SULT2B1b partly abolish LXRa luciferase activity induced by natural ligands but not by the synthetic ligand T1317. HEK293 cells expressing SULT2B1b were selected and used to test LXRa activation by luciferase assay.
  • HepG2 were transduced with lentiviral vectors encoding shLXR selected with puromycin (1 pg/ml) and analyzed for LXRa expression by qPCR.
  • As control we used HepG2 cells transduced with lentiviral vectors encoding a non-targeting shRNA.
  • Figure 21 (a) In vivo OT-I activation following injection of OVApep-loaded DC treated with the natural LXR ligand 22R-HC. B6 mice were adoptively transferred with 3x10 6 purified OT-I cells labeled with CFSE.
  • OT-I cells (1.5x10 5 ) were co-cultured with 0.5x10 5 DC previously treated for 48 hours with 22R-HC, 22S-HC, or left untreated and then pulsed with the SIINFEKL peptide (H-2Kb-OVA-specific epitope recognized by OT-I cells). Forty-eight hours later, supernatants were collected and tested for IFN- ⁇ (c) and TNFa release (d). Four days later, OT-I were collected and counted by FACS (b). DC treated with 22S-HC partly inhibited OT-I proliferation and markedly reduced IFNy secretion. *, P ⁇ 0.05; **, P ⁇ 0.01 (ANOVA).
  • Lxra mRNA is expressed by freshly isolated CD1 1c+ DC and it is up regulated after the treatment of mice with complete Freund's adjuvant. Freshly isolated CD1 1c + and CD11 c " cells express Lxra mRNA. Lxra mRNA turns out to be up regulated 8 hours after CFA treatment of B6 mice. Purified CD3 + cells do not express Lxra transcripts. One representative experiment is shown.
  • FIG. 22 Growth of ZA-treated and SULT2B1 b-expressing tumors in C57BL/6 and NOD-SCID mice, (a) ZA treatment (black circles) does not affect the growth of RMA in NOD-SCID as compared to vehicle treatment (black squares). Seventy-five micrograms of ZA was administered i.p. every 2 days. Arrow indicates the onset of ZA treatment. Results of one experiment with ten mice/group are shown, (b) RMA expressing the SULT2B1 b enzyme (SULT2B1 b, black triangles) grows similarly to RMA untransduced (wild type, black squares) or transduced with the control vector (mock, black circles).
  • Results of one experiment with ten mice/group are shown, (e, f) SULT2B1 b does not modify the growth of the B16 melanoma in B6 (e) as well as in NOD-SCID (f) mice (SULT2B1 b, black triangles; mock, black circles).
  • the insert (e) shows a qPCR analysis for SULT2B1 b mRNA expressed by the tumors. Data are representative of one out of two experiments (mean and s.d. of ten mice/group).
  • Figure 23 (a, b) Inhibition of tumor growth is strictly dependent on the amount of SULT2B1 b expressed by tumors, (a) RMA expressing higher amounts of SULT2B1 b mRNA had a stronger delay of tumor growth. RMA-mock (black circles), RMA- SULT2B1 b #3.1 (black squares), RMA-SULT2B1 b #3.3 (black triangles) and RMA- SULT2B1 b #3.4 (black diamonds). Data are representative of one experiment (mean and s.d. of ten mice/group), (b) qPCR analysis for SULT2B1 b mRNA of the tumors described in Fig.
  • the present invention involves a combination of oxysterol synthesis inhibitors and CD25-depleting monoclonal antibodies, in one aspect.
  • the oxysterol modulator used in the present invention blocks cholesterol synthesis downstream of mevalonic acid.
  • Mevalonate is a precursor of, in particular, squalene, and can be made from acetate.
  • An outline for the pathway for the synthesis of cholesterol from acetate is as follows: acetate C 2 ⁇ mevalonate C 6 ⁇ isopentenyl pyrophosphate C 5 ⁇ squalene C 30 ⁇ cholesterol C 2 j
  • Squalene is a C 30 hydrocarbon and is an intermediate in the synthesis of cholesterol. Squalene consists of six isoprene units. Its formula is as follows:
  • the present invention relates to the use of zaragozic acid.
  • Zaragozic acid is a squalene synthase inhibitor.
  • derivatives of zaragozic acid are also useful in the present invention.
  • the derivative of zaragozic acid is a monocarboxylic derivative.
  • the derivative is a conjugate, a salt, an ester or an amide of zaragozic acid.
  • zaragozic acid derivatives are: wherein X is H, a halogen (F, CI, Br, 1), OH or CH 3 ; Y is halogen (F, CI, Br, I), OH or CH 3 , and wherein ⁇ , Z 2 and Z 3 are each independently H, C1-5 alkyi, C1-5 alkyi substituted with (i) phenyl, (ii) phenyl substituted with methyl, methoxy, halogen (F, CI, Br, I) or hydroxy, (iii) C1 -5 alkylcarbonyloxy, (iv) C6-10 arylcarbonyloxy, (v) C1-5 alkoxycarbonyloxy, (vi) C6-10 aryloxycarbonyloxy, (vii)
  • zaragozic acid derivatives include
  • R is selected from:
  • R 3 is d-salkyl
  • Z is selected from (i) H, (ii) C 1-5 alkyl; (iii) Ci. 5 alkyl substituted with (a) C ⁇ alkylcarbonyloxy, (b) arylcarbonyloxy, (c) d.salkoxycarbonyloxy, (d) aryloxycarbonyloxy; (e) aikyi , (f) t (g) 0 r the groups (a) to (d) form a 5 to 10 membered mono or bicyclic ring with C h alky!, (iv) -e cycloalkyl; or a pharmaceutically acceptable salt thereof.
  • Another example of a squalene synthase inhibitor is a probucol ester of the formula
  • X is H or P0 3 H 2 and X 2 is H or P0 3 H 2 ; and wherein R1 and R2 are H- or - CH3; and R3, R4, R5 and R6 are independently selected from H- or an alkyl group selected from the group consisting of methyl, ethyl, propyl, butyl or tert-butyl; or, a pharmaceutically acceptable salt thereof.
  • Phosphonic acid derivatives are also useful in the present invention.
  • An example of a phosphonic acid derivative is represented by the following general formula (I):
  • Ri represents a hydrogen atom, a hydroxyl group, an acyloxyalkyl group, an alkyloxycarbonyl group, a lower alkyl group which may have a substituent or a lower alkoxy group which may have a substituent;
  • R 2 and R 3 may be the same or different from each other and each represents a hydrogen atom, a lower alkyl group which may have a substituent, an alkali metal or a prodrug ester forming group;
  • RA represents a group represented by the formula: 0 (wherein R4 represents a hydrogen atom, a lower alkyl group, an alkali metal or an acyloxyalkyl
  • R represents a hydrogen atom, a lower alkyl group or an alkali metal
  • R6 wherein R 5 represents a hydrogen atom, a lower alkyl group, an alkali metal or a prodrug ester forming group; and R6 represents a lower alkyl group or a group represented by the formula: -OR7 (wherein R7 represents a hydrogen atom, a lower alkyl group, an alkali metal or a prodrug ester forming group)]; and
  • RB represents a group represented by the formula: S-T-- [wherein S represents an alkenyl group which may have a substituent or a group represented by the formula: (wherein ring A represents an aromatic ring; R8, R9, R10, R11 and R12 may be the same or different from one another and each represents (1) a hydrogen atom, (2) an alkyl group which may have a substituent, (3) an alkenyl group which may have a substituent, (4) a lower alkoxy group which may have a substituent, (5) a carbamoyl group which may have a substituent, (6) a carbamoyloxy group which may have a substituent, (7) a hydroxyl group, (8) an acyl group, (9) a halogen atom, (10) a group represented by the following formula: (wherein R13 and R14 may be the same or different from each other and each represents a lower alkyl group which may have a substituent, or alternatively R13 and R14 may form together
  • R20 S° 2N (wherein R20 represents a hydrogen atom, a lower alkyl group which may have a substituent or a lower alkenyl group which may have a substituent), a
  • J N JL (wherein R22 represents a hydrogen atom, a lower alkyl group which may have a substituent or a lower alkenyl group which may have a substituent); u is an integer of 0 to 3; and v is an integer of 0 to 6); and T represents (1) a single bond, (2) a group represented by the formula: (wherein R23 represents a hydrogen atom, a cycloalkyl group, a cycloalkylalkyi group, a lower alkyl group which may have a substituent or a lower alkenyl group which may have a substituent; W represents a group represented by the formula: -0-, a group represented by the formula: ° , a group represented by
  • N represents a hydrogen atom, a cycloalkyi group, a lower alkyl group which may have a substituent or a lower alkenyl group which may have a substituent
  • squalene synthase inhibitors that are useful in the present invention include lapaquistat (TAK-475), terbinafine, ER-27856 (5- ⁇ /V-[2-butenyl-3-(2- methoxyphenyl)]-A/-methylamino ⁇ -1 , 1 -penthylidenebis(phosphonic acid) tri-sodium salt), RPR-107393 (3-hydroxy-3-[4-(quinolin-6-yl)phenyl]-1 -azabicyclo [2-2-2]octane dihydrochloride) and YM-53601 ((£)-2-[2-fluoro-2-quinuclidin-3-ylidene ethoxy]-9H- carbazole monohydrochloride).
  • CD25-Depleting Monoclonal Antibodies In one aspect the invention involves CD25-depleting monoclonal antibodies.
  • Examples of CD25-depleting monoclonal antibodies include Basiliximab, daclizumab (Zenapax), inolimomab (Leucotac), HuMax-TAC and PC61.
  • the present invention involves LXR ligand inactivators for treating cancer.
  • the LXR ligand inactivator is a sulfotransferase enzyme.
  • the sulfotransferase enzyme is SULT2B1 b.
  • the LXR ligand inactivator is administered using gene therapy.
  • the present invention involves an LXR inhibitor or antagonist for treating cancer.
  • An LXR antagonist includes any agent, which may include any compound, substance or molecule, capable of antagonising any function of an LXR receptor. An antagonist may thus antagonise (down-regulate, inhibit or suppress) any effect of LXR activation.
  • An LXR antagonist may be an antagonist of LXRa or LXRp or both. In one embodiment the LXR antagonist is an LXRa inhibitor or antagonist.
  • the LXR antagonist may be a cholesterol oxide, an oxysterol or a sterol or derivative thereof.
  • the sterol is selected from a hydroxycholesterol and a sulfated oxysterol.
  • the cholesterol oxide may be functionalised.
  • functionalised cholesterol oxides are 7p-hydroxycholesterol, a-epoxycholesterol, ⁇ -epoxycholesterol, 7-keto- cholesterol, cholestane triol, 7a-hydroxycholesterol, 25-hydroxycholesterol, 22(R)- hydroxy-cholesterol, 24(S)-hydroxy-cholesterol, 27-hydroxy-cholesterol.
  • sulfated oxysterols examples include 24-OHChol-3-sulfate and 24-OHChol-3, 24- sulfate.
  • LXR inhibitors or antagonists that are useful in the present invention include a polyunsaturated fatty acid, a geranyl geraniol or geranylgeranyl pyrophosphate, 5a,6a-epoxycholesterol sulphate (ECHS), 7-ketocholesterol-3-
  • R1 represents a hydrogen atom, a lower alkyl group, a lower halogenated alkyl group, an unsubstituted or substituted phenyl group, or an unsubstituted or substituted benzyl group
  • R2 represents a 1 ,1 ,1 ,3,3,3-hexafluoro-2-hydroxypropan-2- yl group or a carboxymethyl group
  • R3 represents a hydrogen atom, a lower alkyl group, a lower alkoxy group or a halogen atom
  • Y represents CO or S02.
  • a further compound modulating LXR function that may be useful in the present invention is of the following formula (I) or a pharmacologically acceptable salt or ester thereof:
  • A represents a C5-C14 aryl group or a 5- to 7-membered heteroaryl group
  • R R2 and R ⁇ are the same or different and each represents a hydrogen atom, a hydroxyl group, a nitro group, a cyano group, an amino group, a halogen atom, a carboxy group, a carbamoyl group, a mercapto group, a C
  • alkyl groups are the same or different
  • a C2-C7 alkylcarbonylamino group an N-(C2-Cy alkylcarbonyl)-N-(C
  • a C2-C7 alkoxycarbonylamino group an N-(C2-Cy alkoxycarbonyl)-N-(C-
  • a C1-C5 alkylsulfonylamino group an N-(C
  • -Cg haloalkylsulfonylamino group (wherein said C
  • R 4 and R ⁇ are the same or different and each represents a hydrogen atom, a hydroxyl group, an amino group, a halogen atom, a mercapto group, a C
  • X represents a hydrogen atom, a hydroxyl group, a halogen atom, a C
  • Y represents a C
  • substituent group a represents a group consisting of a halogen atom, a hydroxyl group, a cyano group, an amino group, a C2-C7 alkylcarbonyloxy group, a C1-C5 alkyl group, a C
  • a further aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of the invention admixed with one or more pharmaceutically acceptable diluents, excipients or carriers.
  • Other active materials may also be present, as may be considered appropriate or advisable for the disease or condition being treated or prevented.
  • the compounds of the present invention can be administered alone, they will generally be administered in admixture with a pharmaceutical carrier, excipient or diluent, particularly for human therapy.
  • a pharmaceutical carrier excipient or diluent
  • the pharmaceutical compositions may be for human or animal usage in human and veterinary medicine.
  • Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).
  • suitable carriers include lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and the like.
  • suitable diluents include ethanol, glycerol and water.
  • compositions may comprise as, or in addition to, the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).
  • Suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose and polyethylene glycol.
  • Suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like.
  • Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition.
  • preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid.
  • Antioxidants and suspending agents may be also used.
  • a process for the preparation of a pharmaceutical or veterinary composition as described above comprising bringing the active compound(s) into association with the carrier, for example by admixture.
  • the formulations are prepared by uniformly and intimately bringing into association the active agent with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.
  • the invention extends to methods for preparing a pharmaceutical composition comprising bringing a compound of general formula (I) in conjunction or association with a pharmaceutically or veterinarily acceptable carrier or vehicle.
  • the compounds of the invention can be present as salts or esters, in particular pharmaceutically and veterinarily acceptable salts or esters.
  • salts of the compounds of the invention include suitable acid addition or base salts thereof.
  • suitable pharmaceutical salts may be found in Berge et al, J Pharm Sci, 66, 1-19 (1977). Salts are formed, for example with strong inorganic acids such as mineral acids, e.g.
  • hydrohalic acids such as hydrochloride, hydrobromide and hydroiodide, sulphuric acid, phosphoric acid sulphate, bisulphate, hemisulphate, thiocyanate, persulphate and sulphonic acids; with strong organic carboxylic acids, such as alkanecarboxylic acids of 1 to 4 carbon atoms which are unsubstituted or substituted (e.g., by halogen), such as acetic acid; with saturated or unsaturated dicarboxylic acids, for example oxalic, malonic, succinic, maleic, fumaric, phthalic or tetraphthalic; with hydroxycarboxylic acids, for example ascorbic, glycolic, lactic, malic, tartaric or citric acid; with aminoacids, for example aspartic or glutamic acid; with benzoic acid; or with organic sulfonic acids, such as (C C 4 )-alkyl- or aryl-sulfonic acids
  • Preferred salts include, for example, acetate, trifluoroacetate, lactate, gluconate, citrate, tartrate, maleate, malate, pantothenate, adipate, alginate, aspartate, benzoate, butyrate, digluconate, cyclopentanate, glucoheptanate, glycerophosphate, oxalate, heptanoate, hexanoate, fumarate, nicotinate, palmoate, pectinate, 3- phenylpropionate, picrate, pivalate, proprionate, tartrate, lactobionate, pivolate, camphorate, undecanoate and succinate, organic sulphonic acids such as methanesulphonate, ethanesulphonate, 2-hydroxyethane sulphonate, camphorsulphonate, 2-naphthalenesulphonate, benzenesulphonate, p- chlorobenzenesulphonate
  • Esters are formed either using organic acids or alcohols/hydroxides, depending on the functional group being esterified.
  • Organic acids include carboxylic acids, such as alkanecarboxylic acids of 1 to 12 carbon atoms which are unsubstituted or substituted (e.g., by halogen), such as acetic acid; with saturated or unsaturated dicarboxylic acid, for example oxalic, malonic, succinic, maleic, fumaric, phthalic or tetraphthalic; with hydroxycarboxylic acids, for example ascorbic, glycolic, lactic, malic, tartaric or citric acid; with aminoacids, for example aspartic or glutamic acid; with benzoic acid; or with organic sulfonic acids, such as (CrC -alkyl- or aryl- sulfonic acids which are unsubstituted or substituted (for example, by a halogen) such as methane- or p-toluen
  • Suitable hydroxides include inorganic hydroxides, such as sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminium hydroxide.
  • Alcohols include alkanealcoho!s of 1-12 carbon atoms which may be unsubstituted or substituted, e.g. by a halogen).
  • the invention includes, where appropriate all enantiomers, diastereoisomers and tautomers of the compounds of the invention.
  • the person skilled in the art will recognise compounds that possess optical properties (one or more chiral carbon atoms) or tautomeric characteristics.
  • the corresponding enantiomers and/or tautomers may be isolated/prepared by methods known in the art.
  • Enantiomers are characterised by the absolute configuration of their chiral centres and described by the R- and S-sequencing rules of Cahn, Ingold and Prelog. Such conventions are well known in the art (e.g. see 'Advanced Organic Chemistry', 3 rd edition, ed. March, J., John Wiley and Sons, New York, 1985).
  • Some of the compounds of the invention may exist as stereoisomers and/or geometric isomers - e.g. they may possess one or more asymmetric and/or geometric centres and so may exist in two or more stereoisomeric and/or geometric forms.
  • the present invention contemplates the use of all the individual stereoisomers and geometric isomers of those inhibitor agents, and mixtures thereof.
  • the terms used in the claims encompass these forms, provided said forms retain the appropriate functional activity (though not necessarily to the same degree).
  • the present invention also includes all suitable isotopic variations of the agent or a pharmaceutically acceptable salt thereof.
  • An isotopic variation of an agent of the present invention or a pharmaceutically acceptable salt thereof is defined as one in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually found in nature.
  • isotopes that can be incorporated into the agent and pharmaceutically acceptable salts thereof include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulphur, fluorine and chlorine such as 2 H, 3 H, 13 C, 14 C, 15 N, 17 0, 18 0, 3 P, 32 P, 35 S, 18 F and 36 CI, respectively.
  • isotopic variations of the agent and pharmaceutically acceptable salts thereof are useful in drug and/or substrate tissue distribution studies. Tritiated, i.e., 3 H, and carbon-14, i.e., 4 C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with isotopes such as deuterium, i.e., 2 H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements and hence may be preferred in some circumstances.
  • the invention includes compounds of general formula (I) where any hydrogen atom has been replaced by a deuterium atom. Isotopic variations of the agent of the present invention and pharmaceutically acceptable salts thereof of this invention can generally be prepared by conventional procedures using appropriate isotopic variations of suitable reagents.
  • the invention further includes the compounds of the present invention in prodrug form, i.e. covalently bonded compounds which release the active parent drug according to general formula (I) in vivo.
  • prodrugs are generally compounds of the invention wherein one or more appropriate groups have been modified such that the modification may be reversed upon administration to a human or mammalian subject. Reversion is usually performed by an enzyme naturally present in such subject, though it is possible for a second agent to be administered together with such a prodrug in order to perform the reversion in vivo. Examples of such modifications include ester (for example, any of those described above), wherein the reversion may be carried out by an esterase etc. Other such systems will be well known to those skilled in the art.
  • the present invention also includes solvate forms of the compounds of the present invention.
  • the terms used in the claims encompass these forms.
  • the invention further relates to the compounds of the present invention in their various crystalline forms, polymorphic forms and (an)hydrous forms. It is well established within the pharmaceutical industry that chemical compounds may be isolated in any of such forms by slightly varying the method of purification and or isolation from the solvents used in the synthetic preparation of such compounds.
  • compositions of the present invention may be adapted for rectal, nasal, intrabronchial, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intraarterial and intradermal), intraperitoneal or intrathecal administration.
  • the formulation is an orally administered formulation.
  • the formulations may conveniently be presented in unit dosage form, i.e., in the form of discrete portions containing a unit dose, or a multiple or sub-unit of a unit dose.
  • the fonmulations may be in the fonn of tablets and sustained release capsules, and may be prepared by any method well known in the art of pharmacy.
  • Formulations for oral administration in the present invention may be presented as: discrete units such as capsules, gellules, drops, cachets, pills or tablets each containing a predetermined amount of the active agent; as a powder or granules; as a solution, emulsion or a suspension of the active agent in an aqueous liquid or a non-aqueous liquid, or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion; or as a bolus etc.
  • these compositions contain from 1 to 250 mg and more preferably from 10-100 mg, of active ingredient per dose.
  • the term "acceptable carrier” includes vehicles such as common excipients e.g. binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone (Povidone), methylcellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, sucrose and starch; fillers and carriers, for example corn starch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride and alginic acid; and lubricants such as magnesium stearate, sodium stearate and other metallic stearates, glycerol stearate stearic acid, silicone fluid, talc waxes, oils and colloidal silica.
  • Flavouring agents such as peppermint, oil of wintergreen, cherry flavouring and the like can also be used. It may be desirable to
  • a tablet may be made by compression or moulding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active agent in a free flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface- active or dispersing agent.
  • Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets may be optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active agent.
  • compositions suitable for oral administration include lozenges comprising the active agent in a flavoured base, usually sucrose and acacia or tragacanth; pastilles comprising the active agent in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active agent in a suitable liquid carrier.
  • compositions or emulsions which may be injected intravenously, intraarterially, intrathecally, subcutaneously, intradermally, intraperitoneally or intramuscularly, and which are prepared from sterile or sterilisable solutions.
  • injectable forms typically contain between 10 - 1000 mg, preferably between 10 - 250 mg, of active ingredient per dose.
  • compositions of the present invention may also be in form of suppositories, pessaries, suspensions, emulsions, lotions, ointments, creams, gels, sprays, solutions or dusting powders.
  • the active ingredient can be incorporated into a cream consisting of an aqueous emulsion of polyethylene glycols or liquid paraffin.
  • the active ingredient can also be incorporated, at a concentration of between 1 and 10% by weight, into an ointment consisting of a white wax or white soft paraffin base together with such stabilisers and preservatives as may be required.
  • a person of ordinary skill in the art can easily determine an appropriate dose of one of the instant compositions to administer to a subject without undue experimentation.
  • a physician will determine the actual dosage which will be most suitable for an individual patient and it will depend on a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy.
  • the dosages disclosed herein are exemplary of the average case. There can of course be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
  • the compounds of this invention which may have good bioavailability, may be tested in one of several biological assays to determine the concentration of a compound which is required to have a given pharmacological effect.
  • the one or more compounds or combinations of the invention are administered in combination with one or more other active agents, for example, existing drugs available on the market.
  • the compounds or combinations of the invention may be administered consecutively, simultaneously or sequentially with the one or more other active agents.
  • a pharmaceutical composition comprising an oxysterol modulator and a chemotherapeutic agent is administered.
  • Drugs in general are more effective when used in combination.
  • combination therapy is desirable in order to avoid an overlap of major toxicities, mechanism of action and resistance mechanism(s).
  • the major advantages of combining chemotherapeutic drugs are that it may promote additive or possible synergistic effects through biochemical interactions and also may decrease the emergence of resistance.
  • one aspect of the present invention further comprises administering another active pharmaceutical ingredient, such as a chemotherapeutic agent, either in combined dosage form with the compound of the present invention or in a separate dosage form.
  • chemotherapeutic agent dosage forms may include solid oral, oral solution, syrup, elixir, injectable, transdermal, transmucosal, or other dosage form.
  • the compound and the other active pharmaceutical ingredient can be combined in one dosage form or supplied in separate dosage forms that are usable together or sequentially.
  • chemotherapeutic agents which may be used in the present invention include, but are not limited to, cytotoxic antibiotics such as aclarubicin, bleomycin, dactinomycin, daunorubicin, doxorubicin, epirubicin, idarubicin, mitomycin, and mitoxantrone (mitozantrone); alkylating agents such as busulfan, carmustine, chlorambucil, chlormethine hydrochloride, mustine hydrochloride, cyclophosphamide, estramustine phosphate, ifosfamide, lomustine, melphalan, thiotepa, and treosulfan; antimetabolites such as capecitabine, cladribine, cytarabine, fludarabine, fluorouracil, gemcitabine, mercaptopurine, methotrexate, raltitrexed, tegafur, and thioguanine; vinca al
  • the one or more compounds or combinations of the invention are administered in combination with immunotherapy.
  • an oxysterol modulator is administered in combination with immunotherapy.
  • the administration may comprise an active immunotherapy and vaccination strategy.
  • Immunotherapy is the treatment of disease by inducing, enhancing, or suppressing an immune response.
  • Immunotherapy for cancer can be considered to be biological therapy, or the application of biologic-response modifiers.
  • biologic-response modifiers can act through one or more mechanisms, such as (i) stimulating the host's antitumor response by increasing the number of effector cells or by producing one or more soluble mediators (eg lymphokines); (ii) decreasing host- suppressor mechanisms; and (iii) altering tumor cells to increase their
  • BRMs may have both immunologic and nonimmunologic effects.
  • cell based immunotherapies using immune effector cells including lymphocytes, macrophages, dendritic cells, natural killer cells, and cytotoxic lymphocytes can be used.
  • Passive cellular immunotherapy is when activated, specific effector cells are directly infused into a patient and are not induced or expanded within the patient.
  • Examples of passive cellular immunotherapy include reinfusion of a patient's lymphocytes after expansion in vitro by exposure to IL-2 (T-cell growth factor). These cells are termed lymphokine-activated killer cells (LAK cells) and may first be exposed to
  • IL-2 phytohemagglutinin, a lymphocyte mitogen.
  • An alternative to infusion of IL-2 after LAK cell infusion is to isolate and expand populations of lymphocytes that have infiltrated tumors in vivo and thus may have tumor specificity, which may allow lower levels of IL-2 to be used.
  • TILs can also be genetically modified to express tumoricidal molecules.
  • Another example of passive cellular immunotherapy is the concurrent use of interferons with infused effector cells. Passive Humoral Immunotherapy relates to the use of antitumor antibodies.
  • Examples include the use of antilymphocyte serum, and conjugation of monoclonal antitumor antibodies with toxins or with radioisotopes so that the antibodies will deliver these toxic agents specifically to the tumor cells.
  • a further example is the use of bispecific antibodies, which link one antibody reacting with the tumor cell to a second antibody reacting with a cytotoxic effector cell, so that the cytotoxic effector cell is targeted more specifically to the tumor.
  • Active specific immunotherapy induces therapeutic cellular immunity in the tumor- bearing host. Intact tumor cells, defined tumor antigens, or general
  • immunostimulants are used.
  • An example is autochthonous tumor cells (taken from the host), which can be used, for example, after irradiation, neuraminidase treatment, hapten conjugation, or hybridization with long-term cell lines in vitro.
  • tumor cells genetically modified to produce immunostimulatory molecules including cytokines such as granulocyte-macrophage colony-stimulating factor or IL-2, costimulatory molecules such as B7-1 , and allogeneic class I HC molecules
  • Allogenic tumor cells can also be used.
  • a further example of active specific immunotherapy is the use of defined tumor antigen-based vaccines.
  • Antigen-specific immunity can also be induced with recombinant viruses (eg adenovirus, vaccinia virus) which express tumor associated antigens such as carcinoembryonic antigen.
  • Nonspecific Immunotherapy includes use of interferons (IFNs) derived from white blood cells (IFN-a or IFN- ⁇ ) or from fibroblasts (IFN- ⁇ ) or synthesized in bacteria by recombinant genetic techniques.
  • IFNs interferons
  • IFN-a or IFN- ⁇ white blood cells
  • IFN- ⁇ fibroblasts
  • bacterial adjuvants which can be used in immunotherapy include attenuated tubercle bacilli (BCG)) and extracts of BCG. These can be used with or without added tumor antigen.
  • Immunotherapy may be combined with other treatments such as chemotherapy or radiotherapy.
  • polypeptide substances such as SULT2B1b
  • they may be administered as the polypeptide itself or by introducing nucleic acid constructs/viral vectors encoding the polypeptide into cells under conditions that allow for expression of the polypeptide in a cell of interest.
  • a polynucleotide for use in the invention in a vector is operably linked to a control sequence that is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector.
  • the term "operably linked” means that the components described are in a relationship permitting them to function in their intended manner.
  • a regulatory sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences.
  • the control sequences may be modified, for example by the addition of further transcriptional regulatory elements to make the level of transcription directed by the control sequences more responsive to transcriptional modulators.
  • Vectors of the invention may be transformed or transfected into a cell as described below to provide for expression of a polypeptide.
  • the present invention also encompasses cells into which the polypeptides, are introduced.
  • Any suitable method of transforming the cell may be used.
  • Non-limiting examples of currently available mechanisms for delivery are via electroporation, calcium phosphate transformation or particle bombardment.
  • transfer of the construct may be performed by any of the methods mentioned which physically or chemically permeabilize the cell membrane. Suitable methods are described in more detail below.
  • the antigen is introduced into the cells via electroporation. Electroporation involves the exposure of a suspension of cells and DNA to a high-voltage electric discharge.
  • electroporation conditions for cells may be optimized. One may particularly with to optimize such parameters as the voltage, the capacitance, the time and the electroporation media composition. The execution of other routine adjustments will be known to those of skill in the art.
  • Particle Bombardment One method for transferring a naked DNA construct into cells involves particle bombardment. This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them.
  • the microprojectiles used have consisted of biologically inert substances such as tungsten, platinum or gold beads.
  • DNA precipitation onto metal particles would not be necessary for DNA delivery to a recipient cell using particle bombardment. It is contemplated that particles may contain DNA rather than be coated with DNA. Hence it is proposed that DNA-coated particles may increase the level of DNA delivery via particle bombardment but are not, in and of themselves, necessary.
  • a Biolistic Particle Delivery System which can be used to propel particles coated with DNA through a screen, such as stainless steel or Nytex screen, onto a filter surface covered with cells in suspension. The screen disperses the particles so that they are not delivered to the recipient cells in large aggregates. It is believed that a screen intervening between the projectile apparatus and the cells to be bombarded reduces the size of projectile aggregates and may contribute to a higher frequency of transformation by reducing the damage inflicted on the recipient cells by projectiles that are too large.
  • cells in suspension are preferably concentrated on filters, or alternatively on solid culture medium.
  • the cells to be bombarded are positioned at an appropriate distance below the macroprojectile stopping plate. If desired, one or more screens are also positioned between the acceleration device and the cells to be bombarded.
  • the execution of other routine adjustments will be known to those of skill in the art.
  • adenovirus expression vector is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to ultimately express a construct that has been cloned therein.
  • the vector comprises a genetically engineered form of adenovirus.
  • Knowledge of the genetic organization or adenovirus, a 36 kb, linear, double-stranded DNA virus, allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kb.
  • retrovirus the adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity.
  • adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification.
  • Adenovirus is particularly suitable for use as a gene transfer vector because of its mid-sized genome, ease of manipulation, high titer, wide target-cell range and high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging.
  • ITRs inverted repeats
  • the early (E) and late (L) regions of the genome contain different transcription units that are divided by the onset of viral DNA replication.
  • the E1 region (E1A and E1B) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes.
  • the expression of the E2 region results in the synthesis of the proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression and host cell shut-off.
  • AAV Adeno-associated virus
  • AAV is an attractive vector system for use in the present invention as it has a high frequency of integration and it can infect nondividing cells, thus making it useful for delivery of genes into mammalian cells in tissue culture.
  • AAV has a broad host range for infectivity.
  • the present invention involves the use of lentiviral vectors.
  • retroviruses A large number of different retroviruses have been identified. Examples include: murine leukemia virus (MLV), human T-cell leukemia virus (HTLV), mouse mammary tumour virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A- MLV), Avian myelocytomatosis virus-29 (MC29), and Avian erythroblastosis virus (AEV).
  • MLV murine leukemia virus
  • HTLV human T-cell leukemia virus
  • MMTV mouse mammary tumour virus
  • RSV Rous sarcoma virus
  • Fujinami sarcoma virus FuSV
  • Moloney murine leukemia virus Mo-MLV
  • Retroviruses are RNA viruses that replicate through an integrated DNA intermediate. Retroviral particles encapsidate two copies of the full-length viral RNA, each copy containing the complete genetic information needed for virus replication. Retroviruses possess a lipid envelope and use interactions between the virally encoded envelope protein that is embedded in the membrane and a cellular receptor to enter the host cells. Using the virally encoded enzyme reverse transcriptase, which is present in the virion, viral RNA is reverse transcribed into a DNA copy. This DNA copy is integrated into the host genome by integrase, another virally encoded enzyme. The integrated viral DNA is referred to as a provirus and becomes a permanent part of the host genome. The cellular transcriptional and translational machinery carries out expression of the viral genes.
  • the host RNA polymerase II transcribes the provirus to generate RNA, and other cellular processes modify and transport the RNA out of the nucleus.
  • a fraction of viral RNAs are spliced to allow expression of some genes whereas other viral RNAs remain full-length.
  • the host translational machinery synthesizes and modifies the viral proteins.
  • the newly synthesized viral proteins and the newly synthesized full-length viral RNAs are assembled together to form new viruses that bud out of the host cells.
  • Retroviruses may be broadly divided into two categories: namely, "simple” and “complex”. Retroviruses may even be further divided into seven groups. Five of these groups represent retroviruses with oncogenic potential. The remaining two groups are the lentiviruses and the spumaviruses. A review of these retroviruses is presented in Coffin et al (1997) ibid.
  • retrovirus and lentivirus genomes share many common features such as a 5' LTR and a 3' LTR, between or within which are located a packaging signal to enable the genome to be packaged, a primer binding site, integration sites to enable integration into a host cell genome and gag, pol and env genes encoding the packaging components - these are polypeptides required for the assembly of viral particles.
  • Lentiviruses have additional features, such as rev and RRE sequences in HIV, which enable the efficient export of RNA transcripts of the integrated provirus from the nucleus to the cytoplasm of an infected target cell.
  • LTRs long terminal repeats
  • the LTRs are responsible for proviral integration, and transcription. LTRs also serve as enhancer-promoter sequences and can control the expression of the viral genes.
  • the LTRs themselves are identical sequences that can be divided into three elements, which are called U3, R and U5. U3 is derived from the sequence unique to the 3' end of the RNA. R is derived from a sequence repeated at both ends of the RNA and U5 is derived from the sequence unique to the 5' end of the RNA. The sizes of the three elements can vary considerably among different retroviruses.
  • pol and env may be absent or not functional.
  • the R regions at both ends of the RNA are repeated sequences.
  • U5 and U3 represent unique sequences at the 5' and 3' ends of the RNA genome respectively.
  • At least part of one or more protein coding regions essential for replication may be removed from the virus. This makes the viral vector replication-defective.
  • Viral vectors for use in the invention may include but are not limited to integration defective retroviral vectors.
  • a vector can be produced, for example, by packaging the vector with catalytically inactive integrase (such as an HIV integrase bearing the D64V mutation in the catalytic site; Naldini et al., Science 1996, and PNAS USA 1996, Leavitt et al. J Virol. 1996) or by deleting essential att sequences from the vector LTR (Nigthingale et al. Mol Ther 2006), or by a combination of the above. These modifications reduce integration to baseline level leaving unaffected the other steps of the transduction process (Naldini et al. Science 1996, Nigthingale et al.
  • Lentivirus vectors are part of a larger group of retroviral vectors.
  • a detailed list of lentiviruses may be found in Coffin et al (1997) "Retroviruses” Cold Spring Harbour Laboratory Press Eds: J Coffin, SM Hughes, HE Varmus pp 758-763).
  • lentiviruses can be divided into primate and non-primate groups.
  • primate lentiviruses include but are not limited to: the human immunodeficiency virus (HIV), the causative agent of human auto-immunodeficiency syndrome (AIDS), and the simian immunodeficiency virus (SIV).
  • the non-primate lentiviral group includes the prototype "slow virus” visna/maedi virus (VMV), as well as the related caprine arthritis-encephalitis virus (CAEV), equine infectious anaemia virus (EIAV) and the more recently described feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV).
  • the lentivirus family differs from retroviruses in that lentiviruses have the capability to infect both dividing and non-dividing cells.
  • a lentiviral vector is a vector which comprises at least one component part derivable from a lentivirus. Preferably, that component part is involved in the biological mechanisms by which the vector infects cells, expresses genes or is replicated.
  • the lentiviral vector may be a "non-primate" vector, i.e., derived from a virus which does not primarily infect primates, especially humans.
  • non-primate lentivirus may be any member of the family of lentiviridae which does not naturally infect a primate and may include a feline immunodeficiency virus (FIV), a bovine immunodeficiency virus (BIV), a caprine arthritis encephalitis virus (CAEV), a Maedi visna virus (MW) or an equine infectious anaemia virus (EIAV).
  • FV feline immunodeficiency virus
  • BIV bovine immunodeficiency virus
  • CAEV caprine arthritis encephalitis virus
  • MW Maedi visna virus
  • EIAV equine infectious anaemia virus
  • viral vectors may be employed as constructs in the methods and compositions described here.
  • Vectors derived from viruses such as vaccinia and herpesviruses may be employed.
  • polypeptide is introduced to the cells using calcium phosphate co-precipitation.
  • the expression construct is delivered into the cell using DEAE-dextran followed by polyethylene glycol.
  • Further embodiments include the introduction of the polypeptide by direct microinjection or sonication loading.
  • the polypetide may be entrapped in a liposome.
  • Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers.
  • a nucleic acid construct complexed with Lipofectamine (Gibco BRL).
  • the liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA.
  • HVJ hemagglutinating virus
  • the liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1).
  • HMG-1 nuclear non-histone chromosomal proteins
  • the nucleic acid construct is introduced into the cell using adenovirus assisted transfection.
  • Increased transfection efficiencies have been reported in cell systems using adenovirus coupled systems, and the inventors contemplate using the same technique to increase transfection efficiencies.
  • the present invention is useful in treating cancer.
  • non-Hodgkin's lymphoma Hodgkin's lymphoma
  • leukemia e.g., acute leukemia such as acute lymphocytic leukemia, acute myelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma
  • colon carcinoma rectal carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, cervical cancer, testicular cancer, lung carcinoma, bladder carcinoma, melanoma, head and neck cancer, brain cancer, cancers of unknown primary site, neoplasms, cancers of the peripheral nervous system, cancers of the central nervous system, tumors (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordom
  • tumors e.g., fibros
  • HEK-293 cells (10 5 cells/well) were transiently transfected with the reporter plasmid TK- HC100-luc (100 ng well) together with 100 ng/well of PCMX-GAL4-RXR, or pCMX-GAL4-PPARy, or pCMX-GAL4-LXRa, or pCMX-GAL4- LXRp plasmids using FuGene 6 Transfection Reagent (Roche). Four hours post- transfection, cells were treated with tumor-conditioned media for 24 h.
  • Luciferase activities were analyzed by luciferase Reporter Assay Systems (Promega) according to the manufacturer's protocol, ⁇ -galactosidase (30 ng/well) was used for transfection normalization.
  • Tumor growth experiments B6 or NOD-SCID mice were injected s.c. with live tumor cells. RMA (0.5 or 1x10 5 ), B16 (5x10 4 ), LLC (2.5-3 x10 5 ), TrampCI (2.5x10 6 ). Tumor size was evaluated by measuring perpendicular diameters by a caliper. Data are reported as the average tumor volume ⁇ SD. Comparison of survival curves was evaluated by log-rank test. Experiments of tumor growth using ZA or T1317 were performed as described here.
  • ZA 75 ⁇ g or vehicle was given i.p. contra laterally every 2 days, starting 8 days post-tumor infusion. T1317 (10 ⁇ ) or vehicle was given intratumor every 2 days, starting 5 days post-tumor infusion.
  • ZA was combined with 500 g of the anti-CD25 depleting mAb PC61 27 or control antibody, given i.p. 4 days before tumor inoculation.
  • shRNA experiments HepG2 cells were transduced with lentiviral vectors encoding hLXRa (SIGMA, MISSIONTM TRC shRNA Target set) or non-targeted shRNA (SIGMA, MISSION Non-Target shRNA Control Vector).
  • mice Lethally irradiated (1 1Gy) B6 mice were transplanted with the bone marrow of CD11c-DTR or LXRa' ' mice (4x10 6 BM cells/mouse). Eight weeks later, transplanted mice were controlled for CD11c ablation or LXRa ' ' ' phenotype in the blood and used for tumor growth experiments. Mice were treated i.p. with 4ng/g body weight of Diphtheria Toxin (DT) or left untreated. Twenty-four hours later mice were injected with 1 x10 5 RMA-mock or RMA-SULT2B1 b and monitored.
  • DT Diphtheria Toxin
  • Endogenous DC migration was induced with 500 ⁇ of Acetone/Dibutylphtalate (1 :1 v/v) containing 5 g/ml FITC applied to the shave skin seventy-two hours upon s.c. tumor injection (0.35 x 10 6 cells/100 ⁇ PBS). Draining lymph nodes were removed 12 hours upon FITC application, as previously mentioned. Enzymatically treated cell suspension was washed and incubated 10 min with Fc blocking solution followed by CD11 c staining and FACS analysis. FITC painting assays were performed on C57BU6 wild type mice or mice transplanted with the bone marrow of LXRa ' or wild type mice. Immunohistochemistry.
  • Tumor samples were either fixed in buffered 4% formalin or embedded in OCT and frozen in liquid nitrogen. Three pm paraffin sections were stained in hematoxylin and eosin for morphological analysis or immunostained with the rat anti-human CD3 (Serotec) after antigen retrieval. Three pm cryosections were fixed with 4% paraformaldheyde and incubated with the rat anti-mouse CD11 b and rat anti mouse CD1 1c.
  • the immunoreactions were revealed by biotinilated- conjugated anti-rat antibody (Vector), horseradish peroxidase (HRP)-conjugated streptavidin, and using 3,3 diaminobenzidine (DAB) as chromogen (Biogenex, SanRamon). Slides were counterstained with hematoxylin. Immunohistochemistry on human tumors was performed on tumor samples fixed in buffered 4% formalin. Serial sections of tumors were then immunostained for CD1 1 c (clone 5D1 1 Novocastra), CCR7 (clone E271 Epitomics) and CD83 (clone 1H4b Novocastra). Mice and reagents.
  • C57BLJ6 (B6) and NOD-SCID mice (6-8 weeks of age) were from Charles River or from Harlan and housed under pathogen-free conditions.
  • OT- I3 36 (Ly 5.1 ) and CD11c-DTR 37 mice have previously been described 38 .
  • the C57BL/6 Lxr ⁇ mice were generated as previously described 39 . Animal studies were approved by the Institutional Animal Care and Use Committee of Istituto Scientifico S.Raffaele. Most human and mouse antibodies were from Becton Dickinson.
  • Anti- CD83 mAb was from Coulter.
  • mAb specific for murine CCR7 was from eBioscience. LNGFr-specific mAb 20.4 was from ATCC.
  • Human iDC at day 4 of culture were harvested and activated (8x10 5 cells) with irradiated (100 Gy) 3T3-CD40L (25x10 4 cells) or with LPS (100 ng/mL) in a 6 wells plate, in the presence or absence of either the tumor CM or natural/synthetic LXR agonists.
  • DC were co-cultured with irradiated (100 Gy) MSR3-CD40L (25x10 4 cells/well).
  • mature DC were harvested and analyzed.
  • Murine bone marrow-derived DC were harvested at day 7 of culture and activated with LPS (500 ng/ml) in the presence of either CM or compounds for 48 hours.
  • mouse DC were isolated by rat anti-mouse CD11c mAb (N418)-coupled magnetic MicroBeads (Miltenyi). Most human melanoma, lung, colon, kidney tumor lines as well as normal renal cells were established in our laboratory, with the exception of MZ2-G43, LG2, LB2033, LB33, LB39, SK29.1 and LB40, kindly provided by Prof. T.
  • CM Tumor-conditioned media
  • Chemotaxis Assays were performed using 5- ⁇ pore polycarbonate filters in a 24-well transwell chamber (Corning Costar Corporation) described elsewhere 40 . Briefly, treated DC (3.5x10 4 /100 ⁇ ) were seeded in the upper chamber, whereas in the lower chamber 600 ⁇ of medium containing CCL19 (R&D systems) was added. The number of DC migrated in the lower chamber was measured by flow cytometer acquisition of a fixed number of beads (Polysciences). The number of treated DC migrating in the absence of chemokine was always subtracted. To evaluate the percentage of migration, the number of migrated DCs was divided by the total number of cells added to the transwell 41 .
  • SULT2B1 b-expressing cell lines were derived by transduction of the wild type cells with the retroviral vector LSULTBI bSAN coding for the mouse SULTBI b gene 42 and for the cell surface marker LANGFr 43 .
  • LSULTBI bSAN retroviral vector was generated by cloning the SULTBI b EcoRI/Xhol fragment into the EcoRI/Xhol site of LXSAN under the long terminal repeat (LTR) transcription control. Primers used to clone SULT2B1b are shown in Table 2. Vector DNA was converted into the corresponding virus by the transinfection protocol.
  • Tumor cell lines were infected by the exposure to virus-containing supernatant in the presence of 8 ⁇ g/ml of polybrene. Transduction efficiency was measured by FACS analysis for LNGFr expression.
  • the SULT2B1 b-ANGFr lentiviral transfer vector was generated by cloning the murine SULT2B1 cDNA in place of the GFP cDNA into the self-inactivating hPGK.GFP.wPRE.mhCMV.ANGFr.SV40PA lentiviral vector (Agel/Sall sites) 44 .
  • the truncated form of the mouse SULT2B1b gene was created by deleting the proline-serine-rich carboxyl terminus of the sequence, as described for the human SULT2B1 b gene in 45 .
  • the deleted form of the mouse SULT2B1 b (aminoacid sequence 1-312 instead of 1-338) was then cloned into the self- inactivating hPGK.GFP.wPRE.mhCMV.ANGFr.SV40PA lentiviral vector (Agel/Sall sites) as described above. Concentrated VSV-G-pseudotyped LV stocks were produced and titered as described previously 44,46 .
  • the comparative Ct method was used to quantify transcripts that were normalized for human or murine GAPDH, murine cyclophillin or human ⁇ -actin.
  • Adoptive transfer and immunization experiments OT-I CD8 + T-cells were purified from spleen and lymph nodes by CD8 negative isolation kit (Dynal, Invitrogen) and labeled with CFSE as described previously 3 . Three x10 6 CFSE labeled CD8 + T- cells/mouse was injected into the tail vein of B6 recipients. After 24 hours, recipients were injected s.c.
  • OVApep-loaded DC 0.3-0.5x10 6
  • OVApep-loaded DC 0.3-0.5x10 6
  • spleen and lymph nodes from immunized mice were collected, digested, stained with CD8a and CD45.1 mAbs and then analyzed by FACS.
  • OT-I activation assay Purified OT-I CD8 + T-cells (1.5 x10 5 ) were cultured with OVApep-loaded DC (0.5 x10 5 ), previously activated with LPS in the presence of 22R-HC, 22S-HC or left untreated. Forty-eight hours later, supernatants were collected to measure the content of mouse IFN- ⁇ and TNFa. Four days later cells were collected, counted by trypan blue exclusion method to evaluate cell number.
  • mice were injected with 10 5 RMA cells. At day 6, as soon as tumor was palpable, mice were injected intratumor with supernatants containing viral particles.
  • iDC immature monocyte-derived DC
  • NIH-3T3 cells expressing the DC activation molecule CD40L (3T3-CD40L) 14 .
  • Activation was performed for 48 hours in the presence or absence of conditioned media (CM) from the human melanoma cell line MSR3.
  • CM conditioned media
  • DC were activated with MSR3 expressing CD40L (MSR3-CD40L).
  • DC expressed the activation marker CD83, up-regulated CD80, CD86, HLA-DR and CD54 molecules, down-regulated CCR5 15,16 and secreted high levels of IL-12 (Fig. 1a and Fig. 17a,e).
  • CM from fourteen out of 21 (66%) melanomas, as well as from some human colon, lung and kidney carcinomas (Fig. 1d), but not from normal tissues inhibited CCR7 expression on DC (Fig. 1c), leaving intact the expression of presenting and co-stimulatory molecules, cytokine release and the allogeneic T-cell stimulatory ability (Fig. 18a-c).
  • the inhibitory factor(s) was also produced by murine tumors (Fig. 1f).
  • human and mouse tumor-CM inhibited CCR7 expression also on murine bone marrow-derived DC (hereafter referred as DC) (Fig. 1g).
  • Agonists of some nuclear receptors have been demonstrated to inhibit the expression of CCR7 and CXCR4 receptors on human and mouse DC 14,17 . Whether these agonists are released by tumors has not been reported yet.
  • MSR3-CM did not activate PPARy but slightly induced RXR activation (Fig. 2a), however we did not find any RXR ligand in MSR3-CM (data not shown). Noteworthy, MSR3-CM activated LXRa, whereas CM from the non-inhibitory tumor M3M001 did not (Fig. 2a).
  • oxysterols 5 22R-HC, 25-HC
  • T1317 synthetic agonist T0901317
  • Oxysterols inhibited CCR7 expression at nanomolar concentrations did not affect costimulatory molecules and impaired also the expression of CXCR4 receptor (Fig. 19a-c).
  • a close correlation between CCR7 inhibition and LXRa activation was found in 60% of the tumor lines tested (21/35, data not shown).
  • CM from ZA-treated tumors (MSR3, MR255, LOVO, RMA and Tramp C1) inhibited CCR7 expression to a lower extent as compared to CM from untreated tumors (Fig. 3b,c).
  • Loss of CCR7 inhibitory activity paralleled the loss of LXRa activation (Fig. 3d), as well as the lack of ABCG1 mRNA induction in DC cultured with ZA-treated MR255-CM (Fig. 3e).
  • CM from SULT2B1 b-transduced cells inhibited CCR7 expression at a significantly lower extent and did not activate LXRa (Fig. 3f,g), as compared to CM from parental cells transduced with vectors coding only for the ANGFR (hereafter referred as mock- transduced).
  • LXRa triggering in DC was responsible for CCR7 inhibition
  • shLXRa expression prevented CCR7 inhibition by 22R-HC and tumor-CM (Fig. 3i), demonstrating that CCR7 inhibition mediated by tumor-CM occurs through LXRa activation in maturing DC.
  • Impaired DC migration affected T cell priming, as H-2K -restricted OVA peptide-pulsed DC treated with 22R- HC-, LOVO- and RMA-CM were poor inducers of the proliferation of adoptively transferred OT-I CD8 + T cells, a transgenic line with a H-2K b -OVA specific TCR 24 (Fig. 21a and Fig. 20b).
  • the inactive oxysterol 22S-HC did not alter CCR7 expression and DC migration, it impaired OT-I activation in vivo (Fig. 4b) and in vitro (Fig. 21 b-d).
  • Tumor control was strictly dependent on the amount of SULT2B1 b expression by tumor cells (Fig. 23a, b) and could be partly abolished by the expression of a deleted form of the murine SULT2B1b (data not shown), which has been shown in a human cellular system to have a reduced sulfurylation activity 25 .
  • this benefit was partly abolished when RMA-SULT2B1b-bearing mice were injected intratu morally with the synthetic LXRa ligand T1317 (Fig. 5g), which is not inactivated by SULT2B1b (Fig. 20a), confirming the inhibitory role of LXRa in the antitumor response.
  • DC and CCR7 play a key role in the LXR-mediated suppression of antitumor immune responses
  • Lymph nodes draining RMA-SULT2B1 b tumors contained a higher number of CD1 1c + FITC + DC compared to RMA-mock (Fig. 5b,c), indicating that the inactivation of tumor-derived LXR agonists restores DC migration to draining lymph nodes.
  • FIG. 5g Histologic and immunohistochemical analyses of the engineered tumors (Fig. 5g) showed a strong infiltrate of granulocytes and CD3 + T cells (Fig. 5h) in RMA- SULT2B1b compared to RMA-mock at 7 and 14 days. This infiltrate was barely detectable in both tumors at day 3.
  • Fig. 5i we found a higher number of infiltrating CD11c + DC in RMA-mock compared to RMA-SULT2B1b at days 3 and 7, whereas an opposite scenario was observed at day 14 (Fig. 5i).
  • the number of CD11 b + cells were similar in both tumors at days 3 and 7, while at day 14 were more frequent in RMA-SULT2B1b.
  • RMA-bearing mice were injected intratumor at day 6 with 10 ⁇ of supernatant containing lentiviral particles encoding either the sulfotransferase enzyme SULT2B1b (LV-SULT2B1 b) or mock (LV-mock).
  • LV-SULT2B1 b the sulfotransferase enzyme SULT2B1b
  • mock LV-mock
  • CD11 b* cells were isolated from mouse bone marrow cells by magnetic beads. Purified CD11 b + cells from bone marrow normally express also the GR1 marker. Purified CD11 b + GR1 + cells (2x10 s ) were plated in the upper chamber of transwell filters and allowed to migrate to either the LXR ligand 22R-HC (15 ⁇ ), the inactive form 22S-HC (15 ⁇ ) or to the medium alone, plated in the lower chamber. After 2 h at 37 " C, the number of cells migrated in the lower chamber was measured by flow cytometer acquisition of a fixed number of beads (Polysciences Inc.). To evaluate the percentage of migration, the number of migrated cells was divided by the total number of cells added to the transwell.
  • CD1 1b + GR1 * cells from LXRs deficient mice or CD1 1 b + GR1 + cells pre-incubated with Pertussis Toxin (100 or 500 ng) for 90' before running the migration assay.
  • CD14 + monocytes were purified from peripheral blood mononuclear cells of healthy donors or melanoma patients. Purified CD14* monocytes were then allowed to migrate overnight at 37°C to 22R-HC (15 ⁇ ) or 22S-HC (15 ⁇ ). The day after, cells were collected and the number of cells migrated in the lower chamber was measured by flow cytometry as described above. Competition assays, and migration experiments using cells deficient for CXCR2 chemokine receptor
  • CD11b + GR1 + cells (2x10 5 ) were incubated for 30' with 22R-HC (50 ⁇ ), CXCL5 (1 ⁇ g/ml) or with the CXCR2 antagonist SB225002 (10 ⁇ ). Then, the cells were washed and allowed to migrate to either 22R-HC (15 ⁇ ), 22S-HC (15 ⁇ ), CXCL5 (100 ng), SDF1a (100 ng), or MIP-1a (100 ng) for 2 h at 37 ° C. In some experiments migration was performed using CD11 b + GR1 + cells from CXCR2 " ' " mice.
  • Flow cytometry MAbs specific for CD1 1 b, GR1 , CD1 15, c-kit, IL ⁇ Ra, CX3CR1 , CCR1 , CXCR4, CXCR2, CD45.1 , CD45.2 and CD31 were from Becton Dickinson, PharMingen, R&D Systems or from eBioscience. Samples were run on a FACS CaliburT (BD) and analyzed by CELLQuestTM software (BD) or by FlowJo.
  • CD45.1 * bone marrow cells (20x10 6 ) were injected into NOD-SCID mice bearing 14 days established tumors releasing LXR ligands (RMA-Mock) or not releasing LXR ligands (RMA-SULT2B1 b). Eighteen hours later, mice were sacrificed, tumors were collected, mechanically disaggregated and treated with collagenase A (1 mg/ml) and DNase (0.4 mg/ml) mixture in HBSS media 20% FBS for 60', and analyzed for the presence of CD45.1 + CD11 b hi9h GR1 i9h cells by flow cytometry.
  • Percentage and number of CD45.rCD11b hl9h GR1 high cells/mg of tumor tissue were evaluated and quantified.
  • 22R-HC (0.5 mM) or 22S-HC (0.5 mM) were mixed in matrigel and injected s.c. into the dorsal flank of mice. Three days later, mice were sacrificed, matrigels were collected, enzymatically disaggregated by dispase, and analyzed for the presence of CD45.2 + CD11 b high GR1 hi9h cells by flow cytometry. The analysis was performed as described above.
  • Data are expressed as mean ⁇ SD and were analyzed for significance by analysis of variance (ANOVA) with Dunnet's or Tukey's multiple comparison test, or when indicated by an unpaired or paired Student's t-test with Prism software.
  • ANOVA analysis of variance
  • mice subcutaneously with 3x10 5 LLC cells. Seven days later, mice were either left untreated, treated with ZA, with tumor vaccination, or with ZA in combination with tumor vaccination and evaluated for tumor growth every 2 days. Tumor vaccination was performed using a nonapeptide derived from the tumor antigen MUT1 emulsified in Incomplete Freund's Adjuvant. ZA (200 ⁇ g) was administered every 2 days. Mice treated with ZA showed a statistically significant delay of tumor growth as compared with untreated tumor-bearing mice. The effect of ZA treatment was superior to the vaccination treatment. The combination therapy (i.e. ZA plus vaccination) was more effective as compared to the single treatments.
  • the combination therapy i.e. ZA plus vaccination
  • Mouse CD11 b+GR1+ bone-marrow derived cells migrate to LXR-releasing tumors in a CXCR2 dependent manner
  • CD1 1 b+GR1 + cells isolated from bone marrow of naive mice have been allowed to migrate to the LXR ligand 22R-Hydroxycholesterol (22R-HC) through transwell filters.
  • 22R-HC 22R-Hydroxycholesterol
  • this migration turns out to be restricted to the CD11 b hi9h GR1 hi9h subset. Moreover, this migration is pertussis toxin sensitive, indicating that the receptor(s) mediating this effect is a G protein coupled receptor, and is independent of LXR, as CD1 1b+GR1 + cells from LXRcf ⁇ , LXR ⁇ ' and LXRa ' mice migrate to 22R-HC similarly to CD1 1 b+GR1 + cells from wild type mice. FACS analysis for chemokine receptors expression by comparing migrating and non migrating CD1 1 b+GR1 + cells, showed the preferential expression of CXCR2, CCR1 and CXCR4 chemokine receptors by migrating cells (Fig.
  • Mouse CD11b+GR1+ bone-marrow derived cells promote neo angiogenesis and tumor growth
  • LXRs modulate immune responses under physiological conditions as well as in different disease models 8,29 .
  • in vitro differentiation of human DC in the presence of LXR agonists and LPS has been shown to affect their T-cell stimulatory ability 18 .
  • LXR plays an important role in cancer.
  • human and murine tumors release cholesterol metabolites dampening the expression of CCR7 receptor on maturing DC by LXR activation; thus, allowing tumor escape from immune surveillance (Fig. 16).
  • tumor- produced l_XR ⁇ agonists appear to paralyze the immune system, as tumors grow similarly both in the presence and in the absence of DC.
  • Oxysterols are substrates for cholesterol sulfotransferase. J Lipid Res 48, 1343-1352 (2007).

Abstract

The invention relates to the discovery that human and murine tumors release LXR ligands (oxysterols) that inhibit CCR7 expression on maturing DC and, therefore, their migration to lymphoid organs. By inhibiting oxysterol synthesis (by Zaragozic Acid, ZA) or by inactivating oxysterols (gene therapy with sulfotransferase SULT2B1 b enzyme) long lasting antigen specific anti-tumor immune response mediated by DC is increased. Surprisingly, we also show that drugs interfering with sterol metabolism (i.e. ZA) in combination with a mAb depleting T regulatory cells potentiate the antitumor effect of the single treatments. This synergic effect is unexpected, providing a new effective combination therapy for the treatment of cancer. The invention also relates to the novel use of LXR ligand inactivators, or of LXR inhibitors/antagonists, for the treatment of cancer. These strategies can also be used in combination with a chemotherapy approach for the treatment of cancer patients.

Description

COMPOSITIONS
FIELD OF THE INVENTION The present invention relates to novel treatment of cancer patients with therapeutics interfering with oxysterol synthesis and metabolism to restore a successful antitumour response. The present invention also relates to novel treatment of cancer patients with therapeutics to prevent migration of pro-angiogenic cells. More particularly, the invention relates to a novel combination of an oxysterol modulator and a CD25-depleting monoclonal antibody for the treatment of cancer. The invention also relates to LXR ligand inactivators for the treatment of cancer. The invention further relates to LXR inhibitors/antagonists for the treatment of cancer. The present invention also relates to the use of an LXR ligand for promoting migration and/ or for isolating mouse CD11b+GR1 + cells or human CD1 + cells from a population.
BACKGROUND OF THE INVENTION
Tumors evade the immune system through mechanisms conditioning their microenvironment. Recently, some immunoescape processes have been molecularly elucidated1. Among them, the activation of different metabolic pathways2,3 leads to immunosuppressive effects.
The nuclear Liver X Receptors (LXR) a and β are involved in lipid and cholesterol homeostasis4,5,6. LXRp is expressed ubiquitously, while LXRa is expressed in liver, adipose tissue, adrenal glands, intestine, lungs and cells of myelomonocytic lineage7. Both receptors are triggered by oxidized cholesterol (oxysterols)5 Recent data indicate that LXRs modulate both innate and adaptive immune responses8.
Dendritic cells (DC) initiate adaptive immune responses9, including antitumor activity following their CCR7-dependent migration to lymphoid organs. Indeed, DC ablation abrogates the induction of antigen-specific effector cells10. The ability of DC to initiate an immune response depends on their migration to lymphoid organs where they present antigens to naive T and B cells 1. This process requires the expression of the lymphoid-homing receptor CCR7 by maturing DC12,13.
Sterol metabolism has recently been linked to innate and adaptive immune responses through LXR signaling. However, whether and how products of sterol metabolism induce immunosuppressive effects has previously remained unknown. Further, whether alteration of cholesterol metabolism in tumor cells and/or triggering of LXR in immune cells may dampen antitumor responses has not previously been investigated.
Thus, there is a need for novel therapeutics that target this area. The present invention addresses this need.
The present invention provides novel therapeutics for use in the treatment of cancer and novel methods of treatment.
SUMMARY OF THE INVENTION
In the present application, we show for the first time that human and murine tumors produce LXR ligands, which inhibit the functional expression of CCR7 on human and murine DC (Fig. 16), and therefore their migration to lymphoid organs. In agreement, CD83*CCR7" DC were detected within human tumors. Mice injected with tumors expressing the LXR ligands inactivating enzyme SULT2B1 b, successfully control tumor growth by regaining DC migration to tumor-draining lymph nodes and by the development of an overt inflammation within tumors. The control of tumor growth was also observed in Lxr« " bone marrow chimeras. Thus, we show a novel mechanism of tumor immunoescape involving products of cholesterol metabolism. Manipulation of this pathway provides a means to restore antitumor immunity in cancer patients.
LXR ligands released by tumor cells could also be active on other cell types such as macrophages or lymphocytes.
In murine models, we demonstrate that interfering with tumor synthesis of LXR ligands or ablating LXRa signaling in DC, leads to an immune-mediated strong inhibition of tumor growth. Noteworthy, immunohistochemical analyses on human tumors provides evidence of infiltrating CCR7 negative mature DC. These results shed light on a novel role of LXRs in antitumor responses.
Accordingly, when we added tumor-CM to monocytes during their differentiation to DC, we noticed a wider dysregulation of DC differentiation and activation (Russo et a/, unpublished observations). Inhibition of CCR7 expression on DC resulted in impaired migration to the draining lymph nodes. Interestingly, impaired migration of monocyte-derived cells from atherosclerotic plaques is one of the factors responsible for progression of atherosclerosis. The mechanism for emigration failure is still under investigation, however, lipid-derived signals have been proposed to dampen migration, suggesting a possible role of LXR also in this context. Importantly, we found that the abrogation of tumor-mediated LXRa activation by either pharmacological compounds able to block cholesterol synthesis, by gene transfer- mediated inactivation of intracellular oxysterols, or by the use of LXRa-/- bone marrow chimeras as tumor recipients, generated effective antitumor responses in different tumor models. Further, we have demonstrated that oxysterols (released by tumor cells) are able to attract a population of mouse pro-angiogenic cells (CD11b+GR1+) of myeloid origin known to be involved in promoting tumor angiogenesis (Shojaei et al., PNAS vol 105 no.7 2640-45, 2008, incorporated herein by reference). We have also shown that mouse CD11 b+GR1 + bone-marrow derived cells migrate to LXR-releasing tumors in a CXCR2 dependent manner, and that mouse CD11b+GR1+ bone-marrow derived cells promote neoangiogenesis and tumor growth. Thus this new pathway, mediated by LXR ligands occurring within the tumor microenvironment, is responsible for the migration of CD11b+GR1+ myeloid cells within the tumor. We have also characterized the human cell counterpart (CD1 + cells) that migrates to LXR ligands.
We have also shown that Zaragozic Acid (ZA) administration delays the growth of the mouse lung tumor LLC, and that the administration of Zaragozic Acid (ZA) is well tolerated and safe.
Surprisingly, we show that drugs interfering with sterol metabolism (such as ZA) in combination with a mAb depleting T regulatory cells potentiate the antitumor effect of the single treatments. This synergic effect is unexpected and therefore it could be a new effective combination therapy for the treatment of cancer.
Thus, the present invention opens new avenues for the treatment of cancer patients with drugs interfering with sterol metabolism to restore a successful antitumour immune response. In addition to enhancing anti-tumor immune response, the use of oxysterol inhibitors has been confirmed to prevent migration of CDl 1 b+GR1+ pro- angiogenic cells.
5 In particular, the present invention relates to the novel use of a combination of an oxysterol modulator and a CD25-depleting monoclonal antibody for the treatment of cancer.
The present invention further relates to the novel use of LXR ligand inactivators for l o the treatment of cancer.
The present invention further relates to the novel use of LXR inhibitors/antagonists for the treatment of cancer.
1 5 Importantly, it should be noted that the oxysterol synthesis inhibitors of the present invention block cholesterol synthesis downstream of mevalonic acid. Indeed, statins (the most used cholesterol-lowering drugs) have been described to have an immune suppressive activity blocking T cell proliferation and dampening DC activation. These discrepancies can be ascribed to the different mechanism of action of the two0 compounds along the pathway of cholesterol synthesis. Indeed, besides mevalonic acid, statins also inhibit the formation of molecules (i.e. farnesyl pyrophosphate and geranylgeranyl pyrophospate) involved in functional posttranslational modification (i.e. prenylation) of small GTPase proteins including Rho, Rac and CdC42, while ZA by acting downstream mevalonic acid synthesis, blocks only cholesterol/oxysterol5 formation.
Importantly, other molecules in development inhibiting cholesterol synthesis downstream of mevalonic acid and along the pathway of cholesterol synthesis (e.g. squalene synthase inhibitors, lanosterol 14-alpha demethylase inhibitor, farnesyl diphosphate synthase (FDPS) inhibitor) may prove to restore a successful antitumour response and prevent migration of pro-angiogenic cells with a mechanism as described above.
STATEMENTS OF THE INVENTION
The present invention is directed towards novel compositions and methods of treating cancer.
The present invention is directed towards therapeutics interfering with oxysterol synthesis and metabolism to restore a successful antitumour response, for treating cancer. According to one aspect of the present invention there is provided a pharmaceutical composition comprising a combination of (i) an oxysterol modulator and (ii) a CD25- depleting monoclonal antibody for treating cancer.
In one embodiment the oxysterol modulator blocks cholesterol synthesis downstream of mevalonic acid.
In one embodiment the oxysterol modulator is selected from a squalene synthase inhibitor and a lanosterol 14-alpha demethylase inhibitor. In one embodiment the lanosterol 14-alpha demethylase inhibitor is an azole.
Examples of azoles include, but are not limited to, fluconazole (Diflucan) and itraconazole (Sporanox). In one embodiment the lanosterol 14-alpha demethylase inhibitor is SKF 104976.
In one embodiment the squalene synthase inhibitor is zaragozic acid or a derivative thereof.
In one embodiment the derivative is a conjugate, a salt, an ester, an amide or a carbamate of zaragozic acid, optionally substituted. Examples of zaragozic acid derivatives include, but are not limited to,
O
Figure imgf000008_0001
wherein X is H, a halogen (F, CI, Br, I), OH or CH3; Y is halogen (F, CI, Br, I), OH or CH3, and wherein Z1 t Z2 and Z3 are each independently H, C1-5 alkyl, C1-5 alkyl substituted with (i) phenyl, (ii) phenyl substituted with methyl, methoxy, halogen (F, CI, Br, I) or hydroxy, (iii) C1-5 alkylcarbonyloxy, (iv) C6-10 arylcarbonyloxy, (v) C1-5
alkoxycarbonyloxy, (vi) C6-10 aryloxycarbonyloxy, (vii) α-s aikyi , (Vjjj)
Figure imgf000009_0001
the groups (iii) to (vi) form a 5 to 10 membered mono- or bicyclic ring with C1- or from
Figure imgf000009_0002
wherein R is selected from:
Figure imgf000009_0003
; R2 is selected from: H, and — ; R3 is C1-5alkyl; Z is selected from (i) H, (ii) Ci.5alkyl; (iii) Chalky! substituted with (a) C^alkylcarbonyloxy, (b) arylcarbonyloxy, (c) Ci.5alkoxycarbonyloxy, (d) aryloxycarbonyloxy; (e)
Figure imgf000009_0004
C1-5 alkyl , (f) , (g) or the groups (a) to (d) form a a 5 to 10 membered mono or bicyclic ring with C1-5alkyl, (iv) cycloalkyl; or a pharmaceutically acceptable salt thereof.
In one embodiment the squalene synthase inhibitor is zaragozic acid.
In one embodiment the squalene synthase inhibitor is selected from lapaquistat (TAK-475), terbinafine, ER-27856 (5-{A/-[2-butenyl-3-(2-methoxyphenyl)]-N- methylamino}-1 ,1-penthylidenebis(phosphonic acid) tri-sodium salt), RPR-107393 (3- hydroxy-3-[4-(quinolin-6-yl)phenyl]-1 -azabicyclo [2-2-2]octane dihydrochloride) and YM-53601 ((E)-2-[2-fluoro-2-quinuclidin-3-ylidene ethoxy]-9H-carbazole monohydrochloride).
In one embodiment the squalene synthase inhibitor is a phosphonic acid derivative. An example of a phosphonic acid derivative is is represented by the following general
RB O
II
RA- -P-OR, formula (I): Rl OR3 wherein R, represents a hydrogen atom, a hydroxyl group, an acyloxyalkyi group, an alkyloxycarbonyl group, a lower alkyl group which may have a substituent or a lower alkoxy group which may have a substituent; R2 and R3 may be the same or different from each other and each represents a hydrogen atom, a lower alkyl group which may have a substituent, an alkali metal or a prodrug ester forming group; RA represents a group represented by the formula: 0 (wherein R4 represents a hydrogen atom, a lower alkyl group, an alkali metal or an acyloxyalkyi group which may have a substituent), a group represented by the
formula:
Figure imgf000010_0001
(wherein RV represents a hydrogen atom, a lower alkyl group or an O
II
— P— OR5
I
alkali metal) or a group represented by the formula: R6 wherein R5 represents a hydrogen atom, a lower alkyl group, an alkali metal or a prodrug ester forming group; and R6 represents a lower alkyl group or a group represented by the formula: -OR7 (wherein R7 represents a hydrogen atom, a lower alkyl group, an alkali metal or a prodrug ester forming group)]; and
RB represents a group represented by the formula: S--T-- [wherein S represents an alkenyl roup which may have a substituent or a group represented by the formula:
Figure imgf000011_0001
(wherein ring A represents an aromatic ring; R8, R9, R10, R11 and
R12 may be the same or different from one another and each represents (1 ) a hydrogen atom, (2) an alkyl group which may have a substituent, (3) an alkenyl group which may have a substituent, (4) a lower alkoxy group which may have a substituent, (5) a carbamoyl group which may have a substituent, (6) a carbamoyloxy group which may have a substituent, (7) a hydroxyl group, (8) an acyl group, (9) a halogen atom, (10) a group represented by the following formula:
Figure imgf000011_0002
(wherein R13 and R14 may be the same or different from each other and each represents a lower alkyl group which may have a substituent, or alternatively R13 and R1 may form together with the nitrogen atom to which they are bonded, a ring which may further contain an oxygen atom, a sulfur atom or a nitrogen atom and which may have one or two, mono- or divalent substituent(s); p is 0 or 1 ; and q is an integer of 0 to 4) or (11 ) a group represented by the formula:
Figure imgf000011_0003
(wherein R15, R16, R17, R18 and R19 may be the same or different from one another and each represents a hydrogen atom, a hydroxyl group, a lower alkyl group or a lower alkoxy group which may have a substituent; ring B represents an aromatic ring; and Y represents an alkylene chain which may have a substituent, an alkenylidene chain which may have a substituent, an alkynylidene chain which may have a substituent, a group represented by the formula: 0 , a group represented by the formula: -0-, or a single bond), or alternatively two adjacent groups of R8, R9, R10, R1 1 and R12 may together form a ring; and X represents a single bond, an alkylene chain which may have a substituent, an alkenylidene chain which may have a substituent or a group represented by the formula: -(CH2)u -Z--(CH2)v - (wherein Z is a group represented by the formula:
( 0 )r
I I r
c
(wherein r is an integer of 0 to 2), a group represented by the formula:
0 a group represented by the formula: -0-, a group represented by the formula:
R20
l _
S°2N (wherein R20 represents a hydrogen atom, a lower alkyl group which may have a substituent or a lower alkenyl group which may have a substituent), a
R21
I
group represented by the formula N— (wherein R21 represents a hydrogen atom, a lower alkyl group which may have a substituent, a lower alkenyl group which
may have a substituent or a group represented by the formula:
Figure imgf000012_0001
R22 o
group represented by the formula: N (wherein R22 represents a hydrogen atom, a lower alkyl group which may have a substituent or a lower alkenyl group which may have a substituent); u is an integer of 0 to 3; and v is an integer of 0 to 6); and T represents (1) a single bond, (2) a group represented by the formula:
Figure imgf000013_0001
(wherein R23 represents a hydrogen atom, a cycioalkyi group, a cycloalkylalkyl group, a lower alkyl group which may have a substituent or a lower alkenyl group which may have a substituent; W represents a group represented by the formula: -0-, a group represented by the formula: 0 , a group represented by
OH
the formula: --NH-, a group represented by the formula: CH— , a group o
represented by the formula: 0 or a single bond; and s and t are independent of each other and are each an integer of 0 to 4), (3) a group represented by the
formula:
Figure imgf000013_0002
(wherein R23, W, s and t are each as defined above; and R29 represents a hydrogen atom, a cycioalkyi group, a cycloalkylalkyl group, a lower alkyl group which may have a substituent or a lower alkenyl group which may have a substituent),
R25
_l
(4) a group represented by the formula: N (wherein R25 represents a hydrogen atom, a cycioalkyi group, a lower alkyl group which may have a substituent or a lower alkenyl group which may have a substituent), or
Figure imgf000013_0003
(5) a group represented by the formula: 2 (wherein D represents a carbon atom or a nitrogen atom, E represents a nitrogen atom or a group represented by the formula: ^ , F represents a group represented by the formula: --0-, a group represented by the formula: ° , a group represented by
OH
I
the formula: -NH-, a group represented by the formula: CH— , a group O
represented by the formula: or a single bond; x and y are independent of each other and are each an integer of 0 to 3.
In one embodiment the squalene synthase inhibitor is a probucol ester of the formula
Figure imgf000014_0001
wherein X, is H or P03H2 and X2 is H or P03H2; and wherein R1 and R2 are H- or - CH3; and R3, R4, R5 and R6 are independently selected from H- or an alkyl group selected from the group consisting of methyl, ethyl, propyl, butyl or tert-butyl; or, a pharmaceutically acceptable salt thereof.
In one embodiment the CD25-depleting monoclonal antibody is selected from Basiliximab (an anti-mouse CD25 antibody), daclizumab (Zenapax), inolimomab (Leucotac), HuMax-TAC and PC61.
In another aspect of the invention there is provided an LXR ligand inactivator for treating cancer.
The LXR ligand inactivator may be an LXRa ligand inactivator or an LXR ligand inactivator.
In one embodiment the LXR ligand inactivator is a sulfotransferase enzyme. In one embodiment the sulfotransferase enzyme is SULT2B1b.
In one embodiment the LXR ligand inactivator is administered using gene therapy. In a further aspect of the present invention there is provided an LXR inhibitor or antagonist for treating cancer.
In one embodiment the LXR inhibitor or antagonist is an LXRa inhibitor or antagonist or an LXRp inhibitor or antagonist.
In one embodiment the LXR antagonist is a cholesterol oxide, an oxysterol or a sterol or derivative thereof.
In one embodiment the sterol is selected from a hydroxycholesterol and a sulfated oxysterol.
In one embodiment the cholesterol oxide is a functionalised cholesterol oxide selected from 7 -hydroxycholesterol, a-epoxycholesterol, β-epoxycholesterol, 7-keto- cholesterol, cholestane triol, 7a-hydroxycholesterol, 25-hydroxycholesterol, 22(R)- hydroxy-cholesterol, 24(S)-hydroxy-cholesterol, 27-hydroxy-cholesterol.
In one embodiment the sulfated oxysterol is selected from 24-OHChol-3-sulfate and 24-OHChol-3, 24-sulfate.
In one embodiment the LXR inhibitor or antagonist is selected from a polyunsaturated fatty acid, a geranyl geraniol or geranylgeranyl pyrophosphate, 5<x,6a-epoxycholesterol sulphate (ECHS), 7-ketocholesterol-3-sulphate, and a
R1
tricyclic compound represented
Figure imgf000015_0001
a pharmacologically acceptable salt thereof, or a hydrate of the compound or the salt in which R1 represents a hydrogen atom, a lower alkyl group, a lower halogenated alkyl group, an unsubstituted or substituted phenyl group, or an unsubstituted or substituted benzyl group; R2 represents a I .I .I .S.S.S-hexafluoro^-hydroxypropan- -yl group or a carboxymethyl group; R3 represents a hydrogen atom, a lower alkyl group, a lower alkoxy group or a halogen atom; X represents a direct bond, an oxygen atom, a sulfur atom, a (CH2)n group or a (CH=CH)n group (wherein n represents an integer of 1-3); and Y represents CO or S02. Further examples of LXR inhibitors or antagonists include, but are not limited to, Liver X Receptor antagonist BMS (Bristol-Myers Squibb Company (BMY)) and Liver X Receptor antagonist EXELIXIS (Exelixis Inc (EXEL)).
In another embodiment the pharmaceutical composition further comprises a pharmaceutically acceptable excipient, diluent or carrier.
According to another aspect we provide a pharmaceutical composition comprising a LXR ligand inactivator and further comprising a pharmaceutically acceptable excipient, diluent or carrier.
According to another aspect we provide a pharmaceutical composition comprising the LXR inhibitor or antagonist and further comprising a pharmaceutically acceptable excipient, diluent or carrier. In one embodiment the pharmaceutical composition, ligand inactivator or LXR inhibitor or antagonist is combined with a chemotherapeutic agent for treating cancer. Examples of the chemotherapeutic agent are cytotoxic antibiotics such as aclarubicin, bleomycin, dactinomycin, daunorubicin, doxorubicin, epirubicin, idarubicin, mitomycin, and mitoxantrone (mitozantrone); alkylating agents such as busulfan, carmustine, chlorambucil, chlormethine hydrochloride, mustine hydrochloride, cyclophosphamide, estramustine phosphate, ifosfamide, lomustine, melphalan, thiotepa, and treosulfan; antimetabolites such as capecitabine, cladribine, cytarabine, fludarabine, fluorouracil, gemcitabine, mercaptopurine, methotrexate, raltitrexed, tegafur, and thioguanine; vinca alkaloids, such as etoposide, vinblastine, vincristine, vindesine, and vinorelbine; other antineoplastic drugs such as amsac ne, altretamine, crisantaspase, dacarbazine, temozolomide, hydroxycarbamide, hydroxyurea, and pentostatin; platinum compounds such as carboplatin, cisplatin, and oxaliplatin; porfimer sodium; procarbazine; razoxane; taxanes such as docetaxel and paclitaxel; topoisomerase I inhibitors such as irinotecan and topotecan; trastuzumab; tretinoin.
There are also provided combination therapies comprising an oxysterol modulator. According to a further aspect there is provided a pharmaceutical composition comprising an oxysterol modulator in combination with a further cancer treatment. According to one embodiment the oxysterol modulator is administered in combination with a chemotherapeutic agent. According one embodiment the oxysterol modulator is administered in combination with immunotherapy (active immunotherapy and vaccination strategy).
According to another aspect of the invention there is provided the use of an LXR ligand for isolating CD11b+GR1 + cells from a population. In a further aspect there is provided the use of an LXR ligand for isolating mouse CD1 1 b+GR1 + and/or human CD14+ cells from a population. According to a further aspect there is provided the use of an LXR ligand for promoting migration of mouse CD11 b+GR1+ and/or human CD14+ cells.
In one embodiment the use involves a migration assay.
In one embodiment the LXR ligand is 22R-HC.
DESCRIPTION OF THE FIGURES Fig. 1 Tumors producing factors inhibiting CCR7 on DC. (a) DC activated by co- culture with 3T3-CD40L cells in the presence of CM from MSR3 melanoma cell line. FACS analysis of one representative experiment, (b) Migration of DC untreated (UT) or activated in the presence or in the absence of MSR3-CM to 100 ng (grey bars) or 10 ng (black bars) of CCL19. *, P < 0.05 (Student's f-test). Mean and s.d. of seven experiments, (c-f) Percentage of CCR7 inhibition induced by CM from several human melanoma (c), colon, lung, kidney tumor cell lines (d), normal fibroblasts and renal cells (e), and murine tumors (f). (g) Percentage of CCR7 inhibition on murine DC treated with CM from human (MR255, LOVO, M3M001 ) and murine (RMA) tumors, (h) Percentage of CCR7 inhibition on DC treated with CM from fresh tumors. *, P < 0.05; **, P < 0.01 ; ***, P < 0.0001 (ANOVA). In panels c-h each symbol corresponds to one DC donor tested and the line represents the mean value, (i) Serial sections of the melanoma MSR3 stained for CD11 c, CCR7 and CD83. CCR7 positive cells are only a fraction of CD83 positive cells (arrows indicate double positive cells; bars = 100 pm).
Fig. 2 Tumor-CM trigger LXRa activation in DC. (a) Luciferase assay for LXRa, RXR and PPARy activation by MSR3-CM, M3M001 -CM, T1317, 9-cis Retinoic Acid (9cRA) and Rosiglitazone (RSG). *, P < 0.05; **, P < 0.01 (Student's f-test). Mean and s.d. of one representative experiment out of three. RLA, Relative Luciferase Activity. (b,c) LXRa activation by CM from tumor lines, mean and s.d. of three experiments *, P < 0.05; ***, P < 0.0001 (ANOVA) (b) or fresh (f) tumors, mean and s.d. of one representative experiment *, P < 0.05; ***, P = 0.0002 (ANOVA) (c). (d) Kinetics of LXRa mRNA expression in maturing DC (D1 and D2 donors). (Insert) RT-PCR for LXRa and LXR expression in immature and mature DC. (e) Expression of ABCG1 mRNA in DC activated in the presence of tumor-CM. *, P < 0.05; ***, P < 0.0001 (ANOVA). Mean and s.d. of one representative experiment out of three. (f,g) CCR7 inhibition at protein (f) and mRNA (g) levels on human DC activated in the presence of natural and synthetic LXRa ligands (D1 and D2 donors). *, P < 0.05; ***, P < 0.0001 (ANOVA). (h,i) Inhibition of CCR7 expression on murine DC by natural and synthetic LXRa ligands. (h) Full and empty histograms represent DC treated with LPS or LPS and LXR ligands, respectively. **, P < 0.01; ***, P < 0.0001 (ANOVA). (f,i) each symbol corresponds to one donor tested and the line represents the mean value.
Fig. 3 Blocking of LXRa signaling abrogates CCR7 inhibition, (a) Diagram showing the experiments with CM from untreated or Zaragozic Acid (ZA)-treated tumors. (b,c) FACS analysis (b) and quantification of the results (c) showing CCR7 expression/inhibition, (d) Luciferase assay for LXRa activation by the tumor-CM described in Fig. 3c *, P < 0.05; **, P < 0.01 (Student's f-test). Mean and s.d. of three experiments (results with MR255-CM in Fig. 3d were from two experiments), (e) ABCG1 mRNA expression in DC activated in the presence of untreated or ZA- treated MR255-CM. *, P < 0.05; ***, P < 0.0001 (Student's f-test). Mean and s.d. of three experiments, (f) CCR7 inhibition in DC activated in the presence of CM from mock-transduced or SULT2B1b-transduced tumors, (g) LXRa activation with the tumor-CM described in Fig. 3f. *, P < 0.05; **, P < 0.01 ; ***, P < 0.0001 (Student's t- test). Mean and s.d. of four (f) and five (g) experiments, (h) qPCR for LXRa mRNA in DC transduced with lentiviral vectors encoding shLXRa or non-targeted shRNA (shRNA NT) and activated with LPS. One experiment out of five, (i) CCR7 inhibition in shLXRa-DCor shRNA NT-DC activated in the presence of 22R-HC or MSR3-, LOVO- or R MA-CM. *, P < 0.05; ***, P < 0.0001 (Student's West). Mean and s.d. of four experiments.
Fig. 4 Dampening of DC migration and T cell priming by tumor-CM and LXR ligands and generation of antitumor responses by avoiding LXRa signaling, (a) Lymph node migration of CD11c+CFSE* DC treated in vitro with LXR ligands or tumor-CM. (b) OT- I T-cell proliferation in mice injected with OVA-loaded DC treated as described in Fig. 4a. *, P < 0.05; **, P < 0.01 (ANOVA). Mean and s.d. of three experiments.(c,d) Tumor growth (c) and survival (d) of RMA-bearing mice treated with ZA or vehicle. Arrow indicates the onset of ZA treatment. **, P < 0.01 (ANOVA). Mean and s.d. of one out of two experiments (ten mice/group). Statistical comparison was performed by the log-rank test. P < 0.0001. (e,f) Tumor growth (e) and survival (f) of mice injected with RMA-SULT2B1 b, RMA untransduced or mock-transduced. SULT2B1b mRNA expression in the insert. **, P < 0.01 (ANOVA). Mean and s.d. of one out of ten experiments (ten mice/group). Statistical comparison was performed by the log- rank test (twenty mice/group). P < 0.0001. (g) Abrogation of RMA-SULT2B1 b rejection by intratumor administration of T1317. (h,i) Delay of TrampC1 -SULT2B1 b (h) and LLC-SULT2B1b (i) tumor growth, (g-i) ***, P < 0.0001 (ANOVA). Mean and s.d. of one out of two experiments (ten mice/group). (j) Survival of RMA-bearing mice treated with ZA and/or PC61 mAb. Arrow indicates the onset of ZA treatment. Statistical comparison (ten mice/group) was performed by the log-rank test (ZA, P < 0.01 ; PC61 , P < 0.004; ZA+PC61 , P= 0.0005; ZA vs ZA+PC61 , P < 0.05; PC61 vs ZA+PC61 , P < 0.05). Delay in tumor growth of R A-bearing mice treated with ZA and/or PC61 mAb.
Fig. 5 Dissection of the role of DC, CCR7 and LXRa, and characterization of infiltrating cells, (a) Growth of RMA-mock and RMA-SULT2B1 b in CD11c-DTR chimeras untreated or treated with diphteria toxin (DT). ***, P < 0.0001 (ANOVA). Mean and s.d. of one experiment (ten mice/group). (b,c) FACS analysis (b) and absolute numbers (c) showing CD11 c+FITC+ DC accumulating in the draining lymph nodes of mice undergoing skin painting. *, P < 0.05; **, P < 0.01 (ANOVA). Mean and s.d. of five experiments, (d) CCR7 expression on CD1 1c+ DC infiltrating RMA-mock and RMA-SULT2B1 b tumors, (e) Absolute numbers of CD11 c+FITC+ DC accumulating in the draining lymph node of Lxra " or wt chimeras undergoing skin painting. **, P < 0.005 (ANOVA); ns: not significant. Mean and s.d. of three experiments, (f) Growth of RMA-mock in Lxra^' or wt chimeras. ***, P < 0.0001 (ANOVA). Mean and s.d. of one out of two experiments (6-7 mice/group), (g) Histology (H&E; bars = 50 pm) and immunohistochemistry of RMA-mock and RMA- SULT2B1 b collected 3, 7 and 14 days after tumor inoculation. Immunohistochemistry for CD3+ (bars = 20 pm), CD11 c+ (bars = 20 pm) and CD11 b+ (bars = 50 pm) cells infiltrating RMA-mock or RMA-SULT2B1 b. RMA lymphoma cells of T origin display a faint expression of CD3 (bars = 20 pm). (h,i) Percentage of CD1 1c+ (h) and CD3+ cells (i) infiltrating RMA-Mock or RMA-SULT2B1 b. *, P < 0.05; **, P < 0.01 (Student's est). Mean and s.d. of three independent experiments.
Fig. 6 Single infusion intratumor of supernatants containing lentiviral vectors encoding SULT2B1 b delays RMA growth.
Fig. 7 Zaragozic Acid (ZA) strongly delays the growth of 7 days established Lewis Lung Carcinoma (LLC) and potentiates the antitumor activity of immunotherapy, (a) **, P < 0.003; ***, P < 0.0008 (/ test). Mean and s.d. of one out of three experiments (4-5 mice/group), (b) Mean tumor weight at sacrifice. The weight of tumors from untreated mice is higher than the weight of tumors collected from ZA-treated mice. ***, P < 0.0009 (/ test). Mean and s.d. of one experiment (4-5 mice/group), (c) Zaragozic Acid improves the antitumor activity of active immunotherapy. Each treatment was statistically significant as compared to untreated mice (LLC). At 17 days p values for LLC vs LLC +VAX, LLC vs LLC +ZA and LLC vs LLC +VAX +ZA were **, P < 0.003; **, P < 0.003; and P < 0.0003, respectively (ANOVA). Mean and s.d. of one out of two experiments (4-5 mice/group), (d) Mean tumor weight at sacrifice. The weight of tumors from untreated mice is higher than those from mice treated with ZA, VAX or ZA+VAX. ***, P < 0.0001 (Anova). Mean and s.d. of one experiment (4-5 mice/group).
Fig. 8 Injection of therapeutic doses of Zaragozic Acid is not associated with general and liver-specific side effects, (a) The weight of LLC-bearing mice left untreated or treated with ZA, VAX, or ZA+VAX did not differ significantly before and after treatments, (b) Blood levels of GOT and GPT enzymes (liver function) were not altered by the treatments (LLC, LLC +ZA, LLC +VAX, LLC +ZA +VAX). Fig. 9 Selective accumulation of CD1 1 bhighGR1h'9h cells in tumors releasing LXR ligands (RMA-Mock) as compared with tumors expressing the LXR inactivating enzyme sulfotransferase 2B1 b (RMA-SULT2B1 b). (a) One representative experiment out of 5 is shown, (b) Quantification of the results shown in (a), showing the percentage of the expression of CD45.1 +CD11bhi9hGR1hi9h cells and the number of cells/mg of tumor tissue.*, P < 0.05 (t test). Mean and s.d. of five experiments with 5 mice/group is shown. Fig. 10 Bone marrow-derived CD11b*GR1 * cells migrate in vitro towards the LXR ligand 22R-HC. This migration is independent of LXRs and is inhibited by Pertussis toxin (PTX). (a) Total bone marrow cells and CD11b+GR1+ cells isolated from the bone marrow of naive mice migrate to the LXR ligand 22R-HC, but not to the inactive isomer 22S-HC. One representative experiment out of three is shown, (b) CD11 b+GR1+ but not CD11b' cells isolated from the bone marrow of naive mice migrate to the LXR ligand 22R-HC. One representative experiment out of three is shown, (c) CD11b+GR1+ from LXRs KO mice migrate to 22R-HC, demonstrating that CD11b+GR1+ migration to LXR ligands is independent of LXR engagement. One representative experiment out of two is shown, (d) The treatment of CD11b*GR1* with PTX (100 or 500 ng) inhibits their migration to the LXR ligand 22R-HC, indicating that the receptor mediating LXR ligand migration is a G Protein Coupled Receptor (GPCR). As expected, PTX control does not affect migration. One representative experiment out of two is shown.
Fig. 11 Selective migration of the CD11 bhighGR1hi9h cells to the LXR ligand 22R-HC. The cells isolated by functional sorting were characterized by qRT-PCR chemokine receptors expression, by flow cytometry for the expression of lineage-specific cell surface markers and functionally. The CD11bhi9f,GR1high cells that migrate to LXR ligands have higher levels of mRNAs and proteins for CCR1, CXCR4 and CXCR2 chemokine receptors than non migrating cells. One representative experiment out of two is shown.
Fig. 12 The migration of bone marrow-derived CD11 b+GR1+ cells towards the LXR ligand 22R-HC is mediated by the CXCR2 chemokine receptor, (a) The pre-treatment of CD11b+GR1+ cells with the LXR ligand 22R-HC blocks their migration to 22R-HC and to the CXCR2 ligand CXCL5, but not to SDF1a and MIP-1a that are ligands of CXCR4 and CCR1 receptors, respectively. One representative experiment out of two is shown, (b) The pre-treatment of CD11 b+GR1 + cells with the CXCR2 ligand CXCL5 blocks their migration to both 22R-HC and CXCL5. One representative experiment out of two is shown, (c) The pre-treatment of CD1 1 b+GR1 + cells with the CXCR2 antagonist SB225002 inhibits their migration to both 22R-HC and CXCL5, indicating that the LXR ligand migration is mediated by the CXCR2 chemokine receptor. One representative experiment out of two is shown, (d) CD1 1 b+GR1 + cells isolated from the bone marrow of CXCRZ' mice do not migrate to both 22R-HC and CXCL5. One representative experiment out of two is shown. Fig. 13 Analysis of in vivo migration of bone marrow-derived CD1 1 b+GR1* cells towards tumor-derived or naturally occurring LXR ligands injected subcute into the dorsal flank of mice. CD1 1 bhi9hGR1 hi9h cells specifically accumulate in tumors releasing LXR ligands and in LXR ligands embedded matrigel. (a) Total bone marrow cells were injected in NOD-SCID mice bearing 14 days established RMA-Mock or RMA-SULT2B1 b mice. The quantification of the results was performed by evaluating the percentage of CD45.1 +CD1 1 bhi9hGR1 high cells within tumors (RMA-Mock or RMA- SULT2B1 b), and the number of cells/mg of tumor tissue.*, P < 0.05; ***, P < 0.0001 (f test). Mean and s.d. of three experiments with 5-7 mice/group is shown, (b) 22R-HC or 22S-HC embedded matrigel were injected subcute in the dorsal flank of mice. Seventy-two hours later, mice were sacrificed, matrigel removed and enzymatically digested. Disaggregated single cells were stained and analyzed by flow cytometry. The quantification of the results was performed by evaluating the percentage of CD45.2+CD11 bhi9hGR1hi9h cells within the matrigel and the number of cells/mg of matrigel. ***, P < 0.0001 (f test). Mean and s.d. of three experiments with 3 mice/group is shown.
Fig. 14 In vivo analysis of the pro-angiogenic ability of CD1 1 bhi9hGR1high cells migrating to LXR ligands. RMA tumor cells co-injected with LXR ligands migrating CD1 1 bhighGR1 hi9h cells display an higher percentage of endothelial CD31 +CD45" cells than RMA tumor cells co-injected with non migrating cells. **, P < 0.001 ; ***, P < 0.0001 (Anova). Mean and s.d. of two experiments with 4-5 mice/group is shown. Fig. 15 Migration of human CD14+ monocytes to LXR ligands. CD14* monocytes isolated from peripheral blood mononuclear cells of a healthy donor or from a melanoma patient, specifically migrate to the LXR ligand 22R-HC but not to 22S-HC. One experiment out of three is shown. Figure 16 Role of LXRa/LXRa ligands in antitumor immune responses. Schematic representation of the effects of tumor-released LXRa ligands on the generation of antitumor immune responses. Tumor cells release LXRa ligands that affect CCR7 expression on maturing DC. As a consequence, DC do not migrate to draining lymph node and fail to elicit antitumor CD8+ T cells.
Figure 17 Phenotypic and mRNA analysis of human DC treated with MSR3-CM. (a) DC co-cultured with either 3T3-CD40L or MSR3-CD40L up regulate CD80, CD86, CD54 and DR molecules and down regulate the chemokine receptor CCR5 at similar levels. One representative experiment out of three is shown, (b) DC co-cultured with MSR3-CD40L show a strong inhibition of CCR7 expression compared to 3T3-CD40L. Results from 5 experiments are shown, (c) CCR7 inhibition is independent of the activation stimulus used. DC co-cultured with MSR3 cells (black bars) in the presence of LPS (100 ng/ml), TNFa (20 ng/ml) or necrotic cells (106 cells) show a strong inhibition of CCR7. On the contrary, DC co-cultured with NIH-3T3 (grey bars) show a normal up regulation of CCR7. One representative experiment out of three is shown, (d) qPCR analysis showing CCR7 mRNA inhibition. Experiments performed in 2 representative donors are shown. DC were left immature, or treated with LPS (100 ng/ml) alone or in the presence of MSR3-CM. (e) DC co-cultured with 3T3- CD40L in the absence or presence of MSR3-CM release similar amounts of IL-12. One representative experiment out of three is shown, (f) DC co-cultured with MSR3- CD40L do not up regulate CXCR4. One representative experiment out of ten is shown.
Figure 18 Phenotypic and functional analysis of DC activated in the presence of CM from the tumors LOVO, RMA, CALU-1 , G43 and Det. (a) DC activated with 3T3- CD40L in the presence of tumor-CM analyzed by FACS for the expression of HLA- DR, CD40, CD80, CD86 and DC-SIGN. One representative experiment out of three is shown, (b) Supernatants from the same groups of DC collected and tested for the content of IL-6. One representative experiment out of three is shown, (c) DC activated in the presence of tumor-CM were co-cultured with allogenic PBMC labeled with 2 μΜ of CFSE. On days 5 and 7 each MLR was stained with an anti-CD3 antibody and analyzed by FACS. The obtained proliferation index is the ratio between the absolute number of CD3+CFSE+ T cells and unstimulated PBMC. One representative experiment out of two is shown.
Figure 19 Effect of natural and synthetic LXR ligands on DC. (a) Natural (22R-HC and 25-HC) and synthetic (T1317) LXR ligands inhibit CCR7 expression at nanomolar concentrations. Data are representative of three experiments (mean and s.d.). (b) 22R-HC, the inactive isomer 22S-HC and the synthetic ligand T1317 do not alter the expression of costimulatory molecules (CD40, CD80, CD83 and CD86) on DC co-cultured with 3T3-CD40L. The 25-HC partly affects the expression of these molecules. One representative experiment out of three is shown, (c) LXR ligands inhibit also the expression of CXCR4 receptor. ***, P < 0.0001 (ANOVA). Data are from three experiments (mean and s.d.). Figure 20 (a) HEK293 cells expressing the SULT2B1b partly abolish LXRa luciferase activity induced by natural ligands but not by the synthetic ligand T1317. HEK293 cells expressing SULT2B1b were selected and used to test LXRa activation by luciferase assay. Activation of LXRa by the addition of natural ligands 22R-HC and 24,25 Epoxycholesterol (24,25-EC) was significantly inhibited in HEK293 cells expressing SULT2B1b.As expected, the synthetic ligand T1317 was not affected by SULT2B1b activity. P < 0.0001 (Student's t-test); RLA, Relative Luciferase Activity. Data are representative of one out of seven experiments (mean and s.d. of experimental replicates), (b) shRNA specific for the human LXRa silences LXRa in human hepatoma HepG2. HepG2 were transduced with lentiviral vectors encoding shLXR selected with puromycin (1 pg/ml) and analyzed for LXRa expression by qPCR. As control, we used HepG2 cells transduced with lentiviral vectors encoding a non-targeting shRNA. Figure 21 (a) In vivo OT-I activation following injection of OVApep-loaded DC treated with the natural LXR ligand 22R-HC. B6 mice were adoptively transferred with 3x106 purified OT-I cells labeled with CFSE. Twenty-four hours later, DC treated for 48 hours with 22R-HC or left untreated, and then pulsed with the SIINFEKL peptide (H- 2Kb-OVA-specific epitope recognized by OT-I cells) were administered s.c. Three days later, spleen and lymph nodes of treated mice were collected and analyzed for OT-I proliferation, evaluated as CFSE dilution. One representative experiment showing the impairment of OT-I proliferation in mice injected with DC treated with 22R-HC. (b-d) In vitro OT-I activation following co-culture with OVApep-loaded DC treated with natural LXR ligands. Purified OT-I cells (1.5x105) were co-cultured with 0.5x105 DC previously treated for 48 hours with 22R-HC, 22S-HC, or left untreated and then pulsed with the SIINFEKL peptide (H-2Kb-OVA-specific epitope recognized by OT-I cells). Forty-eight hours later, supernatants were collected and tested for IFN-γ (c) and TNFa release (d). Four days later, OT-I were collected and counted by FACS (b). DC treated with 22S-HC partly inhibited OT-I proliferation and markedly reduced IFNy secretion. *, P < 0.05; **, P < 0.01 (ANOVA). Data are from at least three experiments (mean and s.d.). (e) Lxra mRNA is expressed by freshly isolated CD1 1c+ DC and it is up regulated after the treatment of mice with complete Freund's adjuvant. Freshly isolated CD1 1c+ and CD11 c" cells express Lxra mRNA. Lxra mRNA turns out to be up regulated 8 hours after CFA treatment of B6 mice. Purified CD3+ cells do not express Lxra transcripts. One representative experiment is shown.
Figure 22 Growth of ZA-treated and SULT2B1 b-expressing tumors in C57BL/6 and NOD-SCID mice, (a) ZA treatment (black circles) does not affect the growth of RMA in NOD-SCID as compared to vehicle treatment (black squares). Seventy-five micrograms of ZA was administered i.p. every 2 days. Arrow indicates the onset of ZA treatment. Results of one experiment with ten mice/group are shown, (b) RMA expressing the SULT2B1 b enzyme (SULT2B1 b, black triangles) grows similarly to RMA untransduced (wild type, black squares) or transduced with the control vector (mock, black circles). TrampCI (c) and LLC (d) expressing the SULT2B1 b enzyme (SULT2B1b, black triangles) grow similarly to tumors transduced with the control vector (mock, black circles). Results of one experiment with ten mice/group are shown, (e, f) SULT2B1 b does not modify the growth of the B16 melanoma in B6 (e) as well as in NOD-SCID (f) mice (SULT2B1 b, black triangles; mock, black circles). The insert (e) shows a qPCR analysis for SULT2B1 b mRNA expressed by the tumors. Data are representative of one out of two experiments (mean and s.d. of ten mice/group). Figure 23 (a, b) Inhibition of tumor growth is strictly dependent on the amount of SULT2B1 b expressed by tumors, (a) RMA expressing higher amounts of SULT2B1 b mRNA had a stronger delay of tumor growth. RMA-mock (black circles), RMA- SULT2B1 b #3.1 (black squares), RMA-SULT2B1 b #3.3 (black triangles) and RMA- SULT2B1 b #3.4 (black diamonds). Data are representative of one experiment (mean and s.d. of ten mice/group), (b) qPCR analysis for SULT2B1 b mRNA of the tumors described in Fig. 8a, showing the different levels of SULT2B1 b transcripts among various SULT2B1 b-transduced RMA tumors, (c) In RMA-bearing B6 mice (mean and s.d. of ten mice/group), the combined treatment with ZA and the PC61 mAb depleting T regulatory cells, significantly delayed tumor growth compared to the single treatments. Mice were treated with PC61 mAb (black squares and triangles; 500 pg) given i.p. 4 days before tumor inoculation. ZA (white squares and black triangles; 75 pg) was administered i.p. every 2 days; arrow indicates the onset of ZA treatment. Control mice were treated with vehicle (black circles). The three treatments significantly delayed tumor growth over controls from day 13 on (P < 0.0001 , at day 19). The combined treatment (i.e. ZA+PC61 ) was statistically significant over the single treatments from day 15 on (ZA vs ZA+PC61 P < 0.0004 and PC61 vs ZA+PC61 P < 0.0045, at day 21 ).
DETAILED DESCRIPTION OF THE INVENTION
Various preferred features and embodiments of the present invention will now be described by way of non-limiting example.
The present invention involves a combination of oxysterol synthesis inhibitors and CD25-depleting monoclonal antibodies, in one aspect. OXYSTEROL SYNTHESIS INHIBITORS
The oxysterol modulator used in the present invention blocks cholesterol synthesis downstream of mevalonic acid. Mevalonate is a precursor of, in particular, squalene, and can be made from acetate. An outline for the pathway for the synthesis of cholesterol from acetate is as follows: acetate C2→ mevalonate C6→ isopentenyl pyrophosphate C5→ squalene C30 → cholesterol C2j
Squalene is a C30 hydrocarbon and is an intermediate in the synthesis of cholesterol. Squalene consists of six isoprene units. Its formula is as follows:
Figure imgf000030_0001
where n = 2.
In particular, the present invention relates to the use of zaragozic acid. Zaragozic acid is a squalene synthase inhibitor.
Figure imgf000030_0002
As described above, derivatives of zaragozic acid are also useful in the present invention. In one embodiment the derivative of zaragozic acid is a monocarboxylic derivative. In one embodiment the derivative is a conjugate, a salt, an ester or an amide of zaragozic acid.
Some examples of zaragozic acid derivatives are:
Figure imgf000031_0001
wherein X is H, a halogen (F, CI, Br, 1), OH or CH3; Y is halogen (F, CI, Br, I), OH or CH3, and wherein Ζ , Z2 and Z3 are each independently H, C1-5 alkyi, C1-5 alkyi substituted with (i) phenyl, (ii) phenyl substituted with methyl, methoxy, halogen (F, CI, Br, I) or hydroxy, (iii) C1 -5 alkylcarbonyloxy, (iv) C6-10 arylcarbonyloxy, (v) C1-5 alkoxycarbonyloxy, (vi) C6-10 aryloxycarbonyloxy, (vii)
Figure imgf000032_0001
the groups (iii) to (vi) form a 5 to 10 membered mono- or bicyclic ring with C1-5 alkyl, or pharmaceutically acceptable salts thereof.
Further zaragozic acid derivatives include
Figure imgf000032_0002
wherein R is selected from:
Figure imgf000032_0003
selected from
Figure imgf000032_0004
and
Figure imgf000032_0005
; R3 is d-salkyl; Z is selected from (i) H, (ii) C1-5alkyl; (iii) Ci.5alkyl substituted with (a) C^alkylcarbonyloxy, (b) arylcarbonyloxy, (c) d.salkoxycarbonyloxy, (d) aryloxycarbonyloxy; (e)
Figure imgf000032_0006
aikyi , (f) t (g) 0r the groups (a) to (d) form a 5 to 10 membered mono or bicyclic ring with Chalky!, (iv) -e cycloalkyl; or a pharmaceutically acceptable salt thereof. Another example of a squalene synthase inhibitor is a probucol ester of the formula
Figure imgf000033_0001
wherein X, is H or P03H2 and X2 is H or P03H2; and wherein R1 and R2 are H- or - CH3; and R3, R4, R5 and R6 are independently selected from H- or an alkyl group selected from the group consisting of methyl, ethyl, propyl, butyl or tert-butyl; or, a pharmaceutically acceptable salt thereof.
Phosphonic acid derivatives are also useful in the present invention. An example of a phosphonic acid derivative is represented by the following general formula (I):
Figure imgf000033_0002
Ri represents a hydrogen atom, a hydroxyl group, an acyloxyalkyl group, an alkyloxycarbonyl group, a lower alkyl group which may have a substituent or a lower alkoxy group which may have a substituent; R2 and R3 may be the same or different from each other and each represents a hydrogen atom, a lower alkyl group which may have a substituent, an alkali metal or a prodrug ester forming group; RA represents a group represented by the formula: 0 (wherein R4 represents a hydrogen atom, a lower alkyl group, an alkali metal or an acyloxyalkyl
group which may have a substituent), a group represented by the formula:
Figure imgf000033_0003
(wherein R represents a hydrogen atom, a lower alkyl group or an alkali metal) or a
o
II
— P— OR5
group represented by the formula: R6 wherein R5 represents a hydrogen atom, a lower alkyl group, an alkali metal or a prodrug ester forming group; and R6 represents a lower alkyl group or a group represented by the formula: -OR7 (wherein R7 represents a hydrogen atom, a lower alkyl group, an alkali metal or a prodrug ester forming group)]; and
RB represents a group represented by the formula: S-T-- [wherein S represents an alkenyl group which may have a substituent or a group represented by the formula:
Figure imgf000034_0001
(wherein ring A represents an aromatic ring; R8, R9, R10, R11 and R12 may be the same or different from one another and each represents (1) a hydrogen atom, (2) an alkyl group which may have a substituent, (3) an alkenyl group which may have a substituent, (4) a lower alkoxy group which may have a substituent, (5) a carbamoyl group which may have a substituent, (6) a carbamoyloxy group which may have a substituent, (7) a hydroxyl group, (8) an acyl group, (9) a halogen atom, (10) a group represented by the following formula:
Figure imgf000034_0002
(wherein R13 and R14 may be the same or different from each other and each represents a lower alkyl group which may have a substituent, or alternatively R13 and R14 may form together with the nitrogen atom to which they are bonded, a ring which may further contain an oxygen atom, a sulfur atom or a nitrogen atom and which may have one or two, mono- or divalent substituent(s); p is 0 or 1 ; and q is an integer of 0 to 4) or (11) a group represented by the formula:
Figure imgf000034_0003
(wherein R15, R16, R17, R18 and R19 may be the same or different from one another and each represents a hydrogen atom, a hydroxyl group, a lower alkyl group or a lower alkoxy group which may have a substituent; ring B represents an aromatic ring; and Y represents an alkylene chain which may have a substituent, an alkenylidene chain which may have a substituent, an alkynylidene chain which may have a substituent, a group represented by the formula: 0 , a group represented by the formula: -O--, or a single bond), or alternatively two adjacent groups of R8, R9, R10, R11 and R12 may together form a ring; and X represents a single bond, an alkylene chain which may have a substituent, an alkenylidene chain which may have a substituent or a group represented by the formula: -(CH2)u -Z-(CH2)v - (wherein Z is a group represented by the formula:
Figure imgf000035_0001
(wherein r is an integer of 0 to 2), a group represented by the formula:
° a group represented by the formula: -0-, a group represented by the formula:
R20 S°2N (wherein R20 represents a hydrogen atom, a lower alkyl group which may have a substituent or a lower alkenyl group which may have a substituent), a
R21
I
group represented by the formula N (wherein R21 represents a hydrogen atom, a lower alkyl group which may have a substituent, a lower alkenyl group which may have a substituent or a group represented by the formula:
Figure imgf000035_0002
R22 o
group represented by the formula: J NJL (wherein R22 represents a hydrogen atom, a lower alkyl group which may have a substituent or a lower alkenyl group which may have a substituent); u is an integer of 0 to 3; and v is an integer of 0 to 6); and T represents (1) a single bond, (2) a group represented by the formula:
Figure imgf000035_0003
(wherein R23 represents a hydrogen atom, a cycloalkyl group, a cycloalkylalkyi group, a lower alkyl group which may have a substituent or a lower alkenyl group which may have a substituent; W represents a group represented by the formula: -0-, a group represented by the formula: ° , a group represented by
OH
J
the formula: -NH-, a group represented by the formula: CH— , a group
o
represented by the formula: ° or a single bond; and s and t are independent of each other and are each an integer of 0 to 4), (3) a group represented by the
R291?23
formula: OH - tw-(-s 2- (wherein R23, W, s and t are each as defined above; and R29 represents a hydrogen atom, a cycloalkyi group, a cycloalkylalkyl group, a lower alkyl group which may have a substituent or a lower alkenyl group which may have a substituent),
R25
_ l
(4) a group represented by the formula: N (wherein R25 represents a hydrogen atom, a cycloalkyi group, a lower alkyl group which may have a substituent or a lower alkenyl group which may have a substituent), or
(5) a group represented by the formula:
Figure imgf000036_0001
(wherein D represents a carbon atom or a nitrogen atom, E represents a nitrogen atom or a group represented by the formula: ^ , F represents a group represented by the formula: -0-, a group represented by the formula: 0 , a group represented by
OH
J
the formula: -NH-, a group represented by the formula: CH— , a group
o
represented by the formula: or a single bond; x and y are independent of each other and are each an integer of 0 to 3. Examples of other squalene synthase inhibitors that are useful in the present invention include lapaquistat (TAK-475), terbinafine, ER-27856 (5-{/V-[2-butenyl-3-(2- methoxyphenyl)]-A/-methylamino}-1 , 1 -penthylidenebis(phosphonic acid) tri-sodium salt), RPR-107393 (3-hydroxy-3-[4-(quinolin-6-yl)phenyl]-1 -azabicyclo [2-2-2]octane dihydrochloride) and YM-53601 ((£)-2-[2-fluoro-2-quinuclidin-3-ylidene ethoxy]-9H- carbazole monohydrochloride).
CD25-Depleting Monoclonal Antibodies In one aspect the invention involves CD25-depleting monoclonal antibodies. Examples of CD25-depleting monoclonal antibodies include Basiliximab, daclizumab (Zenapax), inolimomab (Leucotac), HuMax-TAC and PC61.
In another aspect, the present invention involves LXR ligand inactivators for treating cancer. In one embodiment, the LXR ligand inactivator is a sulfotransferase enzyme. In one embodiment, the sulfotransferase enzyme is SULT2B1 b.
In one embodiment, the LXR ligand inactivator is administered using gene therapy.
In another aspect the present invention involves an LXR inhibitor or antagonist for treating cancer.
An LXR antagonist includes any agent, which may include any compound, substance or molecule, capable of antagonising any function of an LXR receptor. An antagonist may thus antagonise (down-regulate, inhibit or suppress) any effect of LXR activation. An LXR antagonist may be an antagonist of LXRa or LXRp or both. In one embodiment the LXR antagonist is an LXRa inhibitor or antagonist.
The LXR antagonist may be a cholesterol oxide, an oxysterol or a sterol or derivative thereof.
In one embodiment the sterol is selected from a hydroxycholesterol and a sulfated oxysterol.
The cholesterol oxide may be functionalised. Examples of functionalised cholesterol oxides are 7p-hydroxycholesterol, a-epoxycholesterol, β-epoxycholesterol, 7-keto- cholesterol, cholestane triol, 7a-hydroxycholesterol, 25-hydroxycholesterol, 22(R)- hydroxy-cholesterol, 24(S)-hydroxy-cholesterol, 27-hydroxy-cholesterol.
Examples of sulfated oxysterols include 24-OHChol-3-sulfate and 24-OHChol-3, 24- sulfate.
Other examples of LXR inhibitors or antagonists that are useful in the present invention include a polyunsaturated fatty acid, a geranyl geraniol or geranylgeranyl pyrophosphate, 5a,6a-epoxycholesterol sulphate (ECHS), 7-ketocholesterol-3-
R1
sulphate, and a tricyclic compound represented
Figure imgf000038_0001
a pharmacologically acceptable salt thereof, or a hydrate of the compound or the salt in which R1 represents a hydrogen atom, a lower alkyl group, a lower halogenated alkyl group, an unsubstituted or substituted phenyl group, or an unsubstituted or substituted benzyl group; R2 represents a 1 ,1 ,1 ,3,3,3-hexafluoro-2-hydroxypropan-2- yl group or a carboxymethyl group; R3 represents a hydrogen atom, a lower alkyl group, a lower alkoxy group or a halogen atom; X represents a direct bond, an oxygen atom, a sulfur atom, a (CH2)n group or a (CH=CH)n group (wherein n represents an integer of 1-3); and Y represents CO or S02.
A further compound modulating LXR function that may be useful in the present invention is of the following formula (I) or a pharmacologically acceptable salt or ester thereof:
Figure imgf000039_0001
wherein:
A represents a C5-C14 aryl group or a 5- to 7-membered heteroaryl group;
R R2 and R^ are the same or different and each represents a hydrogen atom, a hydroxyl group, a nitro group, a cyano group, an amino group, a halogen atom, a carboxy group, a carbamoyl group, a mercapto group, a C|-C6 alkyl group, a C-|-Cg alkyl group substituted with from 1 to 7 halogen atoms, a C2-C7 alkylcarbonyloxy group, a C\-CQ alkoxy group, a C1-C5 alkylthio group, a C1-C5 alkylsulfinyl group, a
C-j-Cg alkylsulfonyl group, a C|-Cg alkylamino group, a di(C 1-C5 alkyl)amino group
(wherein the alkyl groups are the same or different), a C2-C7 alkylcarbonylamino group, an N-(C2-Cy alkylcarbonyl)-N-(C|-Cg alkyl)amino group, a C2-C7 alkoxycarbonylamino group, an N-(C2-Cy alkoxycarbonyl)-N-(C-|-Cg alkyl)amino group, a C1-C5 alkylsulfonylamino group, an N-(C|-Cg alkylsulfonyl)-N-(C|-Cg alkyl)amino group, a C|-Cg haloalkylsulfonylamino group (wherein said C|-Cg haloalkylsulfonylamino group is a C|-Cg a!kylsulfonylamino group which is substituted with from 1 to 7 halogen atoms), an N-(C-|-C5 haloalkylsulfonyl)-N-(C-| -C5 alkyl)amino group (wherein said C1-C5 haloalkylsulfonyl group is a C1-C5 alkylsulfonyl group which is substituted with from 1 to 7 halogen atoms), a C2-C7 alkylcarbonyl group, a C2-C7 alkoxycarbonyl group, a C2-C 7 alkylaminocarbonyl group or a di(C|- Cg alkyl) aminocarbonyl group (wherein the alkyl groups are the same or different), or R1 and R^ may be taken together to form a C1-C alkylenedioxy group;
R4 and R^ are the same or different and each represents a hydrogen atom, a hydroxyl group, an amino group, a halogen atom, a mercapto group, a C|-C6 alkyl group, a C\-CQ alkyl group substituted with from 1 to 7 halogen atoms, a C\-CQ alkoxy group, a C2-C7 alkoxycarbonyl group or a C\-CQ alkylthio group;
X represents a hydrogen atom, a hydroxyl group, a halogen atom, a C|-Cg alkoxy group or a C|-CQ alkoxy group substituted with from 1 to 7 halogen atoms;
Y represents a C|-Cg alkyl group, a C1-C5 alkyl group substituted with from 1 to 7 substituents (said substituents are the same or different and are selected from substituent group a defined below), a C3-C10 cycloalkyl group, a C3 -C-| Q cycloalkyl group substituted with from 1 to 7 substituents (said substituents are the same or different and are selected from substituent group a as defined below), a 5- to 9- membered heterocyclyl group, a 5- to 9- membered heterocyclyl group substituted with from 1 to 7 substituents (said substituents are the same or different and are selected from substituent group a defined below), a Cg-C-i o ary' group, a Οβ-Ο-ιο aryl group substituted with from 1 to 4 substituents (said substituents are the same or different and are selected from substituent group β defined below), a C4-C14 cycloalkylalkyl group, a C4-C-14 cycloalkylalkyl group substituted with from 1 to 7 substituents (said substituents are the same or different and are selected from substituent group a defined below), a (5- to 9-membered heterocyclyl)-(C|-C4 alkyl) group, a (5- to 9- membered heterocyclyl)-(C-| -C4 alkyl) group substituted with from 1 to 7 substituents (said substituents are the same or different and are selected from substituent group a defined below), a C7-C14 aralkyl group or a C7-C14 aralkyl group substituted with from 1 to 5 substituents (said substituents are the same or different and are selected from substituent group β defined below);
substituent group a represents a group consisting of a halogen atom, a hydroxyl group, a cyano group, an amino group, a C2-C7 alkylcarbonyloxy group, a C1-C5 alkyl group, a C|-C6 alkoxy group, a C|-C alkylthio group, a C1-C5 alkylsulfinyl group, a C^ -CQ alkylsulfonyl group, a phenyl group, a C|-Cg alkylamino group, a di(C|-C6 alkyl)amino group (wherein the alkyl groups are the same or different), a C2- C7 alkylcarbonylamino group, a C|-C6 alkylsulfonylamino group, and a C|-C6 haloalkylsulfonylamino group (wherein said C\-CQ haloalkylsulfonylamino group is a C1-C5 alkylsulfonylamino group which is substituted with from 1 to 7 halogen atoms); and substituent group β represents a group consisting of a halogen atom, a hydroxyl group, a nitro group, a cyano group, an amino group, a C\-CQ alkyl group, a C\-CQ alkyl group substituted with from 1 to 7 halogen atoms, a C2-C7 alkylcarbonyloxy group, a C|-C6 alkoxy group, a C|-C6 alkylthio group, a C1-C5 alkylsulfinyl group, a C1-C5 alkylsulfonyl group, a C1-C5 alkylamino group, a di(C|-C6 alkyl) amino group (wherein the alkyl groups are the same or different), a C2-C7 alkylcarbonylamino group, an N-(C2-C7 alkylcarbonyl)-N-(C-|-C6 alkyl)amino group, a C2-C7 a!koxycarbonylamino group, an -(C2-C7 alkoxycarbonyl)-N-(C|-C5 alkyl)amino group, a C1-C5 alkylsulfonylamino group, an N-(C-|-C0 alkylsulfonyl)-N-(C|-C5 alkyl)amino group, a CJ-CQ haloalkylsulfonylamino group (wherein said haloalkylsulfonylamino group is a C|-Ce alkylsulfonylamino group which is substituted with from 1 to 7 halogen atoms), an N-(C-|-Cg haloalkylsulfonyl)-N-(C|-C6 alkyl)amino group (wherein said haloalkylsulfonyl group is a C1-C5 alkylsulfonyl group which is substituted with from 1 to 7 halogen atoms), a C5-C10 3|7' group, a C7-C14 aralkyloxy group, C1 -C4 alkylenedioxy group, a C2-C7 alkylcarbonyl group, a C 2-C7 alkoxycarbonyl group, a C2-C7 alkylaminocarbonyl group, and a d' Cj-Cg alkyl) aminocarbonyl group (wherein the alkyl groups are the same or different).
Pharmaceutical Compositions
A further aspect of the invention relates to a pharmaceutical composition comprising a compound of the invention admixed with one or more pharmaceutically acceptable diluents, excipients or carriers. Other active materials may also be present, as may be considered appropriate or advisable for the disease or condition being treated or prevented.
Even though the compounds of the present invention (including their pharmaceutically acceptable salts, esters and pharmaceutically acceptable solvates) can be administered alone, they will generally be administered in admixture with a pharmaceutical carrier, excipient or diluent, particularly for human therapy. The pharmaceutical compositions may be for human or animal usage in human and veterinary medicine.
Examples of such suitable excipients for the various different forms of pharmaceutical compositions described herein may be found in the "Handbook of Pharmaceutical Excipients, 2nd Edition, (1994), Edited by A Wade and PJ vVeller. The carrier, or, if more than one be present, each of the carriers, must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient.
Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).
Examples of suitable carriers include lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and the like. Examples of suitable diluents include ethanol, glycerol and water.
The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as, or in addition to, the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).
Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose and polyethylene glycol.
Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like.
Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used.
According to a further aspect of the invention, there is provided a process for the preparation of a pharmaceutical or veterinary composition as described above, the process comprising bringing the active compound(s) into association with the carrier, for example by admixture.
In general, the formulations are prepared by uniformly and intimately bringing into association the active agent with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product. The invention extends to methods for preparing a pharmaceutical composition comprising bringing a compound of general formula (I) in conjunction or association with a pharmaceutically or veterinarily acceptable carrier or vehicle.
Salts/Esters
The compounds of the invention can be present as salts or esters, in particular pharmaceutically and veterinarily acceptable salts or esters.
Pharmaceutically acceptable salts of the compounds of the invention include suitable acid addition or base salts thereof. A review of suitable pharmaceutical salts may be found in Berge et al, J Pharm Sci, 66, 1-19 (1977). Salts are formed, for example with strong inorganic acids such as mineral acids, e.g. hydrohalic acids such as hydrochloride, hydrobromide and hydroiodide, sulphuric acid, phosphoric acid sulphate, bisulphate, hemisulphate, thiocyanate, persulphate and sulphonic acids; with strong organic carboxylic acids, such as alkanecarboxylic acids of 1 to 4 carbon atoms which are unsubstituted or substituted (e.g., by halogen), such as acetic acid; with saturated or unsaturated dicarboxylic acids, for example oxalic, malonic, succinic, maleic, fumaric, phthalic or tetraphthalic; with hydroxycarboxylic acids, for example ascorbic, glycolic, lactic, malic, tartaric or citric acid; with aminoacids, for example aspartic or glutamic acid; with benzoic acid; or with organic sulfonic acids, such as (C C4)-alkyl- or aryl-sulfonic acids which are unsubstituted or substituted (for example, by a halogen) such as methane- or p-toluene sulfonic acid. Salts which are not pharmaceutically or veterinarily acceptable may still be valuable as intermediates.
Preferred salts include, for example, acetate, trifluoroacetate, lactate, gluconate, citrate, tartrate, maleate, malate, pantothenate, adipate, alginate, aspartate, benzoate, butyrate, digluconate, cyclopentanate, glucoheptanate, glycerophosphate, oxalate, heptanoate, hexanoate, fumarate, nicotinate, palmoate, pectinate, 3- phenylpropionate, picrate, pivalate, proprionate, tartrate, lactobionate, pivolate, camphorate, undecanoate and succinate, organic sulphonic acids such as methanesulphonate, ethanesulphonate, 2-hydroxyethane sulphonate, camphorsulphonate, 2-naphthalenesulphonate, benzenesulphonate, p- chlorobenzenesulphonate and p-toluenesulphonate; and inorganic acids such as hydrochloride, hydrobromide, hydroiodide, sulphate, bisulphate, hemisulphate, thiocyanate, persulphate, phosphoric and sulphonic acids.
Esters are formed either using organic acids or alcohols/hydroxides, depending on the functional group being esterified. Organic acids include carboxylic acids, such as alkanecarboxylic acids of 1 to 12 carbon atoms which are unsubstituted or substituted (e.g., by halogen), such as acetic acid; with saturated or unsaturated dicarboxylic acid, for example oxalic, malonic, succinic, maleic, fumaric, phthalic or tetraphthalic; with hydroxycarboxylic acids, for example ascorbic, glycolic, lactic, malic, tartaric or citric acid; with aminoacids, for example aspartic or glutamic acid; with benzoic acid; or with organic sulfonic acids, such as (CrC -alkyl- or aryl- sulfonic acids which are unsubstituted or substituted (for example, by a halogen) such as methane- or p-toluene sulfonic acid. Suitable hydroxides include inorganic hydroxides, such as sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminium hydroxide. Alcohols include alkanealcoho!s of 1-12 carbon atoms which may be unsubstituted or substituted, e.g. by a halogen).
Enantiomers/Tautomers
In all aspects of the present invention previously discussed, the invention includes, where appropriate all enantiomers, diastereoisomers and tautomers of the compounds of the invention. The person skilled in the art will recognise compounds that possess optical properties (one or more chiral carbon atoms) or tautomeric characteristics. The corresponding enantiomers and/or tautomers may be isolated/prepared by methods known in the art.
Enantiomers are characterised by the absolute configuration of their chiral centres and described by the R- and S-sequencing rules of Cahn, Ingold and Prelog. Such conventions are well known in the art (e.g. see 'Advanced Organic Chemistry', 3rd edition, ed. March, J., John Wiley and Sons, New York, 1985).
Compounds of the invention containing a chiral centre may be used as a racemic mixture, an enantiomerically enriched mixture, or the racemic mixture may be separated using well-known techniques and an individual enantiomer may be used alone. Stereo and Geometric Isomers
Some of the compounds of the invention may exist as stereoisomers and/or geometric isomers - e.g. they may possess one or more asymmetric and/or geometric centres and so may exist in two or more stereoisomeric and/or geometric forms. The present invention contemplates the use of all the individual stereoisomers and geometric isomers of those inhibitor agents, and mixtures thereof. The terms used in the claims encompass these forms, provided said forms retain the appropriate functional activity (though not necessarily to the same degree).
The present invention also includes all suitable isotopic variations of the agent or a pharmaceutically acceptable salt thereof. An isotopic variation of an agent of the present invention or a pharmaceutically acceptable salt thereof is defined as one in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually found in nature. Examples of isotopes that can be incorporated into the agent and pharmaceutically acceptable salts thereof include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulphur, fluorine and chlorine such as 2H, 3H, 13C, 14C, 15N, 170, 180, 3 P, 32P, 35S, 18F and 36CI, respectively. Certain isotopic variations of the agent and pharmaceutically acceptable salts thereof, for example, those in which a radioactive isotope such as 3H or 14C is incorporated, are useful in drug and/or substrate tissue distribution studies. Tritiated, i.e., 3H, and carbon-14, i.e., 4C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with isotopes such as deuterium, i.e., 2H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements and hence may be preferred in some circumstances. For example, the invention includes compounds of general formula (I) where any hydrogen atom has been replaced by a deuterium atom. Isotopic variations of the agent of the present invention and pharmaceutically acceptable salts thereof of this invention can generally be prepared by conventional procedures using appropriate isotopic variations of suitable reagents.
Prodrugs
The invention further includes the compounds of the present invention in prodrug form, i.e. covalently bonded compounds which release the active parent drug according to general formula (I) in vivo. Such prodrugs are generally compounds of the invention wherein one or more appropriate groups have been modified such that the modification may be reversed upon administration to a human or mammalian subject. Reversion is usually performed by an enzyme naturally present in such subject, though it is possible for a second agent to be administered together with such a prodrug in order to perform the reversion in vivo. Examples of such modifications include ester (for example, any of those described above), wherein the reversion may be carried out by an esterase etc. Other such systems will be well known to those skilled in the art.
Solvates
The present invention also includes solvate forms of the compounds of the present invention. The terms used in the claims encompass these forms.
Polymorphs
The invention further relates to the compounds of the present invention in their various crystalline forms, polymorphic forms and (an)hydrous forms. It is well established within the pharmaceutical industry that chemical compounds may be isolated in any of such forms by slightly varying the method of purification and or isolation from the solvents used in the synthetic preparation of such compounds.
Administration
The pharmaceutical compositions of the present invention may be adapted for rectal, nasal, intrabronchial, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intraarterial and intradermal), intraperitoneal or intrathecal administration. Preferably the formulation is an orally administered formulation. The formulations may conveniently be presented in unit dosage form, i.e., in the form of discrete portions containing a unit dose, or a multiple or sub-unit of a unit dose. By way of example, the fonmulations may be in the fonn of tablets and sustained release capsules, and may be prepared by any method well known in the art of pharmacy.
Formulations for oral administration in the present invention may be presented as: discrete units such as capsules, gellules, drops, cachets, pills or tablets each containing a predetermined amount of the active agent; as a powder or granules; as a solution, emulsion or a suspension of the active agent in an aqueous liquid or a non-aqueous liquid, or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion; or as a bolus etc. Preferably, these compositions contain from 1 to 250 mg and more preferably from 10-100 mg, of active ingredient per dose.
For compositions for oral administration (e.g. tablets and capsules), the term "acceptable carrier" includes vehicles such as common excipients e.g. binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone (Povidone), methylcellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, sucrose and starch; fillers and carriers, for example corn starch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride and alginic acid; and lubricants such as magnesium stearate, sodium stearate and other metallic stearates, glycerol stearate stearic acid, silicone fluid, talc waxes, oils and colloidal silica. Flavouring agents such as peppermint, oil of wintergreen, cherry flavouring and the like can also be used. It may be desirable to add a colouring agent to make the dosage form readily identifiable. Tablets may also be coated by methods well known in the art.
A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active agent in a free flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface- active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may be optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active agent.
Other formulations suitable for oral administration include lozenges comprising the active agent in a flavoured base, usually sucrose and acacia or tragacanth; pastilles comprising the active agent in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active agent in a suitable liquid carrier.
Other forms of administration comprise solutions or emulsions which may be injected intravenously, intraarterially, intrathecally, subcutaneously, intradermally, intraperitoneally or intramuscularly, and which are prepared from sterile or sterilisable solutions. Injectable forms typically contain between 10 - 1000 mg, preferably between 10 - 250 mg, of active ingredient per dose.
The pharmaceutical compositions of the present invention may also be in form of suppositories, pessaries, suspensions, emulsions, lotions, ointments, creams, gels, sprays, solutions or dusting powders.
An alternative means of transdermal administration is by use of a skin patch. For example, the active ingredient can be incorporated into a cream consisting of an aqueous emulsion of polyethylene glycols or liquid paraffin. The active ingredient can also be incorporated, at a concentration of between 1 and 10% by weight, into an ointment consisting of a white wax or white soft paraffin base together with such stabilisers and preservatives as may be required. Dosage
A person of ordinary skill in the art can easily determine an appropriate dose of one of the instant compositions to administer to a subject without undue experimentation. Typically, a physician will determine the actual dosage which will be most suitable for an individual patient and it will depend on a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy. The dosages disclosed herein are exemplary of the average case. There can of course be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention. No unacceptable toxicological effects are expected when compounds of the present invention are administered in accordance with the present invention. The compounds of this invention, which may have good bioavailability, may be tested in one of several biological assays to determine the concentration of a compound which is required to have a given pharmacological effect.
Combinations In a particularly preferred embodiment, the one or more compounds or combinations of the invention are administered in combination with one or more other active agents, for example, existing drugs available on the market. In such cases, the compounds or combinations of the invention may be administered consecutively, simultaneously or sequentially with the one or more other active agents. In one embodiment, a pharmaceutical composition comprising an oxysterol modulator and a chemotherapeutic agent is administered.
Drugs in general are more effective when used in combination. In particular, combination therapy is desirable in order to avoid an overlap of major toxicities, mechanism of action and resistance mechanism(s). Furthermore, it is also desirable to administer most drugs at their maximum tolerated doses with minimum time intervals between such doses. The major advantages of combining chemotherapeutic drugs are that it may promote additive or possible synergistic effects through biochemical interactions and also may decrease the emergence of resistance.
Beneficial combinations may be suggested by studying the inhibitory activity of the test compounds with agents known or suspected of being valuable in the treatment of a particular disorder. This procedure can also be used to determine the order of administration of the agents, i.e. before, simultaneously, or after delivery. Such scheduling may be a feature of all the active agents identified herein. Thus, one aspect of the present invention further comprises administering another active pharmaceutical ingredient, such as a chemotherapeutic agent, either in combined dosage form with the compound of the present invention or in a separate dosage form. Such separate chemotherapeutic agent dosage forms may include solid oral, oral solution, syrup, elixir, injectable, transdermal, transmucosal, or other dosage form. The compound and the other active pharmaceutical ingredient can be combined in one dosage form or supplied in separate dosage forms that are usable together or sequentially.
Examples of chemotherapeutic agents which may be used in the present invention include, but are not limited to, cytotoxic antibiotics such as aclarubicin, bleomycin, dactinomycin, daunorubicin, doxorubicin, epirubicin, idarubicin, mitomycin, and mitoxantrone (mitozantrone); alkylating agents such as busulfan, carmustine, chlorambucil, chlormethine hydrochloride, mustine hydrochloride, cyclophosphamide, estramustine phosphate, ifosfamide, lomustine, melphalan, thiotepa, and treosulfan; antimetabolites such as capecitabine, cladribine, cytarabine, fludarabine, fluorouracil, gemcitabine, mercaptopurine, methotrexate, raltitrexed, tegafur, and thioguanine; vinca alkaloids, such as etoposide, vinblastine, vincristine, vindesine, and vinorelbine; other antineoplastic drugs such as amsacrine, altretamine, crisantaspase, dacarbazine, temozolomide, hydroxycarbamide, hydroxyurea, and pentostatin; platinum compounds such as carboplatin, cisplatin, and oxaliplatin; porfimer sodium; procarbazine; razoxane; taxanes such as docetaxel and paclitaxel; topoisomerase I inhibitors such as irinotecan and topotecan; trastuzumab; tretinoin. Immunotherapy
In a particularly preferred embodiment, the one or more compounds or combinations of the invention are administered in combination with immunotherapy. In a preferred embodiment, an oxysterol modulator is administered in combination with immunotherapy. The administration may comprise an active immunotherapy and vaccination strategy.
Immunotherapy is the treatment of disease by inducing, enhancing, or suppressing an immune response. Immunotherapy for cancer can be considered to be biological therapy, or the application of biologic-response modifiers. These biologic-response modifiers (BRMs) can act through one or more mechanisms, such as (i) stimulating the host's antitumor response by increasing the number of effector cells or by producing one or more soluble mediators (eg lymphokines); (ii) decreasing host- suppressor mechanisms; and (iii) altering tumor cells to increase their
immunogenicity or make them more susceptible to damage by immunologic processes. BRMs may have both immunologic and nonimmunologic effects. In particular, cell based immunotherapies using immune effector cells including lymphocytes, macrophages, dendritic cells, natural killer cells, and cytotoxic lymphocytes can be used.
Passive cellular immunotherapy is when activated, specific effector cells are directly infused into a patient and are not induced or expanded within the patient. Examples of passive cellular immunotherapy include reinfusion of a patient's lymphocytes after expansion in vitro by exposure to IL-2 (T-cell growth factor). These cells are termed lymphokine-activated killer cells (LAK cells) and may first be exposed to
phytohemagglutinin, a lymphocyte mitogen. An alternative to infusion of IL-2 after LAK cell infusion is to isolate and expand populations of lymphocytes that have infiltrated tumors in vivo and thus may have tumor specificity, which may allow lower levels of IL-2 to be used. TILs can also be genetically modified to express tumoricidal molecules. Another example of passive cellular immunotherapy is the concurrent use of interferons with infused effector cells. Passive Humoral Immunotherapy relates to the use of antitumor antibodies.
Examples include the use of antilymphocyte serum, and conjugation of monoclonal antitumor antibodies with toxins or with radioisotopes so that the antibodies will deliver these toxic agents specifically to the tumor cells. A further example is the use of bispecific antibodies, which link one antibody reacting with the tumor cell to a second antibody reacting with a cytotoxic effector cell, so that the cytotoxic effector cell is targeted more specifically to the tumor.
Active specific immunotherapy induces therapeutic cellular immunity in the tumor- bearing host. Intact tumor cells, defined tumor antigens, or general
immunostimulants are used. An example is autochthonous tumor cells (taken from the host), which can be used, for example, after irradiation, neuraminidase treatment, hapten conjugation, or hybridization with long-term cell lines in vitro. Also, tumor cells genetically modified to produce immunostimulatory molecules (including cytokines such as granulocyte-macrophage colony-stimulating factor or IL-2, costimulatory molecules such as B7-1 , and allogeneic class I HC molecules) can be used. Allogenic tumor cells can also be used. A further example of active specific immunotherapy is the use of defined tumor antigen-based vaccines.
Antigen-specific immunity can also be induced with recombinant viruses (eg adenovirus, vaccinia virus) which express tumor associated antigens such as carcinoembryonic antigen. Nonspecific Immunotherapy includes use of interferons (IFNs) derived from white blood cells (IFN-a or IFN-γ) or from fibroblasts (IFN-β) or synthesized in bacteria by recombinant genetic techniques. Examples of bacterial adjuvants which can be used in immunotherapy include attenuated tubercle bacilli (BCG)) and extracts of BCG. These can be used with or without added tumor antigen.
Immunotherapy may be combined with other treatments such as chemotherapy or radiotherapy. Introduction of polypeptides and nucleic acid sequences into cells
Where the invention makes use of polypeptide substances such as SULT2B1b, they may be administered as the polypeptide itself or by introducing nucleic acid constructs/viral vectors encoding the polypeptide into cells under conditions that allow for expression of the polypeptide in a cell of interest.
Preferably, a polynucleotide for use in the invention in a vector is operably linked to a control sequence that is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector. The term "operably linked" means that the components described are in a relationship permitting them to function in their intended manner. A regulatory sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences. The control sequences may be modified, for example by the addition of further transcriptional regulatory elements to make the level of transcription directed by the control sequences more responsive to transcriptional modulators. Vectors of the invention may be transformed or transfected into a cell as described below to provide for expression of a polypeptide.
The present invention also encompasses cells into which the polypeptides, are introduced.
Convenient non-limiting methods for introducing both genes and polypeptides into cells are discussed below.
Any suitable method of transforming the cell may be used. Non-limiting examples of currently available mechanisms for delivery are via electroporation, calcium phosphate transformation or particle bombardment. However, transfer of the construct may be performed by any of the methods mentioned which physically or chemically permeabilize the cell membrane. Suitable methods are described in more detail below. 1. Electroporation
In certain preferred embodiments of the present invention, the antigen is introduced into the cells via electroporation. Electroporation involves the exposure of a suspension of cells and DNA to a high-voltage electric discharge.
It is contemplated that electroporation conditions for cells may be optimized. One may particularly with to optimize such parameters as the voltage, the capacitance, the time and the electroporation media composition. The execution of other routine adjustments will be known to those of skill in the art.
2. Particle Bombardment One method for transferring a naked DNA construct into cells involves particle bombardment. This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them. The microprojectiles used have consisted of biologically inert substances such as tungsten, platinum or gold beads.
It is contemplated that in some instances DNA precipitation onto metal particles would not be necessary for DNA delivery to a recipient cell using particle bombardment. It is contemplated that particles may contain DNA rather than be coated with DNA. Hence it is proposed that DNA-coated particles may increase the level of DNA delivery via particle bombardment but are not, in and of themselves, necessary.
Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force. Another method involves the use of a Biolistic Particle Delivery System, which can be used to propel particles coated with DNA through a screen, such as stainless steel or Nytex screen, onto a filter surface covered with cells in suspension. The screen disperses the particles so that they are not delivered to the recipient cells in large aggregates. It is believed that a screen intervening between the projectile apparatus and the cells to be bombarded reduces the size of projectile aggregates and may contribute to a higher frequency of transformation by reducing the damage inflicted on the recipient cells by projectiles that are too large.
For the bombardment, cells in suspension are preferably concentrated on filters, or alternatively on solid culture medium. The cells to be bombarded are positioned at an appropriate distance below the macroprojectile stopping plate. If desired, one or more screens are also positioned between the acceleration device and the cells to be bombarded.
It is contemplated that one may wish to adjust various of the bombardment parameters in small scale studies to fully optimize the conditions. One may particularly wish to adjust physical parameters such as gap distance, flight distance, tissue distance and helium pressure. One may also optimize the trauma reduction factors by modifying conditions which influence the physiological state of the recipient cells and which may therefore influence transformation and integration efficiencies. For example, the osmotic state, tissue hydration and the subculture stage or cell cycle of the recipient cells may be adjusted for optimum transformation. The execution of other routine adjustments will be known to those of skill in the art.
3. Viral Transformation
a. Adenoviral Infection
One method for delivery of the nucleic acid constructs involves the use of an adenovirus expression vector. Although adenovirus vectors are known to have a low capacity for integration into genomic DNA, this feature is counterbalanced by the high efficiency of gene transfer afforded by these vectors. "Adenovirus expression vector" is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to ultimately express a construct that has been cloned therein.
The vector comprises a genetically engineered form of adenovirus. Knowledge of the genetic organization or adenovirus, a 36 kb, linear, double-stranded DNA virus, allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kb. In contrast to retrovirus, the adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity. Also, adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification.
Adenovirus is particularly suitable for use as a gene transfer vector because of its mid-sized genome, ease of manipulation, high titer, wide target-cell range and high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging. The early (E) and late (L) regions of the genome contain different transcription units that are divided by the onset of viral DNA replication. The E1 region (E1A and E1B) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes. The expression of the E2 region (E2A and E2B) results in the synthesis of the proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression and host cell shut-off. The products of the late genes, including the majority of the viral capsid proteins, are expressed only after significant processing of a single primary transcript issued by the major late promoter (MLP). The MLP, (located at 16.8 m.u.) is particularly efficient during the late phase of infection, and all the mRNA's issued from this promoter possess a 5'-tripartite leader (TPL) sequence which makes them preferred mRNA's for translation. b. AAV Infection Adeno-associated virus (AAV) is an attractive vector system for use in the present invention as it has a high frequency of integration and it can infect nondividing cells, thus making it useful for delivery of genes into mammalian cells in tissue culture. AAV has a broad host range for infectivity. Details concerning the generation and use of rAAV vectors are described in U.S. Pat. No. 5,139,941 and U.S. Pat. No. 4,797,368, each incorporated herein by reference. Recombinant AAV vectors have been used successfully for in vitro and in vivo transduction of marker genes and genes involved in human diseases.
c. Retroviral and lentiviral vectors
In one embodiment the present invention involves the use of lentiviral vectors.
A large number of different retroviruses have been identified. Examples include: murine leukemia virus (MLV), human T-cell leukemia virus (HTLV), mouse mammary tumour virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A- MLV), Avian myelocytomatosis virus-29 (MC29), and Avian erythroblastosis virus (AEV). A detailed list of retroviruses may be found in Coffin et al. (1997) "Retroviruses", Cold Spring Harbour Laboratory Press Eds: JM Coffin, SM Hughes, HE Varmus pp 758-763.
Retroviruses are RNA viruses that replicate through an integrated DNA intermediate. Retroviral particles encapsidate two copies of the full-length viral RNA, each copy containing the complete genetic information needed for virus replication. Retroviruses possess a lipid envelope and use interactions between the virally encoded envelope protein that is embedded in the membrane and a cellular receptor to enter the host cells. Using the virally encoded enzyme reverse transcriptase, which is present in the virion, viral RNA is reverse transcribed into a DNA copy. This DNA copy is integrated into the host genome by integrase, another virally encoded enzyme. The integrated viral DNA is referred to as a provirus and becomes a permanent part of the host genome. The cellular transcriptional and translational machinery carries out expression of the viral genes. The host RNA polymerase II transcribes the provirus to generate RNA, and other cellular processes modify and transport the RNA out of the nucleus. A fraction of viral RNAs are spliced to allow expression of some genes whereas other viral RNAs remain full-length. The host translational machinery synthesizes and modifies the viral proteins. The newly synthesized viral proteins and the newly synthesized full-length viral RNAs are assembled together to form new viruses that bud out of the host cells.
Retroviruses may be broadly divided into two categories: namely, "simple" and "complex". Retroviruses may even be further divided into seven groups. Five of these groups represent retroviruses with oncogenic potential. The remaining two groups are the lentiviruses and the spumaviruses. A review of these retroviruses is presented in Coffin et al (1997) ibid. The basic structure of retrovirus and lentivirus genomes share many common features such as a 5' LTR and a 3' LTR, between or within which are located a packaging signal to enable the genome to be packaged, a primer binding site, integration sites to enable integration into a host cell genome and gag, pol and env genes encoding the packaging components - these are polypeptides required for the assembly of viral particles. Lentiviruses have additional features, such as rev and RRE sequences in HIV, which enable the efficient export of RNA transcripts of the integrated provirus from the nucleus to the cytoplasm of an infected target cell.
In the provirus, these genes are flanked at both ends by regions called long terminal repeats (LTRs). The LTRs are responsible for proviral integration, and transcription. LTRs also serve as enhancer-promoter sequences and can control the expression of the viral genes. The LTRs themselves are identical sequences that can be divided into three elements, which are called U3, R and U5. U3 is derived from the sequence unique to the 3' end of the RNA. R is derived from a sequence repeated at both ends of the RNA and U5 is derived from the sequence unique to the 5' end of the RNA. The sizes of the three elements can vary considerably among different retroviruses.
In a defective retroviral vector genome gag, pol and env may be absent or not functional. The R regions at both ends of the RNA are repeated sequences. U5 and U3 represent unique sequences at the 5' and 3' ends of the RNA genome respectively.
In a typical retroviral vector of the present invention, at least part of one or more protein coding regions essential for replication may be removed from the virus. This makes the viral vector replication-defective.
Viral vectors for use in the invention may include but are not limited to integration defective retroviral vectors. Such a vector can be produced, for example, by packaging the vector with catalytically inactive integrase (such as an HIV integrase bearing the D64V mutation in the catalytic site; Naldini et al., Science 1996, and PNAS USA 1996, Leavitt et al. J Virol. 1996) or by deleting essential att sequences from the vector LTR (Nigthingale et al. Mol Ther 2006), or by a combination of the above. These modifications reduce integration to baseline level leaving unaffected the other steps of the transduction process (Naldini et al. Science 1996, Nigthingale et al. Mol Ther 2006, Vargas et al. Hum Gene Ther 2004, Yafiez-Munoz et al. Nat Med 2006; Philippe et al. PNAS 2006). Lentivirus vectors are part of a larger group of retroviral vectors. A detailed list of lentiviruses may be found in Coffin et al (1997) "Retroviruses" Cold Spring Harbour Laboratory Press Eds: J Coffin, SM Hughes, HE Varmus pp 758-763). In brief, lentiviruses can be divided into primate and non-primate groups. Examples of primate lentiviruses include but are not limited to: the human immunodeficiency virus (HIV), the causative agent of human auto-immunodeficiency syndrome (AIDS), and the simian immunodeficiency virus (SIV). The non-primate lentiviral group includes the prototype "slow virus" visna/maedi virus (VMV), as well as the related caprine arthritis-encephalitis virus (CAEV), equine infectious anaemia virus (EIAV) and the more recently described feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV).
The lentivirus family differs from retroviruses in that lentiviruses have the capability to infect both dividing and non-dividing cells.
A lentiviral vector, as used herein, is a vector which comprises at least one component part derivable from a lentivirus. Preferably, that component part is involved in the biological mechanisms by which the vector infects cells, expresses genes or is replicated.
The lentiviral vector may be a "non-primate" vector, i.e., derived from a virus which does not primarily infect primates, especially humans.
The examples of non-primate lentivirus may be any member of the family of lentiviridae which does not naturally infect a primate and may include a feline immunodeficiency virus (FIV), a bovine immunodeficiency virus (BIV), a caprine arthritis encephalitis virus (CAEV), a Maedi visna virus (MW) or an equine infectious anaemia virus (EIAV). d. Other Viral Vectors
Other viral vectors may be employed as constructs in the methods and compositions described here. Vectors derived from viruses such as vaccinia and herpesviruses may be employed.
4. Calcium Phosphate Co-Precipitation or DEAE-Dextran Treatment
In other preferred embodiments, polypeptide is introduced to the cells using calcium phosphate co-precipitation. In another embodiment, the expression construct is delivered into the cell using DEAE-dextran followed by polyethylene glycol.
5. Direct Microinjection or Sonication Loading
Further embodiments include the introduction of the polypeptide by direct microinjection or sonication loading.
6. Liposome Mediated Transformation
In a further embodiment, the polypetide may be entrapped in a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers. Also contemplated is a nucleic acid construct complexed with Lipofectamine (Gibco BRL). In certain embodiments of the invention, the liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA. In other embodiments, the liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1). In yet further embodiments, the liposome may be complexed or employed in conjunction with both HVJ and HMG-1.
7. Adenoviral Assisted Transfection
In certain embodiments, the nucleic acid construct is introduced into the cell using adenovirus assisted transfection. Increased transfection efficiencies have been reported in cell systems using adenovirus coupled systems, and the inventors contemplate using the same technique to increase transfection efficiencies.
Cancers
The present invention is useful in treating cancer.
Examples of types of cancer, include, but are not limited to, non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemia (e.g., acute leukemia such as acute lymphocytic leukemia, acute myelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma), colon carcinoma, rectal carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, cervical cancer, testicular cancer, lung carcinoma, bladder carcinoma, melanoma, head and neck cancer, brain cancer, cancers of unknown primary site, neoplasms, cancers of the peripheral nervous system, cancers of the central nervous system, tumors (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, seminoma, embryonal carcinoma, Wilms' tumor, small cell lung carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, and retinoblastoma.
The following Examples further illustrate, but do not limit, the invention. EXAMPLE I METHODS
Luciferase assay. HEK-293 cells (105 cells/well) were transiently transfected with the reporter plasmid TK- HC100-luc (100 ng well) together with 100 ng/well of PCMX-GAL4-RXR, or pCMX-GAL4-PPARy, or pCMX-GAL4-LXRa, or pCMX-GAL4- LXRp plasmids using FuGene 6 Transfection Reagent (Roche). Four hours post- transfection, cells were treated with tumor-conditioned media for 24 h. Luciferase activities were analyzed by luciferase Reporter Assay Systems (Promega) according to the manufacturer's protocol, β-galactosidase (30 ng/well) was used for transfection normalization. Tumor growth experiments. B6 or NOD-SCID mice were injected s.c. with live tumor cells. RMA (0.5 or 1x105), B16 (5x104), LLC (2.5-3 x105), TrampCI (2.5x106). Tumor size was evaluated by measuring perpendicular diameters by a caliper. Data are reported as the average tumor volume ± SD. Comparison of survival curves was evaluated by log-rank test. Experiments of tumor growth using ZA or T1317 were performed as described here. ZA (75 μg) or vehicle was given i.p. contra laterally every 2 days, starting 8 days post-tumor infusion. T1317 (10 μΜ) or vehicle was given intratumor every 2 days, starting 5 days post-tumor infusion. In some experiments ZA was combined with 500 g of the anti-CD25 depleting mAb PC6127 or control antibody, given i.p. 4 days before tumor inoculation. shRNA experiments. HepG2 cells were transduced with lentiviral vectors encoding hLXRa (SIGMA, MISSION™ TRC shRNA Target set) or non-targeted shRNA (SIGMA, MISSION Non-Target shRNA Control Vector). Forty-eight hours later, cells were selected by puromycin (1 μg/ml). Upon selection, cells were collected and analyzed for LXRa mRNA expression by qRT-PCR. The sequence 4 (HU4) was then chosen to transduce DC. pLKO.1 containing HU4 shLXRa or non-target shRNA plasmids were then modified (KpnI/BamHI sites) to introduce the cell surface marker LANGFr for experiments on DC. DC transduction ranged from 40 to 90% as evaluated by FACS for the marker ANGFR encoded by both the vectors.
Tumor growth in CD11c-DTR and LXRa'' bone marrow chimera. Lethally irradiated (1 1Gy) B6 mice were transplanted with the bone marrow of CD11c-DTR or LXRa'' mice (4x106 BM cells/mouse). Eight weeks later, transplanted mice were controlled for CD11c ablation or LXRa''' phenotype in the blood and used for tumor growth experiments. Mice were treated i.p. with 4ng/g body weight of Diphtheria Toxin (DT) or left untreated. Twenty-four hours later mice were injected with 1 x105 RMA-mock or RMA-SULT2B1 b and monitored. Treatment with DT or vehicle was performed every 2 days until follow-up was completed. In vivo DC migration and skin painting assays. Murine bone marrow-derived DC, treated with tumor-CM or the compounds, or left untreated were labeled with 5 μΜ of CFSE and injected (1x106) s.c. in the hind leg footpad. Popliteal lymph nodes were recovered 36 h later, mechanically disaggregated and treated with collagenase A (1 mg/ml) and DNase (0.4 mg/ml) mixture in HBSS media 20% FBS for 60 min. Endogenous DC migration (FITC painting assay) was induced with 500 μΙ of Acetone/Dibutylphtalate (1 :1 v/v) containing 5 g/ml FITC applied to the shave skin seventy-two hours upon s.c. tumor injection (0.35 x 106 cells/100 μΙ PBS). Draining lymph nodes were removed 12 hours upon FITC application, as previously mentioned. Enzymatically treated cell suspension was washed and incubated 10 min with Fc blocking solution followed by CD11 c staining and FACS analysis. FITC painting assays were performed on C57BU6 wild type mice or mice transplanted with the bone marrow of LXRa ' or wild type mice. Immunohistochemistry. Tumor samples were either fixed in buffered 4% formalin or embedded in OCT and frozen in liquid nitrogen. Three pm paraffin sections were stained in hematoxylin and eosin for morphological analysis or immunostained with the rat anti-human CD3 (Serotec) after antigen retrieval. Three pm cryosections were fixed with 4% paraformaldheyde and incubated with the rat anti-mouse CD11 b and rat anti mouse CD1 1c. The immunoreactions were revealed by biotinilated- conjugated anti-rat antibody (Vector), horseradish peroxidase (HRP)-conjugated streptavidin, and using 3,3 diaminobenzidine (DAB) as chromogen (Biogenex, SanRamon). Slides were counterstained with hematoxylin. Immunohistochemistry on human tumors was performed on tumor samples fixed in buffered 4% formalin. Serial sections of tumors were then immunostained for CD1 1 c (clone 5D1 1 Novocastra), CCR7 (clone E271 Epitomics) and CD83 (clone 1H4b Novocastra). Mice and reagents. C57BLJ6 (B6) and NOD-SCID mice (6-8 weeks of age) were from Charles River or from Harlan and housed under pathogen-free conditions. OT- I336 (Ly 5.1 ) and CD11c-DTR37 mice have previously been described38. The C57BL/6 Lxr ^ mice were generated as previously described39. Animal studies were approved by the Institutional Animal Care and Use Committee of Istituto Scientifico S.Raffaele. Most human and mouse antibodies were from Becton Dickinson. Anti- CD83 mAb was from Coulter. We also used an anti-CCR7 mAb from MBL. mAb specific for murine CCR7 was from eBioscience. LNGFr-specific mAb 20.4 was from ATCC. Most synthetic compounds and fluorescent dyes were from Sigma-Aldrich. Rosiglitazone (RSG) was from Cayman. 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester, mixed isomer (5-(6)-CFDA, SE (CFSE) was from Molecular Probes Inc. Human IL-12 and IL-6 ELISA kits and Cytometric Bead Assay for measuring murine IFNy and TNFa were from BD biosciences. DC and tumor cell lines. Human DC were generated and cultured as described elsewhere40. Human iDC at day 4 of culture were harvested and activated (8x105 cells) with irradiated (100 Gy) 3T3-CD40L (25x104 cells) or with LPS (100 ng/mL) in a 6 wells plate, in the presence or absence of either the tumor CM or natural/synthetic LXR agonists. In some experiments DC were co-cultured with irradiated (100 Gy) MSR3-CD40L (25x104 cells/well). At day 6, mature DC were harvested and analyzed. Murine bone marrow-derived DC were harvested at day 7 of culture and activated with LPS (500 ng/ml) in the presence of either CM or compounds for 48 hours. Percentage of CCR7 inhibition (I) was calculated by using the following formula: I = [(C-T) *100]/C, in which C stands for the percentage of CD83+CCR7+ control DC and T stands for the percentage of CD83+CCR7+ treated DCs. In some experiments, mouse DC were isolated by rat anti-mouse CD11c mAb (N418)-coupled magnetic MicroBeads (Miltenyi). Most human melanoma, lung, colon, kidney tumor lines as well as normal renal cells were established in our laboratory, with the exception of MZ2-G43, LG2, LB2033, LB33, LB39, SK29.1 and LB40, kindly provided by Prof. T. Boon (Ludwig Institute for Cancer Research, Brussels), and Me10221 provided by Prof. G. Parmiani (Scientific Institute S.Raffaele, Milan). Human fibroblasts were kindly provided by Dr. M.P. Protti (Scientific Institute S.Raffaele, Milan). LS180, LOVO, HT29, KJ29, Calu-1 and LLC were from ATCC. RMA was obtained from Dr. M. Bellone (Scientific Institute S.Raffaele, Milan), B6.1 and B6.7 were obtained from Dr. M.P. Colombo (Istituto Nazionale dei Tumori, Milan). TrampCI was obtained from Dr. M. Bellone. Tumor-conditioned media (CM) were obtained by seeding a fixed number of cells in flasks and harvesting the supematants after 48-72 hours, when the cells are confluent. CM from fresh specimens were obtained by culturing resected tumors for 48 hours w/o FBS. MSR3 and RMA fresh tumors were obtained by growing these cells in NOD-SCID mice. Generation of 3T3-CD40L cell line was described elsewhere40.
Chemotaxis Assays. Chemotaxis assays were performed using 5-μιτι pore polycarbonate filters in a 24-well transwell chamber (Corning Costar Corporation) described elsewhere40. Briefly, treated DC (3.5x104/100 μΙ) were seeded in the upper chamber, whereas in the lower chamber 600 μΙ of medium containing CCL19 (R&D systems) was added. The number of DC migrated in the lower chamber was measured by flow cytometer acquisition of a fixed number of beads (Polysciences). The number of treated DC migrating in the absence of chemokine was always subtracted. To evaluate the percentage of migration, the number of migrated DCs was divided by the total number of cells added to the transwell41.
Mixed lymphocytes reaction. DC activated with 3T3-CD40L in the presence of tumor-CM (1x104 cells/well) were co-cultured with allogenic PBMC (1x10s cells/well) labeled with 2μΜ of CFSE. After 72 hours rhlL-2 (10 U/ml) was added. On days 5 and 7 each MLR was stained with an anti-CD3 antibody and analyzed by FACS. The obtained proliferation index is the ratio between the absolute number of CD3*CFSE+ T cells and unstimulated PBMC.
Viral vectors and transduction procedures. SULT2B1 b-expressing cell lines were derived by transduction of the wild type cells with the retroviral vector LSULTBI bSAN coding for the mouse SULTBI b gene42 and for the cell surface marker LANGFr43. LSULTBI bSAN retroviral vector was generated by cloning the SULTBI b EcoRI/Xhol fragment into the EcoRI/Xhol site of LXSAN under the long terminal repeat (LTR) transcription control. Primers used to clone SULT2B1b are shown in Table 2. Vector DNA was converted into the corresponding virus by the transinfection protocol. Tumor cell lines were infected by the exposure to virus-containing supernatant in the presence of 8 μg/ml of polybrene. Transduction efficiency was measured by FACS analysis for LNGFr expression. The SULT2B1 b-ANGFr lentiviral transfer vector was generated by cloning the murine SULT2B1 cDNA in place of the GFP cDNA into the self-inactivating hPGK.GFP.wPRE.mhCMV.ANGFr.SV40PA lentiviral vector (Agel/Sall sites)44. The truncated form of the mouse SULT2B1b gene was created by deleting the proline-serine-rich carboxyl terminus of the sequence, as described for the human SULT2B1 b gene in45. The deleted form of the mouse SULT2B1 b (aminoacid sequence 1-312 instead of 1-338) was then cloned into the self- inactivating hPGK.GFP.wPRE.mhCMV.ANGFr.SV40PA lentiviral vector (Agel/Sall sites) as described above. Concentrated VSV-G-pseudotyped LV stocks were produced and titered as described previously44,46. Cells were transduced with 1 x108 or 1x109 transduction units (TU)/ml VSV-G pseudotyped LV stocks, corresponding to 1.5 or 15 MOI47. Real-Time RT-PCR. Total RNA was isolated with TRIZOL reagent (Invitrogen). Reverse transcription was performed from 1 -2 μg of total RNA, incubating 1 hour at 42'C with MLV-reverse transcriptase (Invitrogen). Quantitative PCR was performed using real-time PCR (ABI PRISM 7700, Applied biosystems), 40 cycles of 95'C for 12s and 60°C for 1 min using Sybr Green. Primers used are shown in Table 2. All PCR reactions were done in triplicate. The comparative Ct method was used to quantify transcripts that were normalized for human or murine GAPDH, murine cyclophillin or human β-actin. Adoptive transfer and immunization experiments. OT-I CD8+ T-cells were purified from spleen and lymph nodes by CD8 negative isolation kit (Dynal, Invitrogen) and labeled with CFSE as described previously3. Three x106 CFSE labeled CD8+ T- cells/mouse was injected into the tail vein of B6 recipients. After 24 hours, recipients were injected s.c. with OVApep-loaded DC (0.3-0.5x106), previously treated for 48 h with LPS in the presence of 22R-HC, 22S-HC, LOVO-CM, M3M001-CM, R A-CM or left untreated. Three days later, spleen and lymph nodes from immunized mice were collected, digested, stained with CD8a and CD45.1 mAbs and then analyzed by FACS. In vitro OT-I activation assay. Purified OT-I CD8+ T-cells (1.5 x105) were cultured with OVApep-loaded DC (0.5 x105), previously activated with LPS in the presence of 22R-HC, 22S-HC or left untreated. Forty-eight hours later, supernatants were collected to measure the content of mouse IFN-γ and TNFa. Four days later cells were collected, counted by trypan blue exclusion method to evaluate cell number.
FACS analysis of CCR7 expression by DC infiltrating murine tumors. Twenty mice were infused with either RMA-mock or RMA-SULT2B1b (3x10s) s.c. Three days later tumors (ten tumors/group) were collected, pooled and mechanically/enzymatically disaggregated. Cells were then washed, counted and stained by CD11b, CD11c, CCR7 mAbs. Analysis for CCR7 expression was performed on the gated CD11c+ DC. Quantification of tumor-infiltrating CD11c* and CD3+ cells. The amount of immunoreactive CD11c+ and CD3+ cells was calculated as the percentage of positive cells/1000 nucleated elements, as determined at 400x magnification in randomly chosen high-power fields. Gene Therapy Experiment
Mice were injected with 105 RMA cells. At day 6, as soon as tumor was palpable, mice were injected intratumor with supernatants containing viral particles. We used LV-SULT2B1b and LV-mock concentrated supernatants with 1.07 x10"9 and 4.5 x10"9 TU/ml, respectively.
Statistical Analyses. Data are expressed as mean ± SD and were analyzed for significance by analysis of variance (ANOVA) with Dunnet's or Tukey's multiple comparison test, or when indicated by an unpaired or paired Student's t-test with Prism software. Survival curves were analyzed by log-rank test with Prism software.
EXPERIMENTAL RESULTS
Tumor-derived factors inhibit CCR7 expression on maturing DC
To investigate the influence of tumor-conditioned environment on DC maturation, we activated immature monocyte-derived DC (iDC) with NIH-3T3 cells expressing the DC activation molecule CD40L (3T3-CD40L)14. Activation was performed for 48 hours in the presence or absence of conditioned media (CM) from the human melanoma cell line MSR3. In some experiments DC were activated with MSR3 expressing CD40L (MSR3-CD40L). In all culture conditions, DC expressed the activation marker CD83, up-regulated CD80, CD86, HLA-DR and CD54 molecules, down-regulated CCR515,16 and secreted high levels of IL-12 (Fig. 1a and Fig. 17a,e). Conversely, we found a strong inhibition of CCR7 expression in DC cultured in the presence of SR3-CM or SR3-CD40L (Fig. 1a and Fig. 17b). This inhibition was functional as SR3-exposed DC migrated much less efficiently to the CCR7 ligand CCL19 (P <0.05; Fig. 1b). Inhibition of CCR7 expression was independent of the maturation stimulus used and occurred at transcriptional level (Fig. 17c,d). SR3 inhibited also the expression of CXCR4 (Fig. 17f); thus, demonstrating that SR3 cells induce a functional inhibition of chemokine receptors involved in DC migration to lymphoid organs. CM from fourteen out of 21 (66%) melanomas, as well as from some human colon, lung and kidney carcinomas (Fig. 1d), but not from normal tissues inhibited CCR7 expression on DC (Fig. 1c), leaving intact the expression of presenting and co-stimulatory molecules, cytokine release and the allogeneic T-cell stimulatory ability (Fig. 18a-c). Thus, indicating that in our setting the inhibitory activity does not interfere with other steps of DC activation and is a common feature among human tumors. The inhibitory factor(s) was also produced by murine tumors (Fig. 1f). Furthermore, human and mouse tumor-CM inhibited CCR7 expression also on murine bone marrow-derived DC (hereafter referred as DC) (Fig. 1g). To rule out the possibility of an in vitro artifact, we tested CM from specimens surgically removed from a melanoma patient (DEP) or from NOD-SCID mice (i.e. MSR3 and RMA). Fresh tumors inhibited CCR7 expression (Fig. 1 h), suggesting that the production of this inhibitory factor(s) would occur also in vivo. Then, we analyzed by immunohistochemistry serial sections of the original melanoma MSR3, showing the presence of both CD11 c+CD83+CCR7+ and CD1 1 c+CD83+CCR7" cells (Fig. 1i, arrows). This phenotype was also observed in about 40% of 61 tumors analyzed, while in the remaining tumors we detected only CD83+CCR7+ cells (Table 1). Altogether, these data indicate that human and murine tumors produce a factor(s) capable of inhibiting the expression of CCR7 receptor on human and mouse DC undergoing activation, an observation confirmed in vivo in a relevant proportion of human tumors. Tumors trigger LXRa activation on DC
Agonists of some nuclear receptors (i.e. RXR, PPARy) have been demonstrated to inhibit the expression of CCR7 and CXCR4 receptors on human and mouse DC14,17. Whether these agonists are released by tumors has not been reported yet. We investigated the ability of tumor-CM to activate nuclear receptors by using luciferase reporter assays. MSR3-CM did not activate PPARy but slightly induced RXR activation (Fig. 2a), however we did not find any RXR ligand in MSR3-CM (data not shown). Noteworthy, MSR3-CM activated LXRa, whereas CM from the non-inhibitory tumor M3M001 did not (Fig. 2a). Then, we evaluated whether the other CCR7 inhibitory tumors activated LXRa. With exception of B16, CM from all other tumors (MR255, LOVO and RMA) were able to activate LXRa (Fig. 2b), as well as CM from fresh tumors (Fig. 2c).
Accordingly to other studies18, we detected LXRa and β transcripts in immature and mature DC by RT-PCR (Fig. 2d, insert), and an increase of LXRa transcripts, reaching the peak 7 hours upon LPS stimulation (Fig. 2d). To investigate whether LXRa expressed by DC was triggered by tumor-CM, we analyzed the expression of the LXRa target gene ABCG1 in DC activated in the presence of tumor-CM. Seven hours upon activation in the presence of MSR3-, LOVO-, and MR255-CM, DC showed a significant increase of the ABCG1 transcripts, 2 to 7-fold higher than untreated DC (Fig. 2e). In agreement with these results, the natural LXR ligands, i.e. oxysterols5 (22R-HC, 25-HC), and the synthetic agonist T0901317 (hereafter referred as T1317) were, indeed, able to inhibit CCR7 expression at protein and mRNA levels (Fig. 2f,g), whereas the inactive isoform (22S-HC) did not (Fig. 2f). Oxysterols inhibited CCR7 expression at nanomolar concentrations, did not affect costimulatory molecules and impaired also the expression of CXCR4 receptor (Fig. 19a-c). These ligands also inhibited CCR7 expression on LPS-activated murine DC (Fig. 2h,i). Finally, a close correlation between CCR7 inhibition and LXRa activation was found in 60% of the tumor lines tested (21/35, data not shown).
Inactivation of LXRa signaling abrogates the inhibition of CCR7 expression
To gain insight into the mechanism linking tumor-derived LXRa ligands and CCR7 inhibition, we used different strategies. We sought to block cholesterol/oxysterol synthesis in tumors using Zaragozic Acid (ZA)19, an inhibitor of the squalene synthase enzyme (Fig. 3a). CM from ZA-treated tumors (MSR3, MR255, LOVO, RMA and Tramp C1) inhibited CCR7 expression to a lower extent as compared to CM from untreated tumors (Fig. 3b,c). Loss of CCR7 inhibitory activity paralleled the loss of LXRa activation (Fig. 3d), as well as the lack of ABCG1 mRNA induction in DC cultured with ZA-treated MR255-CM (Fig. 3e).
To further support the role of LXRa in tumor-mediated CCR7 inhibition, we took advantage of the sulfotransferase SULT2B1 b enzyme20. This enzyme inactivates natural oxysterols by sulfurylation21 , thus preventing LXRa activation in vitro and in vivo22. We developed retroviral and lentiviral vectors coding for the mouse SULT2B1 b enzyme and for a cell surface marker (i.e. ANGFR)23 to engineer MSR3, LOVO and RMA tumor cells. The enzyme was fully functional as demonstrated by luciferase reporter assays in HEK-293 cells expressing SULT2B1 b (Fig. 20a). CM from SULT2B1 b-transduced cells inhibited CCR7 expression at a significantly lower extent and did not activate LXRa (Fig. 3f,g), as compared to CM from parental cells transduced with vectors coding only for the ANGFR (hereafter referred as mock- transduced). To finally prove that LXRa triggering in DC was responsible for CCR7 inhibition, we transduced DC with lentiviral vectors encoding a short hairpin RNA specific to LXRa (shLXRa), as demonstrated in the human hepatoma HepG2 (Fig. 20b). Silencing efficiency ranged from 65 to 90% (Fig. 3h). In several experiments, shLXRa expression prevented CCR7 inhibition by 22R-HC and tumor-CM (Fig. 3i), demonstrating that CCR7 inhibition mediated by tumor-CM occurs through LXRa activation in maturing DC.
LXR engagement impairs DC migration in vivo dampening antigen-specific T cell priming
Since tumor-CM as well as natural and synthetic LXR agonists inhibit CCR7 expression on murine DC (Fig. 1g and Fig. 2i), we evaluated whether these DC had an impaired migration to draining lymph nodes. We analyzed by FACS the draining lymph nodes harvested from mice injected with DC activated with LPS in the presence of 22R-HC, 22S-HC, MR255-, LOVO-, RMA- or M3M001-CM, and stained with the dye CFSE. DC treated with 22R-HC-, MR255-, LOVO- and R MA-CM migrated poorly to the draining lymph node (Fig. 4a), whereas 22S-HC and Μ3Μ0Ό1- treated DC migrated as efficiently as untreated controls (UT). Impaired DC migration affected T cell priming, as H-2K -restricted OVA peptide-pulsed DC treated with 22R- HC-, LOVO- and RMA-CM were poor inducers of the proliferation of adoptively transferred OT-I CD8+ T cells, a transgenic line with a H-2Kb-OVA specific TCR24 (Fig. 21a and Fig. 20b). Although the inactive oxysterol 22S-HC did not alter CCR7 expression and DC migration, it impaired OT-I activation in vivo (Fig. 4b) and in vitro (Fig. 21 b-d). These results indicate that the tumor-mediated functional impairment of CCR7 expression on DC dampens the priming phase of the immune response.
Avoiding LXR activation promotes tumor rejection in vivo We observed the up regulation of Lxra mRNA in CD11c+ DC from mice treated for 8 or 24 hours with complete Freund's adjuvant (Fig. 21 e). Therefore, we evaluated whether avoiding tumor-mediated LXRa activation by blocking cholesterol synthesis could restore antitumor responses. ZA treatment of R A-bearing mice significantly delayed tumor growth and prolonged survival compared to controls (Fig. 5c, d). Importantly, ZA treatment of RMA-bearing immunodeficient NOD-SCID mice did not interfere with tumor growth, indicating that a fully competent immune system was required to delay tumor growth and that this delay was not due to the effect of ZA on the tumor (Fig. 22a). We then carried out similar experiments using tumors expressing the SULT2B1b enzyme. RMA transduced with vectors encoding DNGFR (Mock) grew similarly to wild type RMA (Wild type). On the contrary, 60% of mice injected with RMA expressing SULT2B1b (SULT2B1b, Fig. 5e insert; n=120 mice) rejected the tumor and showed a prolonged survival (Fig. 5e,f). Again, engineered tumors grew similarly in immunodeficient NOD-SCID mice (Fig. 22b). Mice rejecting RMA-SULT2B1b rejected a second challenge of wild type RMA, demonstrating the induction of long-lasting immune responses (data not shown). Tumor control was strictly dependent on the amount of SULT2B1 b expression by tumor cells (Fig. 23a, b) and could be partly abolished by the expression of a deleted form of the murine SULT2B1b (data not shown), which has been shown in a human cellular system to have a reduced sulfurylation activity25. Importantly, this benefit was partly abolished when RMA-SULT2B1b-bearing mice were injected intratu morally with the synthetic LXRa ligand T1317 (Fig. 5g), which is not inactivated by SULT2B1b (Fig. 20a), confirming the inhibitory role of LXRa in the antitumor response. An immune- mediated delay of tumor growth was also observed with TrampCI and LLC carcinomas expressing SULT2B1b (Fig. 5h,i and Fig. 22c, d) Noteworthy, SULT2B1b transduction of B16, a melanoma inhibiting CCR7 in an LXRa independent manner, did not modify tumor growth in both immunocompetent and NOD-SCID mice (Fig. 22e-f and insert).
To potentiate the antitumor effect observed with ZA, we treated R A-bearing mice with ZA and the CD25-depleting mAb PC6126,27, which ablates all CD4+ T cells, including the CD4+CD25+ T regulatory cells. The combined treatment significantly delayed tumor growth (Fig. 4k) and prolonged survival compared to the single treatments (Fig. 4j). Indeed, 40% of mice treated with the combination therapy were still alive after 52 days. All together these results suggest that drugs interfering with sterol metabolism {i.e. ZA) in combination with a mAb depleting T regulatory cells potentiate the antitumor effect of the single treatments.
DC and CCR7 play a key role in the LXR-mediated suppression of antitumor immune responses
To demonstrate the main role of DC in this antitumor effect, we evaluated tumor growth in mice transplanted with bone marrow of CD1 1c-DTR transgenic mice10, where CD1 1c+ DC can be conditionally deleted by diphteria toxin (DT) treatments. As expected, CD1 1c-DTR bone marrow chimeras controlled the growth of RMA-
SULT2B1b similarly to what observed in wt mice (Fig. 5a and data not shown).
Following DT treatment, R A-SULT2B1 b grew as fast as RMA- ock (Fig. 5a). Noteworthy, we did not observe a further increase of RMA-Mock growth in the absence of DC (Fig. 5a), suggesting that tumor-produced LXRa agonists paralyze the immune system.
The ability of DC to migrate to draining lymph nodes in the presence of tumor- producing SULT2B1 b was evaluated by FITC painting experiments on the skin overlying the established tumors. Lymph nodes draining RMA-SULT2B1 b tumors contained a higher number of CD1 1c+FITC+ DC compared to RMA-mock (Fig. 5b,c), indicating that the inactivation of tumor-derived LXR agonists restores DC migration to draining lymph nodes. FACS analysis for CD1 1 c and CCR7 expression on DC harvested from 3 days-established RMA-SULT2B1b and RMA-mock tumors showed a higher number of CD11c+CCR7+ DC infiltrating the RMA-SULT2B1b as compared to RMA-mock tumors (29.5% and 19.6%, respectively; Fig. 5d), with a CCR7 MFI of 1355 and 605 in RMA-SULT2B1b and RMA-mock, respectively. Thus, indicating that the inactivation of LXR ligands increases both the percentage of CD11c DC expressing CCR7 and the number of CCR7 molecules on DC surface.
Finally, to investigate the role of LXR in vivo, we performed FITC painting and tumor challenge experiments in mice transplanted with bone marrow of Lxra "28 or wild type mice. In wild type chimeras, the number of CD11c+FITC+ cells in the draining lymph nodes of RMA-bearing mice was lower than in tumor free mice (Fig. 5e). As expected, in the Lxra"'" chimeras, the presence of RMA-mock did not affect CD11c+FITC+ cell migration (Fig. 5e). Accordingly, Lxr ^' chimeras controlled the growth of RMA-mock (Fig. 5f). RMA-SULT2B1b grew similarly in both chimeras (data not shown).
Histologic and immunohistochemical analyses of the engineered tumors (Fig. 5g) showed a strong infiltrate of granulocytes and CD3+ T cells (Fig. 5h) in RMA- SULT2B1b compared to RMA-mock at 7 and 14 days. This infiltrate was barely detectable in both tumors at day 3. As expected, we found a higher number of infiltrating CD11c+ DC in RMA-mock compared to RMA-SULT2B1b at days 3 and 7, whereas an opposite scenario was observed at day 14 (Fig. 5i). The number of CD11 b+ cells were similar in both tumors at days 3 and 7, while at day 14 were more frequent in RMA-SULT2B1b.
These results suggest that blocking tumor-mediated LXRa signaling re-gains DC migration to draining lymph nodes, where DC activate effective antitumor T cells, which directly and by the induction of an overt inflammation, are eventually responsible for tumor rejection. Gene therapy experiment: a single infusion intratumor of supernatants containing lentiviral vectors encoding SULT2B1b delays R A growth
RMA-bearing mice were injected intratumor at day 6 with 10 μΙ of supernatant containing lentiviral particles encoding either the sulfotransferase enzyme SULT2B1b (LV-SULT2B1 b) or mock (LV-mock). We observed a statistically significant delay of tumor growth only in mice treated with LV-SULT2B1 b (Fig. 6). Mean and s.d. of one experiment with 5 mice/group. **, P < 0.005 (ANOVA). RMA wt and RMA-SULT2B1b were used as controls.
EXAMPLE H
METHODS
Further tumor growth experiments
B6 mice were injected s.c. with live RMA tumor cells (1x105). Tumor size was evaluated by measuring perpendicular diameters by a caliper. Data are reported as the average tumor volume ± SD. Experiments of tumor growth using Zaragozic Acid (ZA) were performed as described here. We used two routes of ZA administration: intraperitoneal (i.p.) (200 μg) contra laterally, or intravenous (i.v.) (100 μg) every 2 days alternating i.p. infusions with i.v. infusions, and starting ZA treatments 7 days after tumor infusion. In experiments combining ZA plus active immunotherapy, the same schedule of ZA was used in combination with subcutaneous injection of the MUT1 peptide (4 μg) emulsified in Incomplete Freunds' Adjuvant (IFA). Active immunotherapy was performed 7 and 14 days after tumor injection. We measured tumor weights of the different experimental groups at mice sacrifice.
Experiments evaluating Zaragozic Acid (ZA) safety B6 mice injected s.c. with live R A tumor cells (1x105) left untreated or treated with ZA, active immunotherapy or the combined therapy were weighed before and after treatments. GPT and GOT enzymes that evaluate the liver function were measured in the blood of mice at the end of the treatments.
Analysis of cells infiltrating tumors releasing and not releasing LXR ligands
CD45.1 + B6 mice injected s.c. with live tumor cells (1x10s) releasing (RMA-Mock) or not releasing (RMA-SULT2B1 b) LXR ligands. Two weeks later, mice were sacrificed, tumors were collected, mechanically disaggregated and treated with collagenase A (1 mg/ml) and DNase (0.4 mg/ml) mixture in HBSS media 20% FBS for 60', and analyzed for the presence of CD45.1+CD11 bhi9hGR1hi9h cells by flow cytometry. Percentages and number of CD45.1+CD1 1 bhi9hGR1 hi9h cells/mg of tumor tissue were evaluated and quantified. In vitro migration assays
CD11 b* cells were isolated from mouse bone marrow cells by magnetic beads. Purified CD11 b+ cells from bone marrow normally express also the GR1 marker. Purified CD11 b+GR1 + cells (2x10s) were plated in the upper chamber of transwell filters and allowed to migrate to either the LXR ligand 22R-HC (15 μΜ), the inactive form 22S-HC (15 μΜ) or to the medium alone, plated in the lower chamber. After 2 h at 37"C, the number of cells migrated in the lower chamber was measured by flow cytometer acquisition of a fixed number of beads (Polysciences Inc.). To evaluate the percentage of migration, the number of migrated cells was divided by the total number of cells added to the transwell. In some experiments we used CD1 1b+GR1* cells from LXRs deficient mice, or CD1 1 b+GR1+ cells pre-incubated with Pertussis Toxin (100 or 500 ng) for 90' before running the migration assay. For human cells, CD14+ monocytes were purified from peripheral blood mononuclear cells of healthy donors or melanoma patients. Purified CD14* monocytes were then allowed to migrate overnight at 37°C to 22R-HC (15 μΜ) or 22S-HC (15 μΜ). The day after, cells were collected and the number of cells migrated in the lower chamber was measured by flow cytometry as described above. Competition assays, and migration experiments using cells deficient for CXCR2 chemokine receptor
CD11b+GR1+ cells (2x105) were incubated for 30' with 22R-HC (50 μ ), CXCL5 (1 μg/ml) or with the CXCR2 antagonist SB225002 (10 μΜ). Then, the cells were washed and allowed to migrate to either 22R-HC (15 μΜ), 22S-HC (15 μΜ), CXCL5 (100 ng), SDF1a (100 ng), or MIP-1a (100 ng) for 2 h at 37°C. In some experiments migration was performed using CD11 b+GR1+ cells from CXCR2"'" mice.
Mouse chemokines and receptors RT2 Profiler™ PCR array and validation of the results
We performed qRT-PCR analysis on migrated and non-migrated CD11b+GR1+ cells, using the mouse chemokines and receptors RT2 Profiler™ PCR array. To this aim, we performed migration assays using six-well transwell filters by plating 2x106 cells. Cells collected from the lower chamber of the transwell filters were pooled and mRNA extracted for the subsequent analyses. We also extracted mRNA from the fraction of cells that did not migrate to the LXR ligands {i.e. the fraction remaining in the upper chamber of the transwell filters). A comparative analysis of mRNAs fold change between migrated and non-migrated cells was then carried out following manufacturer's Instructions and a software released by manufacturers. Validation of the results was performed by analyzing cell surface markers by flow cytometry.
Flow cytometry MAbs specific for CD1 1 b, GR1 , CD1 15, c-kit, IL^Ra, CX3CR1 , CCR1 , CXCR4, CXCR2, CD45.1 , CD45.2 and CD31 were from Becton Dickinson, PharMingen, R&D Systems or from eBioscience. Samples were run on a FACS CaliburT (BD) and analyzed by CELLQuestTM software (BD) or by FlowJo.
In vivo migration assays to tumor-derived or synthetic LXR ligands
CD45.1 * bone marrow cells (20x106) were injected into NOD-SCID mice bearing 14 days established tumors releasing LXR ligands (RMA-Mock) or not releasing LXR ligands (RMA-SULT2B1 b). Eighteen hours later, mice were sacrificed, tumors were collected, mechanically disaggregated and treated with collagenase A (1 mg/ml) and DNase (0.4 mg/ml) mixture in HBSS media 20% FBS for 60', and analyzed for the presence of CD45.1+CD11 bhi9hGR1 i9h cells by flow cytometry. Percentage and number of CD45.rCD11bhl9hGR1high cells/mg of tumor tissue were evaluated and quantified. 22R-HC (0.5 mM) or 22S-HC (0.5 mM) were mixed in matrigel and injected s.c. into the dorsal flank of mice. Three days later, mice were sacrificed, matrigels were collected, enzymatically disaggregated by dispase, and analyzed for the presence of CD45.2+CD11 bhighGR1 hi9h cells by flow cytometry. The analysis was performed as described above.
Tumor angiogenesis assay and microvessel density counts (MVD)
Tumor cells (RMA) and CD45.1 +CD11 bhi9hGR1hi9h cells (2: 1 ratio) functionally isolated from the bone marrow of naive mice were resuspended together in matrigel and co- injected subcutaneously into the dorsal flank of mice. Six days later, we quantified MVD measuring by flow cytometry the percentage of CD31 +CD45" endothelial cells collected from tumors mechanically disaggregated and treated with dispase for 60'. As control, we calculated MVD in tumors co-injected with CD1 1b'GR1" cells or injected alone. Statistical Analyses
Data are expressed as mean ± SD and were analyzed for significance by analysis of variance (ANOVA) with Dunnet's or Tukey's multiple comparison test, or when indicated by an unpaired or paired Student's t-test with Prism software.
EXPERIMENTAL RESULTS
Zaragozic Acid (ZA) administration delays the growth of the mouse lung tumor LLC
LLC tumor is an aggressive lung transplantable tumor that normally induce the death of tumor-bearing mice in 3-4 weeks from tumor infusion. To evaluate the effect of ZA given alone or in combination with tumor vaccination, we injected mice subcutaneously with 3x105 LLC cells. Seven days later, mice were either left untreated, treated with ZA, with tumor vaccination, or with ZA in combination with tumor vaccination and evaluated for tumor growth every 2 days. Tumor vaccination was performed using a nonapeptide derived from the tumor antigen MUT1 emulsified in Incomplete Freund's Adjuvant. ZA (200 μg) was administered every 2 days. Mice treated with ZA showed a statistically significant delay of tumor growth as compared with untreated tumor-bearing mice. The effect of ZA treatment was superior to the vaccination treatment. The combination therapy (i.e. ZA plus vaccination) was more effective as compared to the single treatments.
The administration of Zaragozic Acid (ZA) is well tolerated and safe
We evaluated whether the prolonged administration of ZA could induce hepatic damage. Mice were weighed before and after treatments, and no difference was observed among the groups of untreated or treated mice at the therapeutic doses administered (Fig. 8). Additionally, we did not observe any relevant modification of serum levels of the hepatic enzymes AST and ALT, two enzymes released in the blood when liver necrosis occurs. These data were also supported by histologic analysis showing a conserved hepatic structure and histology among the groups of untreated and treated mice.
Mouse CD11 b+GR1+ bone-marrow derived cells migrate to LXR-releasing tumors in a CXCR2 dependent manner
We analyzed the immune cells infiltrating the mouse lymphoma RMA, which produces LXR ligands (RMA-wild type), and the RMA engineered to express the LXR-inactivating enzyme SULT2B1 b (RMA-SULT2B1 b). We observed a significantly higher number of infiltrating CD1 1 b+GR1 + cells in RMA-wild type, as compared with RMA-SULT2B1 b (Fig. 9). These results can be explained by either the tumor-derived LXR ligand-mediated recruitment of these cells at tumor sites, or by the in situ induction of cell expansion, or both. Studies performed in parabiotic mice (performed in collaboration with Dr. EJ Villablanca at MGH, Boston), which are surgically joined and therefore have a common blood circulation, have shown that these cells are actively recruited by tumors; therefore, suggesting that tumor-derived LXR ligands may behave as chemokines. To demonstrate that these cells are recruited by LXR ligands, we performed some in vitro migration assays (Fig. 10). CD1 1 b+GR1 + cells isolated from bone marrow of naive mice have been allowed to migrate to the LXR ligand 22R-Hydroxycholesterol (22R-HC) through transwell filters. We observed a specific migration of CD1 1 b+GR1 + cells to 22R-HC, but not to the inactive isomer 22S-HC. Noteworthy, this migration turns out to be restricted to the CD11 bhi9hGR1hi9h subset. Moreover, this migration is pertussis toxin sensitive, indicating that the receptor(s) mediating this effect is a G protein coupled receptor, and is independent of LXR, as CD1 1b+GR1 + cells from LXRcf^, LXR^' and LXRa ' mice migrate to 22R-HC similarly to CD1 1 b+GR1 + cells from wild type mice. FACS analysis for chemokine receptors expression by comparing migrating and non migrating CD1 1 b+GR1 + cells, showed the preferential expression of CXCR2, CCR1 and CXCR4 chemokine receptors by migrating cells (Fig. 11 ). Migration competition assays demonstrated that only CXCR2 is involved in the migration of CD11 b+GR1 + cells to 22R-HC (Fig. 12). In vivo, we have observed that the injection of total bone marrow cells in mice bearing 14 days-established RMA wild type tumors, turns out in the selective migration of the CD11 b+GR1 + cells in the tumor 18 hours after the infusion. On the contrary, there is no preferential migration of CD1 1 b+GR1 + cells in 14 days-established RMA tumors engineered to express the LXR ligands inactivating enzyme SULT2B1b (RMA-SULT2B1 b). Finally, when we injected 22R-HC or the inactive isomer 22S-HC embedded in matrigel s.c. in mice, we observed the accumulation of CD11 b+GR1 + cells mainly in the 22R-HC embedded matrigel (Fig. 13), indicating that LXR ligands are able to recruit CD1 1 b+GR1+ cells in vivo.
Mouse CD11b+GR1+ bone-marrow derived cells promote neo angiogenesis and tumor growth
These cells have been demonstrated to promote tumor angiogenesis. Thus, to demonstrate that in our conditions the CD11 b+GR1 + cells migrating to LXR ligands are indeed able to promote tumor neoangiogenesis, we isolated them functionally (i.e. through the collection of the cells migrating through transwell filters in response to LXR ligands), mixed them with tumor cells and co-injected this mix s.c. in mice. Six days later, tumors were harvested and analyzed for the presence of CD31 +DC45- endothelial cells. We observed a higher percentage of CD31+ cells within tumors co- injected with migrating CD11 b+GR1 + cells as compared to tumors alone or co- injected with non migrating CD1 1b+GR1 + cells (Fig. 14). This indicates that CD11 b+GR1 + cells migrating to LXR ligands are able to promote tumor angiogenesis.
Characterization of the human cell counterpart migrating to LXR ligands
To identify the human counterpart endowed with the ability to migrate in response to LXR ligands, we tested peripheral blood mononuclear cells. A fraction of CD14+ monocytes (10-15%) migrated to the LXR ligand 22R-HC (Fig. 15). This migration was obtained using monocytes from tumor patients as well as from healthy donors.
DISCUSSION LXRs modulate immune responses under physiological conditions as well as in different disease models8,29. In addition, in vitro differentiation of human DC in the presence of LXR agonists and LPS has been shown to affect their T-cell stimulatory ability18. Here we demonstrate that LXR plays an important role in cancer. We show that human and murine tumors release cholesterol metabolites dampening the expression of CCR7 receptor on maturing DC by LXR activation; thus, allowing tumor escape from immune surveillance (Fig. 16). Indeed, in the RMA model, tumor- produced l_XR< agonists appear to paralyze the immune system, as tumors grow similarly both in the presence and in the absence of DC. We found that inhibition of CCR7 expression in DC by tumor-derived or synthetic LXR ligands occurs independently of the maturation stimulus used, while leaving intact DC immunostimulatory properties. The discrepancy with studies18,30 showing the impairment of other molecules/functions of synthetic LXR ligand-treated DC may be due to the experimental conditions. We performed LXR triggering in already differentiated DC, in which transcripts encoding some DC activation molecules are ready to be translated31,32. Instead, synthesis of CCR7 mRNA, which starts 4-6 hours upon stimulation may be actively blocked. Accordingly, we noticed a wider dysregulation of DC differentiation when tumor-CM was added during DC differentiation (data not shown). Inhibition of CCR7 expression on DC resulted in impaired migration to the draining lymph nodes. Interestingly, impaired migration of monocyte-derived cells from atherosclerotic plaques is one of the factors responsible for progression of atherosclerosis33. The mechanism for emigration failure is still unknown, however, lipid-derived signals have been proposed to dampen migration33, suggesting a possible role of LXR also in this context.
Importantly, we found that abrogation of tumor-mediated LXR( activation generated effective antitumor responses. These treatments, preventing CCR7 inhibition on DC, restored their migration to draining lymph nodes.
Tumor rejection did not occur in mice devoid of DC and in immunedeficient mice, highlighting the central role of DC and T cells in this mechanism. Whether additional cells, including macrophages and lymphocytes, may contribute to tumor rejection is currently under investigation. The recently described role of LXR( in T and B cells34, suggests that proliferation of antitumor T cells might be dampened by tumor-derived LXR ligands, further contributing to inhibiting antitumor immune responses. Differently from ZA, which promotes antitumor immune responses, statins have been described to impair both DC and T cells35. This discrepancy can be ascribed to their inhibition mechanisms. ZA, by inhibiting sterol synthesis downstream of mevalonic acid, would block only cholesterol/oxysterol formation35. Instead, statins inhibit also the formation of farnesyl and geranylgeranyl pyrophosphate that are involved in the functional posttranslational modification of small GTPase proteins (Rho, etc.)35.
Our findings highlight a novel mechanism of tumor immunoescape in which tumors release LXR ligands that impair DC migration to draining lymph nodes. Importantly, we found CCR7 negative mature DC infiltrating several human tumors. The inactivation of this immunoescape mechanism restored antitumor responses and, noteworthy, improved the effect of other compounds counteracting suppressive mechanisms. Our findings also highlight a novel mechanism of preventing the migration of pro-angiogenic cells. Altogether, these findings encourage the development of new strategies of cancer treatment based on drugs interfering with the synthesis of cholesterol.
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Claims

1. A pharmaceutical composition comprising a combination of (i) an oxysterol modulator and (ii) a CD25-depleting monoclonal antibody for treating cancer.
2. The pharmaceutical composition of claim 1 wherein the oxysterol modulator blocks cholesterol synthesis downstream of mevalonic acid.
3. The pharmaceutical composition of claim 1 or 2 wherein the oxysterol modulator is selected from a squalene synthase inhibitor and a lanosterol 14-alpha demethylase inhibitor.
4. The pharmaceutical composition of claim 3 wherein the lanosterol 14-alpha demethylase inhibitor is SKF 104976.
5. The pharmaceutical composition of claim 3 wherein the lanosterol 14-alpha demethylase inhibitor is an azole.
6. The pharmaceutical composition of claim 5 wherein the azole is selected from fluconazole and itraconazole.
7. The pharmaceutical composition of claim 3 wherein the squalene synthase inhibitor is zaragozic acid or a derivative thereof.
8. The pharmaceutical composition of claim 7 wherein the derivative is a conjugate, a salt, an ester, an amide or a carbamate of zaragozic acid, optionally substituted.
9. The pharmaceutical composition of claim 7 or 8 wherein the derivative of zaragozic acid is selected from
Figure imgf000096_0001
Figure imgf000097_0001
wherein X is H, a halogen (F, CI, Br, I), OH or CH3; Y is halogen (F, CI, Br, I), OH or CH3, and wherein Z Z2 and Z3 are each independently H, C1 -5 alkyl, C1-5 alkyl substituted with (i) phenyl, (ii) phenyl substituted with methyl, methoxy, halogen (F, CI, Br, I) or hydroxy, (iii) C1 -5 alkylcarbonyloxy, (iv) C6-10 arylcarbonyloxy, (v) C1-5
alkoxycarbonyloxy, (vi) C6-10 aryloxycarbonyloxy, (vii) A° ci-s aikyi | (Vijj)i 0® ( 0r the groups (iii) to (vi) form a 5 to 10 membered mono- or bicyclic ring with C1 -5 alkyl; or from
Figure imgf000098_0001
wherein R is selected from:
Figure imgf000098_0002
selected from : , CH3-CH=CH-(CH2)4-CH=CH-(CH2)4-C(0)-0-, and
Figure imgf000098_0003
R2 is selected from: H, and— R3 is d.5alkyl; Z is selected from (i) H, (ii) C^alkyl; (iii) C^alkyl substituted with (a) CLsalkylcarbonyloxy, (b)
Figure imgf000098_0004
arylcarbonyloxy, (c) Ci.5alkoxycarbonyloxy, (d) aryloxycarbonyloxy; (e)
(f)
Figure imgf000098_0005
, (g) or the groups (a) to (d) form a a 5 to 10 membered mono or bicyclic ring with C1-5alkyl, (iv) C3.6 cycloalkyl; or a pharmaceutically acceptable salt thereof.
10. The pharmaceutical composition of claim 7 wherein the squalene synthase inhibitor is zaragozic acid.
11. The pharmaceutical composition of claim 3 wherein the squalene synthase inhibitor is selected from lapaquistat (TAK-475), terbinafine, ER-27856 (5-{Λ/-[2- butenyl-3-(2-methoxyphenyl)]-/V-methylamino}-1 , 1-penthylidenebis(phosphonic acid) tri-sodium salt), RPR-107393 (3-hydroxy-3-[4-(quinolin-6-yl)phenyl]-1-azabicyclo [2- 2-2]octane dihydrochloride) and YM-53601 ((£)-2-[2-fluoro-2-quinuclidin-3-ylidene ethoxy]-9H-carbazole monohydrochloride).
12. The pharmaceutical composition of claim 3 wherein the squalene synthase inhibitor is a phosphonic acid derivative.
13. The pharmaceutical composition of claim 12 wherein the phosphonic acid
derivative is represented by the following general formula (I):
Figure imgf000099_0001
wherein represents a hydrogen atom, a hydroxyl group, an acyloxyalkyl group, an alkyloxycarbonyl group, a lower alkyl group which may have a substituent or a lower alkoxy group which may have a substituent; R2 and R3 may be the same or different from each other and each represents a hydrogen atom, a lower alkyl group which may have a substituent, an alkali metal or a prodrug ester forming group; RA represents a group represented by the formula: 0 (wherein R4 represents a hydrogen atom, a lower alkyl group, an alkali metal or an acyloxyalkyl group which
may have a substituent), a group represented by the formula:
Figure imgf000099_0002
(wherein R represents a hydrogen atom, a lower alkyl group or an alkali metal) or a group o
II
— P— OR5
represented by the formula: R6 wherein R5 represents a hydrogen atom, a lower alkyl group, an alkali metal or a prodrug ester forming group; and R6 represents a lower alkyl group or a group represented by the formula: -OR7 (wherein R7 represents a hydrogen atom, a lower alkyl group, an alkali metal or a prodrug ester forming group)]; and
RB represents a group represented by the formula: S--T-- [wherein S represents an alkenyl group which may have a substituent or a group represented by the formula:
Figure imgf000100_0001
(wherein ring A represents an aromatic ring; R8, R9, R10, R11 and
R12 may be the same or different from one another and each represents (1) a hydrogen atom, (2) an alkyl group which may have a substituent, (3) an alkenyl group which may have a substituent, (4) a lower alkoxy group which may have a substituent, (5) a carbamoyl group which may have a substituent, (6) a carbamoyloxy group which may have a substituent, (7) a hydroxyl group, (8) an acyl group, (9) a halogen atom, (10) a group represented by the following formula:
Figure imgf000100_0002
(wherein R13 and R14 may be the same or different from each other and each represents a lower alkyl group which may have a substituent, or alternatively R13 and R14 may form together with the nitrogen atom to which they are bonded, a ring which may further contain an oxygen atom, a sulfur atom or a nitrogen atom and which may have one or two, mono- or divalent substituent(s); p is 0 or 1; and q is an integer of 0 to 4) or (11 ) a group represented by the formula:
Figure imgf000100_0003
(wherein R15, R16, R17, R18 and R19 may be the same or different from one another and each represents a hydrogen atom, a hydroxyl group, a lower alkyl group or a lower alkoxy group which may have a substituent; ring B represents an aromatic ring; and Y represents an alkylene chain which may have a substituent, an alkenylidene chain which may have a substituent, an alkynylidene
II
chain which may have a substituent, a group represented by the formula: 0 , a group represented by the formula: --0--, or a single bond), or alternatively two adjacent groups of R8, R9, R10, R11 and R12 may together form a ring; and X represents a single bond, an alkylene chain which may have a substituent, an alkenylidene chain which may have a substituent or a group represented by the formula: --(CH2)u -Z--(CH2)v -- (wherein Z is a group represented by the formula:
( o )r
e—
(wherein r is an integer of 0 to 2), a group represented by the formula:
0 a group represented by the formula: -0-, a group represented by the formula:
R20
S°2N (wherein R20 represents a hydrogen atom, a lower alkyl group which may have a substituent or a lower alkenyl group which may have a substituent), a
R21
I
group represented by the formula— N— (wherein R21 represents a hydrogen atom, a lower alkyl group which may have a substituent, a lower alkenyl group which
may have a substituent or a group represented by the formula:
Figure imgf000101_0001
R22 O
group represented by the formula: — N (wherein R22 represents a hydrogen atom, a lower alkyl group which may have a substituent or a lower alkenyl group which may have a substituent); u is an integer of 0 to 3; and v is an integer of 0 to 6); and T represents (1) a single bond, (2) a group represented by the formula:
Figure imgf000101_0002
(wherein R23 represents a hydrogen atom, a cycloalkyl group, a cycloalkylalkyi group, a lower alkyl group which may have a substituent or a lower alkenyl group which may have a substituent; W represents a group represented by the formula: -0-, a group represented by the formula: 0 , a group represented by
OH
I
the formula: --ΝΗ--, a group represented by the formula: CH— , a group o
represented by the formula: ° or a single bond; and s and t are independent of each other and are each an integer of 0 to 4), (3) a group represented by the
formula:
Figure imgf000102_0001
2 (wherein R23, W, s and t are each as defined above; and R29 represents a hydrogen atom, a cycloalkyl group, a cycloalkylalkyl group, a lower alkyl group which may have a substituent or a lower alkenyl group which may have a substituent),
R25
I
(4) a group represented by the formula: N (wherein R25 represents a hydrogen atom, a cycloalkyl group, a lower alkyl group which may have a substituent or a lower alkenyl group which may have a substituent), or
(5) a group represented by the formula:
Figure imgf000102_0002
(wherein D represents a carbon atom or a nitrogen atom, E represents a nitrogen atom or a group represented by the formula:
Figure imgf000102_0003
, F represents a group represented by the formula: --0--, a group represented by the formula: 0 , a group represented by
OH
J
the formula: -ΝΗ--, a group represented by the formula: CH— , a group o
represented by the formula: 0 or a single bond; x and y are independent of each other and are each an integer of 0 to 3.
14. The pharmaceutical composition of claim 3 wherein the squalene synthase
inhibitor is a probucol ester of the formula
Figure imgf000102_0004
wherein ^ is H or P03H2 and X2 is H or P03H2; and wherein R1 and R2 are H- or - CH3; and R3, R4, R5 and R6 are independently selected from H- or an alkyl group selected from the group consisting of methyl, ethyl, propyl, butyl or tert-butyl; or, a pharmaceutically acceptable salt thereof.
15. The pharmaceutical composition of any of claims 1 to 14 wherein the CD25- depleting monoclonal antibody is selected from Basiliximab, daclizumab (Zenapax), inolimomab (Leucotac), HuMax-TAC and PC61.
16. An LXR ligand inactivator for treating cancer.
17. The LXR ligand inactivator of claim 16 wherein the LXR ligand inactivator is an LXRa ligand inactivator or an LXRp ligand inactivator.
18. The LXR ligand inactivator of claim 16 or 17 wherein the LXR ligand inactivator is a sulfotransferase enzyme.
19. The LXR ligand inactivator of claim 8 wherein the sulfotransferase enzyme is SULT2B1b.
20. The LXR ligand inactivator of any one of claims 16 to 19 wherein the LXR ligand inactivator is administered using gene therapy.
21. An LXR inhibitor or antagonist for treating cancer.
22. The LXR inhibitor or antagonist of claim 21 wherein the LXR inhibitor or antagonist is an LXRa inhibitor or antagonist or an LXRp inhibitor or antagonist.
23. The LXR inhibitor or antagonist of claim 21 or 22 wherein the LXR antagonist is a cholesterol oxide, an oxysterol or a sterol or derivative thereof.
24. The LXR inhibitor or antagonist of claim 23 wherein the sterol is selected from a hydroxycholesterol and a sulfated oxysterol.
25. The LXR inhibitor or antagonist of claim 23 wherein the cholesterol oxide is a functionalised cholesterol oxide selected from 7p-hydroxycholesterol, a- epoxycholesterol, β-epoxycholesterol, 7-keto-cholesterol, cholestane triol, 7a- hydroxycholesterol, 25-hydroxycholesterot, 22(R)-hydroxy-cholesterol, 24(S)- hydroxy-cholesterol, 27-hydroxy-cholesterol.
26. The LXR inhibitor or antagonist of claim 24 wherein the sulfated oxysterol is selected from 24-OHChol-3-sulfate and 24-OHChol-3, 24-sulfate.
27. The LXR inhibitor or antagonist of claim 21 wherein the LXR inhibitor or antagonist is selected from a polyunsaturated fatty acid, a geranyl geraniol or geranylgeranyl pyrophosphate, 5a,6a-epoxycholesterol sulphate (ECHS), 7- ketocholesterol-3-sulphate, and a tricyclic compound represented
R1
Figure imgf000104_0001
, a pharmacologically acceptable salt thereof, or a hydrate of the compound or the salt in which R1 represents a hydrogen atom, a lower alkyl group, a lower halogenated alkyl group, an unsubstituted or substituted phenyl group, or an unsubstituted or substituted benzyl group; R2 represents a 1 ,1 ,1 ,3,3,3- hexafluoro-2-hydroxypropan-2-yl group or a carboxymethyl group; R3 represents a hydrogen atom, a lower alkyl group, a lower alkoxy group or a halogen atom; X represents a direct bond, an oxygen atom, a sulfur atom, a (CH2)n group or a (CH=CH)n group (wherein n represents an integer of 1-3); and Y represents CO or S02.
28. The LXR inhibitor or antagonist of claim 21 wherein the LXR inhibitor or antagonist is selected from Liver X Receptor antagonist BMS and Liver X Receptor antagonist EXELIXIS.
29. The pharmaceutical composition of any of claims 1 to 15 further comprising a pharmaceutically acceptable excipient, diluent or carrier.
30. A pharmaceutical composition comprising the LXR ligand inactivator of any of claims 16 to 20 and further comprising a pharmaceutically acceptable excipient, diluent or carrier.
31. A pharmaceutical composition comprising the LXR inhibitor or antagonist of any of claims 21 to 28 and further comprising a pharmaceutically acceptable excipient, diluent or carrier.
32. The pharmaceutical composition of any of claims 1 to 15 and 29 to 31 , the ligand inactivator of any of claims 16 to 20 or the LXR inhibitor or antagonist of any of claims 21 to 28 in combination with a chemotherapeutic agent for treating cancer.
33. The pharmaceutical composition of any of claims 1 to 15 and 29 to 31 , the ligand inactivator of any of claims 16 to 20 or the LXR inhibitor or antagonist of any of claims 21 to 28 in combination with a chemotherapeutic agent wherein the chemotherapeutic agent is selected from cytotoxic antibiotics such as aclarubicin, bleomycin, dactinomycin, daunorubicin, doxorubicin, epirubicin, idarubicin, mitomycin, and mitoxantrone (mitozantrone); alkylating agents such as busulfan, carmustine, chlorambucil, chlormethine hydrochloride, mustine hydrochloride, cyclophosphamide, estramustine phosphate, ifosfamide, lomustine, melphalan, thiotepa, and treosulfan; antimetabolites such as capecitabine, cladribine, cytarabine, fludarabine, fluorouracil, gemcitabine, mercaptopurine, methotrexate, raltitrexed, tegafur, and thioguanine; vinca alkaloids, such as etoposide, vinblastine, vincristine, vindesine, and vinorelbine; other antineoplastic drugs such as amsacrine, altretamine, crisantaspase, dacarbazine, temozolomide, hydroxycarbamide, hydroxyurea, and pentostatin; platinum compounds such as carboplatin, cisplatin, and oxaliplatin; porfimer sodium; procarbazine; razoxane; taxanes such as docetaxel and paclitaxel; topoisomerase I inhibitors such as irinotecan and topotecan; trastuzumab; tretinoin.
34. A pharmaceutical composition comprising an oxysterol modulator in combination with a further cancer treatment.
35. A pharmaceutical composition according to claim 34 wherein the oxysterol modulator is administered in combination with a chemotherapeutic agent.
36. A pharmaceutical composition according to claim 34 wherein the oxysterol modulator is administered in combination with immunotherapy.
37. Use of an LXR ligand for isolating CD11b+GR1+ cells from a population.
38. Use of an LXR ligand for isolating mouse CD11b+GR1+ and/or human CD1 + cells from a population.
39. Use according to claim 37 or 38 wherein said use involves a migration assay.
40. Use according to any of claims 37 to 39 wherein the LXR ligand is 22R-HC.
41. Use of an LXR ligand for promoting migration of mouse CD11 b+GR1 + and/or human CD14+ cells.
42. Use according to claim 41 wherein the LXR ligand is 22R-HC.
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