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1 Régulation de lexpression des enzymes du métabolisme des médicaments par les xénorécepteurs CAR et PXR et conséquences physiopathologiques. Jean Marc.

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Présentation au sujet: "1 Régulation de lexpression des enzymes du métabolisme des médicaments par les xénorécepteurs CAR et PXR et conséquences physiopathologiques. Jean Marc."— Transcription de la présentation:

1 1 Régulation de lexpression des enzymes du métabolisme des médicaments par les xénorécepteurs CAR et PXR et conséquences physiopathologiques. Jean Marc Pascussi Institut de Génomique Fonctionnelle Inserm U661, Montpellier Institut national de la santé et de la recherche médicale

2 Xenobiotics Xenobiotics: foreign chemicals Inhaled Ingested absorbed Pollutants Industrial chemicals Pesticides Toxins produced by molds, plants and animals Drugs

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5 Les xénorécepteurs, sentinelles moléculaires de limmunité métabolique PXR AhR CAR Phase I Phase II Phase III transporteurs métabolisme élimination

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7 Different strategies for activating transcription factors

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9 9

10 Nuclear Hormone Receptors superfamily

11 11 The Nuclear Receptor Gene superfamily

12 Nuclear receptors

13 The paradigm … General scheme for activation of gene transcription by NRs Robyr, Wolffe, Wahli Mol. Endocrinol 2000

14 Les xénorécepteurs CAR et PXR Expression Organes de la détoxication: foie (hépatocytes) et tracus digestif (entérocytes) PXRCAR

15 CAR and PXR coordinate the metabolic immunity in response to xenochemicals or endogenous activators

16 Nuclear Receptor PXR (NR1I2) … the master xenosensor

17 17 Nuclear receptor CAR (NR1I3)

18 Schéma de lhétérodimère CAR-RXR ou PXR-RXR en présence de leurs ligands et différentes organisations des HREs. Lannotation x correspond au nombre de nucléotides entre les deux motifs (daprès Handschin C & Meyer UA. Pharmacol Rev, 2003; 55, ) CAR ou PXR RXR Acide rétinoïque 9-cisLigand Gène cible

19 Les xénorécepteurs CAR et PXR partagent les mêmes NRE 2B6 3A4 2B6 3A4

20 CAR and PXR regulate the bodies 'garbage-disposal system.' excretion Uptake Solubilisation Phase I CYP2A,2B,3A Carboxylesterases Phase II GST SULT2 UGT1A PAPSSP2 Phase III Transport MDR1A,1B MRP3… Phase 0 OATP2

21 Les xénorécepteurs CAR et PXR CITCO 437 g.mol g.mol -1 Ligands/activateurs PXR (Polygamic Xenobiotic Receptor ?) PXR (Polygamic Xenobiotic Receptor ?) Médicaments: Antibiotiques (rifampicine,etc.) Glucocorticoides, RU486, PCN Antifongiques (clotrimazole) Anticancéreux (paclitaxel, codermolide, ixabepilone, tamoxifene, cyclophosphamide) Inhibiteur de protéases virales (amprenavir, ritonavir) Inhibiteurs des pompes à protons (Benzimidazoles) Bloqueurs des canaux calciques (nifedipine) Alimentation/Phytothérapie: Mille pertuis gluglustérone b-carotène Ginko Biloba Pesticides Contaminants environnementaux CAR CAR Médicaments: Clotrimazole (agoniste inverse) Phenobarbital, phénytoïne Phytothérapies: Artémise Pesticides CITCO (agoniste) phénobarbital g.mol -1 rifampicine

22 hPXR LBD crystal structure (2.6 A resolution) Watkins et al. Science 2001; Chrencik et al. Mol Endocrinol 2005 Large and flexible ligand-binding pocket Presence of 2 additional strands of b-sheet Hydrophobic ligand-binding pocket Can accommodates a single hydrophobic ligand in multiple conformation Absence of a highly constrained pocket allows for molecular flexibility and plasticity in ligand recognition Bound ligand= SR12813

23 Plasticity in the PXR Binding Pocket hyperforinSR Å1544 Å ReceptorVolume of ligand pocket (A 3 ) ER 476 VDR871 PXR CAR1120

24 hCAR/RXR LBD heterodimer crystal structure (2.6 A resolution) Bound ligand: CITCO RXRCAR Xu et al. Mol Cell 2004 SRC-1 peptide Vol= A

25 CAR activators provoke CAR nuclear translocation NT PB CITCO CAR-GFP in human hepatocytes In vivo in mice

26 26 Mécanismes dactivation des xénorécepteurs CAR et PXR CARPXR RXR cofactors CAR cytoplasme noyau PB CCRP OA AMPc/AMP AICAR AMPK LKB1 HSP CAR PXR RIF Citco Thr-38-P Thr-38 PP2A PKC ?

27 CAR PXR, CAR Complementary roles of CAR and PXR toward xenobiotics recognition ?

28 28

29 29

30 Species differences in CAR and PXR activation

31 (CYP3A1 DR3) 2 -tk-CAT Variation in LBD consistent with in vivo species differences in response to inducers

32 CAR and PXR humanized mice Wolf et al. J Clin Invest. 2008

33 xenobiotic RXR CAR/PXR Endocrine disruption VitD3, T3 and lipids metabolism Xenosensors that protect the body from a multitude of foreign chemicals (xenobiotics) and endogenous toxic compounds Drug-drug and food-drug interactions Interindividual variability in drug response Ying and Yang of CAR and PXR

34 Major Cytochrome P450s involved in the metabolism of clinically used drugs are PXR and CAR target genes

35 Nuclear receptor Activating drugs PXR amprenavir, avasimibe, bosentan, carbamazepine, ciglitazone, clotrimazole, cortisone, corticosterone, cyclophosphamide, dexamethasone, efavirenz, exemestane, hydrocortisone, hyperforin, lovastatin, mifepristone, nelfinavir, nifedipine, omeprazole, paclitaxel, phenobarbital, phenytoin, rifabutin, rifampicin, ritonavir, simvastatin, spironolactone, tamoxifen, 4- hydroxytamoxifen, troglitazone, troleandomycin, St Johns wort, Kava, Sophora flavescens CAR CITCO, phenobarbital, phenytoin Garlic, Ginkgo CAR and PXR are activated by widely used drugs and top selling phytochemicals

36 CAR and PXR xenosensors are involved in drug-drug interactions CYPs CYP enzymes XeR RXR Drug A OH Drug B Inactivation activation

37 Xenosensors and drug-drug interactions «Loss of analgesic effect of morphine due to coadministration of rifampin » Fromm et al Pain «Fatal paracetamol poisoning in an epileptic (phenytoin)» Minton et al Hum Toxicol

38 «Profound drop of cyclosporin A whole blood through levels caused by St. Johns wort (Hypericum perforatum)» Breidenbach et al Transplantation Xenosensors and diet-drug interactions

39 «Osteomalacia associated with carbamazepine/valproate» Karaaslan et al Ann Pharmacother «Rifampicin induced osteomalacia» Shah et al Tubercle «Calcium metabolism during rifampicin and isoniazid therapy for tuberculosis» Brodie et al J R Soc Med. «Antiretroviral therapy and the prevalence of osteopenia and osteoporosis : a meta-analytic review» Brown et al AIDS. Role of CAR and PXR in drugs-induced osteomalacia and osteopenia ?

40 CYP3A4 (DR3, ER6) CYP2B6 (DR4) CYP2C9 (DR4) SULT2A1 (IR0) PXR RXR VDR RXR RXRCAR NRE Thummel et al. Mol Pharm 2001 Drocourt et al. J Biol Chem 2002 Echchgadda et al. Mol Pharmacol (OH)D3 1,25(OH)D3 1,24,25(OH)D3 (inactive) 7 déhydroxycholestérol CYP27A1 CYP27B1 CYP24A1 VDR RXR Déficits en vitamine D et ostéomalacies consécutives à la prise prolongée de médicaments… sur la piste de CAR et PXR

41 Les activateurs de CAR et PXR augmentent lexpression et l'activité de la CYP24 dans lhépatocyte humain Lambert 2008 TAP Pascussi, 2005, JCI

42 CYP24 : nouveau gène cible de CAR et PXR

43 HepG2HuH7 CAR et PXR transactivent les VDREs du gène CYP24 Konno 2008 Mol. Pharmacology HuH7

44 The VDR-PXR cross-talk CYP3A4 CYP24 CYP3A4 CYP24 XeR CYP3A4 CYP24 Drocourt 2002 JBC, Xu 2006 Mol P, Pascussi 2005 JCI, Moreau 2007 BBRC, K onno 2008 MP, Lambert 2008 TAP

45 CAR, PXR et la stéatose hépatique non alcoolique induite par certains médicaments. Nakamura 2007 JBC - Rifampicine traitement contre la tuberculose Stéatose hépatique Morere 1975 Sem Hop - Carbamazepine traitement antiépileptique Stéatose hépatique Oscarson 2006 CPT - Phenobarbital traitement antiépileptique Stéatose hépatique Calandre 1991 ANS - Nifedipine traitement contre lhypertension Stéatose hépatique Babary 1989 J Hep

46 Protocole double couleur - ARN de cultures traitées - ARN de référence Extraction ARN Culture primaire dhépatocytes humains Traitements (8 & 40h): DMSO 0.1% Phénobarbital (0.5mM) Rifampicine (10 M) CITCO (100nM) Etudes comparatives de transcriptomes dhépatocytes humains... sur la piste dun acteur peu connu de la lipogénese : Spot14 Spot14, THRSP (Chr. 11q13.5/14.1) 143AA, 17kDa, Pi=4.75

47 Les activateurs de PXR augmentent lexpression de Spot14 dans lhépatocyte humain.

48 Spot14 est un gène cible de PXR SFN

49 Lélément TRE de promoteur du gène Spot14 est nécessaire à laction de CAR et PXR

50 Les activateurs de PXR augmentent lexpression de la FASN dans lhépatocyte humain.

51 Lexpression de Spot14 est nécessaire à linduction de la FASN par PXR

52 Lactivation de PXR provoque une accumulation dacides gras dans lhépatocyte humain. Analyses lipidomiques: Quantification des acides gras dans les hépatocytes humains chromatographie liquide à ultra haute performance (UPLC) et spectrométrie de masse (Q-TRAP) nmoles/g protéines +/- RIF (72h)

53 Palmitate IE MPE +/- RIF (72h) 3% D2O (6h) Mesure de la lipogenèse de novo : Quantification de lincorporation de deutérium dans C16:0 par chromatographie en phase gazeuse et spectrométrie de masse Lactivation de PXR provoque une augmentation de la lipogenèse de novo Michel Beylot INSERM ERI22

54 FG12 hS14 Ectopic overexpression of S14 increases lipogenesis in HepaRG cells Palmitate IE MPE FG12 hS14FG12 6mM Glucose 27mM Glucose FA quantifications De novo lipogenesis

55 En conclusion: Lactivation des xénorécepteurs induit une forte perturbation du métabolisme lipidique provoquant une accumulation des acides gras et des triglycérides hépatiques.

56 XS Role of CAR and PXR in interindividual variability in response to cancer therapy ? Toxicity Efficacy Narrow therapeutic index

57 CAR and PXR regulate genes involved in biotransformation and clearance of widely used anticancer drugs MRP1 Arsenic trioxide, chlorambucil, daunorubicin, doxorubicin, epirubicin, etoposide, melphalan, methotrexate, mitoxantrone, paclitaxel, vinblastine, vincristine MRP2 Cisplatin, irinotecan doxorubicin, etoposide, methotrexate, SN-38, vinblastine, vincristine MRP3 Carboplatin, cisplatin, doxorubicin, epirubicin, etoposide, methotrexate, teniposide, vinblastine, vincristine BCRP Imatinib, methotrexate, mitoxantrone, SN-38, topotecan CYP2A6 cyclophosphamide, ifosmamide, flutamine, tegafur CYP2B6 altretamine, cyclophosphamide, ifosmamide, tomoxifen CYP2C8 cyclophosphamide, docetaxel, ifosmamide, paclitaxel, tegafur, tretinoin CYP3A4/5 Bexarotene, busulfan, cisplatin, cyclophosphamide, cytarabine, dexamethasone, docetaxel, doxorubicin, erlotinib, etoposide, exemestane, flutamide,fulvestrant, gefitinib, ifosfamide, imatinib, irinotecan, letrozole, medroxyprogresterone acetate, mitoxantrone, paclitaxel, tamoxifen, targretin, teniposide, topotecan, toremifene, tretinoin, vinblastine, vincristine, vindesine, vinorelbine UGT doxorubicine, epirubicin, etoposide, irinotecan, topotecan, tamoxifen SULT tamoxifen GST busulfan, chlorambucil, cyclophosphamide, doxorubicin, ifosphamide, melphalan, nitrosurea

58 Role of CAR and PXR in pharmacokinetic drug-drug interaction in oncology, some examples Rifampicin Phenobarbital Phenytoin Decrease CPT11 & SN38 and increase SN38-G plasma concentrations St Johns wort Rifampicin Phenytoin Decrease cyclophosphamide and increase 4-hydroxyclyclophosphamide plasma concentrations phenobarbital Severe toxicity Therapeutic escape and risk of relapse. rifampicin Increases of erlotinib clearance and reduces the AUC by 66¨%

59 Role of CAR and PXR in cyclophosphamide activation Chang, Yu, Maurel, Waxman. Cancer Res., 1997 De Jonge, Cancer Chemother Pharmacol phenytoin

60 Role of CAR and PXR on peripheral metabolism of irinotecan (Campto ®)

61 CAR and PXR are expressed in several neoplastic human tissues Neuroblastoma (PXR) Misawa, Cancer Res, 2005 Endomedrial cancer cells (PXR) Masuyama, Mol. Pharm., 2007 Hepatocarcinoma (CAR, PXR) Huang, Mol Endocrinol, 2005 Pascussi Hepatology 2007 Intestinal & colon cancer cells (CAR, PXR) Jiang, J Gastrointest Surg, 2009 Raynal, Mol.Cancer, 2010 Ouyang, Br J Cancer, 2010 Breast tissues (PXR) Dotzlaw, 1999 Miki, Cancer Res, 2006 Lung cancer cells (PXR) Miki, Mol Cell Endocrinol, 2005 Ovarian cancer tissues (PXR) Gupta, Human Cnacer Biology, 2008 Osteosarcoma (PXR) Mensah-Osman, Cancer, 2007 Prostate cancer cells (PXR) Chen, Cancer res., 2008

62 Expression of PXR in Human Breast Carcinoma PXR OATP-A Miki et al, Cancer Res 2006; 66: (1). January 1, 2006 LCM/RT-PCR (C) carcinoma cells (S) stromal cells

63 PXR expression in normal and cancerous human prostate tissues Chen Y et al., Cancer Res 2007;67:

64 Increased chemoresistance in PC-3 cells by PXR agonist, SR Chen Y et al., Cancer Res 2007;67: CTRL 0.2 μM SR μM SR12813

65 Increased chemosensitivity of PC-3 cells with PXR expression knocked down Chen Y et al., Cancer Res 2007;67:

66 Increased chemosensitivity of endothelial HEC-1cells with PXR expression knocked down Masuyama, Mol. Pharm., 2007

67 SKOV-3 D. Gupta et al. Human cancer Biology, 2008 Increased chemoresistance in ovarian carcinoma cells by PXR agonist

68 Systemic drug clearanceCancer cells ? Role of CAR and PXR in intra-tumoral metabolism of irinotecan ?

69 Raynal C. et al. Mol. Cancer Res 2010 PXR expression in normal and cancerous human colon tissues

70 SW620 LS174T PXR expression level restricts SN38 chemosensibility in colon cancer cells

71

72 Intracellular metabolites profiles (HPLC PXR expression level enhances SN38 glucuronidation in colon cancer cells

73 SN38-G (inactive metabolite) SN38 (cytotoxic metabolite) PXR UGT1As PXR increases UGT1As-mediated SN38 inactivation in colon cancer cells

74 SN38 SN38-G SN38-G (inactive metabolite) SN38 (cytotoxic metabolite) CAR/PXR UGT1A1

75 Pro-drug activationCancer cells ? Role of CAR and PXR in intra-tumoral metabolism of cyclophosphamide ?

76 PXR regulates ALDH1A1 gene expression and Aldefluor® activity

77 Hepatic cells Cancer cells CAR and PXR may increase cancer cell resistance to 4-OH-CPA while promoting severe toxicity

78 Aldefluor®-positive cells are associated with cancer initiation properties, chemotherapy-resistance and poor clinical outcome a

79 Aldehyde Dehydrogenase 1 is a marker for normal and malignant human colonic stem cells and have tumor initiating properties CD44 ALDH1 Nuclei

80 Aldefluor®-positive Colorectal cancer stem cells are enriched in xenogeneic tumors following chemotherapy

81 Aldefluor®-positive cells display higher expression of PXR and PXR target genes PXR ALDH1A1 ABCG2 CYP3A4 OCT4 LGR5 CD26 BMI1 NANOG PXR

82 Aldefluor®-positive cells display enhanced clonogenic, sphere forming activity and magnétorésistance ALDHbrALDHlow Nb sphere / 100 cells * Soft-agar (1000 cells) – 3 weeks

83 siRNA (sibGAL / siPXR) 100nM Cell viability (ATP content) PXR knock-down decreases chemoresistance of Aldefluor®-positive cells

84 ONCO TALK - 29/01/2013 PXR knock-down decreases xenogeneic tumor recurrence Tumor formation Treatment response Relapse Folfiri: 50mg/kg 5-FU 30mg/kg irinotecan

85 cells 3D Tumor cells dissociation PXR knock-down decreases Aldefluor®-positive cells enrichment and tumor initiating activity after cytotoxics treatment FOLFIRI 1500 cells reinjection

86 PXR inhibition as a new strategy for Chemoresistant and Tumor Initiating Cells (CTIC) re-sensitization to conventional therapies ? PXR inhibition & conventional therapy Conventional therapy

87 Les xénorécepteurs CAR et PXR Gènes Cibles Enzymes et transpoteurs majeurs de la fonction de détoxication entérohépatique OATP2 CYP3A4,5,7 CYP2B6 CYP2C8,9 CYP1A2 UGT1A1,6,9 GSTs SULT2A1 MDR1 MRP2-4

88 Adduit - Hépatite fulminante - HCC Xénobiotique CAR/PXR Transporteurs Métabolites CYP450Transférases Interactions médicamenteuses Molécule thérapeutique inactif actif Perturbations métaboliques Perturbations endocriniennes - Lipides - Vitamine D Interactions croisées avec dautres voies cellulaires

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90 90

91 Conclusion Nuclear receptors PXR and CAR play a major part in this process by controlling a network of signaling pathways that regulate the expression of specific batteries of genes involved in the detoxication machinery Cells and organisms are able to increase and adapt their capacity of detoxication in response to some xenobiotics and drugs Because 1) many endocrine hormones are metabolised by CAR and PXR target genes, and 2) they interfere with other signalling pathways, chronic activation of these NRs (drugs, industrial or natural contaminants) could alter endocrine physiology and disease promotion. According to their role as masters xenobiotic responsive receptors linking DME genes expression to environment stimuli, CAR and PXR might contribute to the well-known intra- and inter-subject variability in anticancer drugs response. Environmental and genetic factors affecting CAR or PXR (expression or activation levels) may affect the cytotoxic threshold of tumor cells to chemotherapy which can consequently mask or attenuate pharmacogenetic associations.


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