WO1996013256A1 - Drugs containing nitric oxide synthase inhibitors for treating immunodeficiency diseases - Google Patents
Drugs containing nitric oxide synthase inhibitors for treating immunodeficiency diseases Download PDFInfo
- Publication number
- WO1996013256A1 WO1996013256A1 PCT/FR1995/001419 FR9501419W WO9613256A1 WO 1996013256 A1 WO1996013256 A1 WO 1996013256A1 FR 9501419 W FR9501419 W FR 9501419W WO 9613256 A1 WO9613256 A1 WO 9613256A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- cells
- pathologies
- inhibitor
- nitric oxide
- thymocytes
- Prior art date
Links
- 239000003814 drug Substances 0.000 title claims abstract description 20
- 239000000236 nitric oxide synthase inhibitor Substances 0.000 title claims abstract description 9
- 208000029462 Immunodeficiency disease Diseases 0.000 title claims description 5
- 229940079593 drug Drugs 0.000 title abstract description 5
- CSYSULGPHGCBQD-UHFFFAOYSA-N s-ethylisothiouronium diethylphosphate Chemical compound CCSC(N)=N.CCOP(O)(=O)OCC CSYSULGPHGCBQD-UHFFFAOYSA-N 0.000 title description 3
- 210000001744 T-lymphocyte Anatomy 0.000 claims abstract description 38
- 230000006907 apoptotic process Effects 0.000 claims abstract description 32
- 239000003112 inhibitor Substances 0.000 claims abstract description 27
- 230000003612 virological effect Effects 0.000 claims abstract description 15
- 230000005764 inhibitory process Effects 0.000 claims abstract description 13
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 11
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 9
- 230000006378 damage Effects 0.000 claims abstract description 7
- 229940123921 Nitric oxide synthase inhibitor Drugs 0.000 claims abstract description 6
- NTWVQPHTOUKMDI-YFKPBYRVSA-N N-Methyl-arginine Chemical compound CN[C@H](C(O)=O)CCCN=C(N)N NTWVQPHTOUKMDI-YFKPBYRVSA-N 0.000 claims abstract description 3
- 230000007170 pathology Effects 0.000 claims description 35
- 238000002360 preparation method Methods 0.000 claims description 18
- 239000000203 mixture Substances 0.000 claims description 15
- 102000019197 Superoxide Dismutase Human genes 0.000 claims description 13
- 108010012715 Superoxide dismutase Proteins 0.000 claims description 13
- 230000035755 proliferation Effects 0.000 claims description 11
- 102000016938 Catalase Human genes 0.000 claims description 10
- 108010053835 Catalase Proteins 0.000 claims description 10
- 102000008299 Nitric Oxide Synthase Human genes 0.000 claims description 10
- 108010021487 Nitric Oxide Synthase Proteins 0.000 claims description 10
- 230000001419 dependent effect Effects 0.000 claims description 9
- -1 peroxide ions Chemical class 0.000 claims description 9
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 claims description 8
- 210000002540 macrophage Anatomy 0.000 claims description 7
- 230000002538 fungal effect Effects 0.000 claims description 6
- 230000036039 immunity Effects 0.000 claims description 6
- 108010024636 Glutathione Proteins 0.000 claims description 4
- 206010061598 Immunodeficiency Diseases 0.000 claims description 4
- 206010028980 Neoplasm Diseases 0.000 claims description 4
- 239000013543 active substance Substances 0.000 claims description 4
- 229960003180 glutathione Drugs 0.000 claims description 4
- 230000007813 immunodeficiency Effects 0.000 claims description 4
- 208000015181 infectious disease Diseases 0.000 claims description 4
- 208000030159 metabolic disease Diseases 0.000 claims description 4
- 241000700605 Viruses Species 0.000 claims description 3
- 230000003211 malignant effect Effects 0.000 claims description 3
- 230000002093 peripheral effect Effects 0.000 claims description 3
- 201000011510 cancer Diseases 0.000 claims 1
- 239000008194 pharmaceutical composition Substances 0.000 claims 1
- 201000010099 disease Diseases 0.000 abstract description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 4
- 230000006052 T cell proliferation Effects 0.000 abstract 1
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 description 116
- 210000004027 cell Anatomy 0.000 description 52
- NCGICGYLBXGBGN-UHFFFAOYSA-N 3-morpholin-4-yl-1-oxa-3-azonia-2-azanidacyclopent-3-en-5-imine;hydrochloride Chemical compound Cl.[N-]1OC(=N)C=[N+]1N1CCOCC1 NCGICGYLBXGBGN-UHFFFAOYSA-N 0.000 description 14
- 210000001541 thymus gland Anatomy 0.000 description 14
- 238000000034 method Methods 0.000 description 12
- 238000000338 in vitro Methods 0.000 description 10
- 239000002609 medium Substances 0.000 description 10
- 102000017420 CD3 protein, epsilon/gamma/delta subunit Human genes 0.000 description 8
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 description 8
- 229930064664 L-arginine Natural products 0.000 description 8
- 235000014852 L-arginine Nutrition 0.000 description 8
- 238000009472 formulation Methods 0.000 description 8
- 230000008569 process Effects 0.000 description 8
- 230000034994 death Effects 0.000 description 7
- 230000004913 activation Effects 0.000 description 6
- 238000001994 activation Methods 0.000 description 6
- 238000003782 apoptosis assay Methods 0.000 description 5
- 230000007423 decrease Effects 0.000 description 5
- 210000002919 epithelial cell Anatomy 0.000 description 5
- 239000002555 ionophore Substances 0.000 description 5
- 230000000236 ionophoric effect Effects 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 230000005522 programmed cell death Effects 0.000 description 5
- 230000002992 thymic effect Effects 0.000 description 5
- 208000030507 AIDS Diseases 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 230000001640 apoptogenic effect Effects 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 229940088598 enzyme Drugs 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 210000000265 leukocyte Anatomy 0.000 description 4
- 150000002826 nitrites Chemical class 0.000 description 4
- 239000001301 oxygen Substances 0.000 description 4
- 229910052760 oxygen Inorganic materials 0.000 description 4
- 230000037361 pathway Effects 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 108010002350 Interleukin-2 Proteins 0.000 description 3
- 102000000588 Interleukin-2 Human genes 0.000 description 3
- 102000003992 Peroxidases Human genes 0.000 description 3
- 239000000427 antigen Substances 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 239000003963 antioxidant agent Substances 0.000 description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 239000000543 intermediate Substances 0.000 description 3
- 108040007629 peroxidase activity proteins Proteins 0.000 description 3
- 108020003175 receptors Proteins 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- IQFYYKKMVGJFEH-OFKYTIFKSA-N 1-[(2r,4s,5r)-4-hydroxy-5-(tritiooxymethyl)oxolan-2-yl]-5-methylpyrimidine-2,4-dione Chemical compound C1[C@H](O)[C@@H](CO[3H])O[C@H]1N1C(=O)NC(=O)C(C)=C1 IQFYYKKMVGJFEH-OFKYTIFKSA-N 0.000 description 2
- 206010003445 Ascites Diseases 0.000 description 2
- 102000000584 Calmodulin Human genes 0.000 description 2
- 108010041952 Calmodulin Proteins 0.000 description 2
- ODKSFYDXXFIFQN-SCSAIBSYSA-N D-arginine Chemical compound OC(=O)[C@H](N)CCCNC(N)=N ODKSFYDXXFIFQN-SCSAIBSYSA-N 0.000 description 2
- 229930028154 D-arginine Natural products 0.000 description 2
- 102000004388 Interleukin-4 Human genes 0.000 description 2
- 108090000978 Interleukin-4 Proteins 0.000 description 2
- RHGKLRLOHDJJDR-BYPYZUCNSA-N L-citrulline Chemical compound NC(=O)NCCC[C@H]([NH3+])C([O-])=O RHGKLRLOHDJJDR-BYPYZUCNSA-N 0.000 description 2
- 235000006708 antioxidants Nutrition 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 230000030833 cell death Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 210000002889 endothelial cell Anatomy 0.000 description 2
- 239000008187 granular material Substances 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 230000001590 oxidative effect Effects 0.000 description 2
- 230000003071 parasitic effect Effects 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 230000000638 stimulation Effects 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 230000026727 thymocyte apoptotic process Effects 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 241000228212 Aspergillus Species 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 1
- 102000029816 Collagenase Human genes 0.000 description 1
- 108060005980 Collagenase Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 241000701022 Cytomegalovirus Species 0.000 description 1
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- 102000009024 Epidermal Growth Factor Human genes 0.000 description 1
- 101800003838 Epidermal growth factor Proteins 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- 206010017533 Fungal infection Diseases 0.000 description 1
- 101001002657 Homo sapiens Interleukin-2 Proteins 0.000 description 1
- 101001002709 Homo sapiens Interleukin-4 Proteins 0.000 description 1
- 101000878605 Homo sapiens Low affinity immunoglobulin epsilon Fc receptor Proteins 0.000 description 1
- XQFRJNBWHJMXHO-RRKCRQDMSA-N IDUR Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(I)=C1 XQFRJNBWHJMXHO-RRKCRQDMSA-N 0.000 description 1
- OWYWGLHRNBIFJP-UHFFFAOYSA-N Ipazine Chemical compound CCN(CC)C1=NC(Cl)=NC(NC(C)C)=N1 OWYWGLHRNBIFJP-UHFFFAOYSA-N 0.000 description 1
- 102000011782 Keratins Human genes 0.000 description 1
- 108010076876 Keratins Proteins 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- 229930182816 L-glutamine Natural products 0.000 description 1
- 208000004554 Leishmaniasis Diseases 0.000 description 1
- 102100038007 Low affinity immunoglobulin epsilon Fc receptor Human genes 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 208000002720 Malnutrition Diseases 0.000 description 1
- 208000031888 Mycoses Diseases 0.000 description 1
- NTNWOCRCBQPEKQ-YFKPBYRVSA-N N(omega)-methyl-L-arginine Chemical compound CN=C(N)NCCC[C@H](N)C(O)=O NTNWOCRCBQPEKQ-YFKPBYRVSA-N 0.000 description 1
- NTNWOCRCBQPEKQ-UHFFFAOYSA-N NG-mono-methyl-L-arginine Natural products CN=C(N)NCCCC(N)C(O)=O NTNWOCRCBQPEKQ-UHFFFAOYSA-N 0.000 description 1
- 229940123134 Nitric oxide inhibitor Drugs 0.000 description 1
- 208000030852 Parasitic disease Diseases 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 208000001647 Renal Insufficiency Diseases 0.000 description 1
- ZIIQCSMRQKCOCT-YFKPBYRVSA-N S-nitroso-N-acetyl-D-penicillamine Chemical compound CC(=O)N[C@@H](C(O)=O)C(C)(C)SN=O ZIIQCSMRQKCOCT-YFKPBYRVSA-N 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical class [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 1
- 230000033540 T cell apoptotic process Effects 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- 102000013127 Vimentin Human genes 0.000 description 1
- 108010065472 Vimentin Proteins 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 230000003305 autocrine Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 229960002173 citrulline Drugs 0.000 description 1
- 229960002424 collagenase Drugs 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 210000004443 dendritic cell Anatomy 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 229910001882 dioxygen Inorganic materials 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 230000000546 effect on cell death Effects 0.000 description 1
- 210000003038 endothelium Anatomy 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 229940116977 epidermal growth factor Drugs 0.000 description 1
- 210000003386 epithelial cell of thymus gland Anatomy 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 210000000285 follicular dendritic cell Anatomy 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 229960002743 glutamine Drugs 0.000 description 1
- 238000001631 haemodialysis Methods 0.000 description 1
- 201000005787 hematologic cancer Diseases 0.000 description 1
- 208000024200 hematopoietic and lymphoid system neoplasm Diseases 0.000 description 1
- 230000000322 hemodialysis Effects 0.000 description 1
- 102000055229 human IL4 Human genes 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000004941 influx Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 201000006370 kidney failure Diseases 0.000 description 1
- 210000001821 langerhans cell Anatomy 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 201000004792 malaria Diseases 0.000 description 1
- 230000001071 malnutrition Effects 0.000 description 1
- 235000000824 malnutrition Nutrition 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 229940126601 medicinal product Drugs 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 210000005087 mononuclear cell Anatomy 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 230000006654 negative regulation of apoptotic process Effects 0.000 description 1
- 239000002840 nitric oxide donor Substances 0.000 description 1
- 150000002831 nitrogen free-radicals Chemical class 0.000 description 1
- 208000015380 nutritional deficiency disease Diseases 0.000 description 1
- 239000007764 o/w emulsion Substances 0.000 description 1
- 239000007800 oxidant agent Substances 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 1
- 150000002978 peroxides Chemical class 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000009696 proliferative response Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 239000002562 thickening agent Substances 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 210000005048 vimentin Anatomy 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 239000007762 w/o emulsion Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000008215 water for injection Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/06—Tripeptides
- A61K38/063—Glutathione
-
- 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
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
- A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
- A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/44—Oxidoreductases (1)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/44—Oxidoreductases (1)
- A61K38/446—Superoxide dismutase (1.15)
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the invention relates to medicaments containing, as active substance, inhibitors of nitric oxide synthase. and the use of nitric oxide synthase inhibitors for the preparation of medicaments for inhibiting the formation of nitric oxide.
- Programmed cell death by apoptosis is a morphologically distinct form of cell death that is involved in many immune responses such as thymic selection, cell-induced cytotoxicity, activation-induced death and the pathogenesis of acquired immune deficiency syndrome (Korsmeyer, SJ (1992), Immunol. Today, 13: 285-288; Cohen, JJ (1993) Immunol. Today 14: 126-130: Smith, CA, Williams, GT, guitarist. R .. Jenkinson. EJ and Owen, JJT (1989) Nature 337: 181-184; Krammer, PH. Behrmann. I .. Daniel. P ..
- NO is itself considered to be a free radical with . . . powerful anti-microbial effects (Moncada, S. and Higgs, EA (1993) N. Engl. J. Med. 329: 2002-2012; Nathan, C. (1992) FASEB J. 6: 3051-3064; Nussler, A . and Billiar, TR (1993) J. Leuk. Biol. 54: 171-178).
- molecular oxygen to form peroxynitrites, a powerful oxidizing component (Moncada. S. and Higgs, EA (1993) above; Nathan. C.
- Intrathymic negative selection by apoptosis is implicated in the suppression of self-reactive thymocytes following the ligation of TCR / CD3 by self antigens (Smith, C.A., et al. (1989) above; Von Boehmer,
- NO is synthesized from L-arginine by the enzyme NO-synthase.
- NO-synthase enzymes There are at least three types of NO-synthase enzymes:
- One aspect of the invention is to provide drugs for the prevention or treatment of apoptosis of normal and infected T cells.
- One aspect of the invention is to provide medicaments to prevent preventively or curatively the inhibition of the proliferation of T cells.
- At least one inhibitor of any molecule or group of molecules capable of participating in the synthesis of NO in the organism in particular an inhibitor of nitric oxide synthase, for the preparation of a drug intended for the treatment of pathologies involving either the inhibition by NO of the proliferation of T cells, or the destruction by NO. in particular by apoptosis, of T cells, in particular of thymocytes, in particular of T lymphocytes, the abovementioned pathologies being in particular viral pathologies.
- the invention relates to the use of at least one inhibitor of any molecule or group of molecules capable of participating in the synthesis of NO in the organism, in particular an inhibitor of nitric oxide synthase, for the preparation of a medicinal product intended for the treatment of pathologies involving either NO inhibition with the exception of
- Exogenous NO originating from macrophages from the proliferation of T cells, ie the destruction by NO, in particular by apoptosis, of T cells, in particular of thymocytes, in particular of T lymphocytes, the aforementioned pathologies being in particular viral pathologies.
- the object of the invention is therefore to avoid the formation of either endogenous NO
- this exogenous NO which can come from thymus cells other than T cells, or from cells other than those of the thymus, for example macrophages or to inhibit their proliferation, this exogenous NO can come from thymus cells other than T cells, or from cells other than thymus cells, excluding macrophages.
- - J With regard more particularly to the proliferation of T cells, the aim is of the invention to avoid their inhibition either by endogenous NO or by exogenous NO, it being understood that by exogenous NO, exogenous NO originating from of macrophages.
- exogenous NO mention may be made of NO originating from epithelial cells, from endothelial cells. Langerhans cells, and dendritic or follicular dendritic cells.
- the invention relates to the use of at least one inhibitor as defined above, for the preparation of a medicament intended for the treatment of non-bacterial pathologies.
- the invention relates to the use of at least one inhibitor as defined above, for the preparation of a medicament intended for the treatment of human pathologies.
- the invention relates to the use of at least one inhibitor, for the preparation of a medicament intended for the treatment of pathologies involving T-dependent immunity, such as pathologies of immunodeficiency d viral, parasitic or fungal origin, or pathologies of viral, parasitic or fungal origin involving T-dependent immunity, or metabolic disorders, cancers, in particular malignant hepatopathies.
- pathologies involving T-dependent immunity such as pathologies of immunodeficiency d viral, parasitic or fungal origin, or pathologies of viral, parasitic or fungal origin involving T-dependent immunity, or metabolic disorders, cancers, in particular malignant hepatopathies.
- the invention relates to the use of at least one inhibitor, for the preparation of a medicament intended for the treatment of pathologies involving T-dependent immunity, such as pathologies of immunodeficiency d 'viral or fungal origin, or pathologies of viral or fungal origin involving T-dependent immunity, or metabolic disorders, cancers, especially malignant hematopathies.
- pathologies the following can be cited:
- - viral infections such as those caused by cytomegalovirus or herpes
- the invention relates to the use of at least one nitric oxide synthase inhibitor, for the preparation of a medicament intended for the treatment of pathologies involving the apoptosis of thymocytes, in particular of lymphocytes Peripheral T, in particular viral pathologies, in particular those resulting from the infection of an individual by a virus of the HIV type.
- the nitric oxide synthase inhibitor is L-N-monomethyl-arginine (L-NMMA).
- the medicaments according to the invention are presented in an oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous and intra-articular), and topical (including oral, nasal) form, although the administration form is more appropriate depends on the patient's condition and the disease to be treated.
- the formulations suitable for oral administration can be presented in unit form such as capsules, tablets or cachets each containing a predetermined quantity of active substance; in the form of powder or granules; in the form of a solution or suspension in an aqueous liquid or a non-aqueous liquid; or in the form of a liquid oil in water emulsion or a liquid water in oil emulsion.
- Formulations for parenteral administration include sterile aqueous or non-aqueous injections which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the patient's blood; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
- the formulations can be presented in unit dose or multi-dose containers, for example sealed ampoules, and can be stored in lyophilized form requiring only the addition of sterile liquid, for example saline, water for injection, immediately before use.
- the formulations can be presented for continuous injection.
- Extemporaneous solutions and suspensions for injection can be prepared from sterile powder, granules and cachets of the kind described above.
- the formulations of the invention include other agents which are conventional in the fields with respect to the formulations in question.
- the compounds of the invention can be administered orally or by injection at a dose of
- Tablets or other forms of unit dosage form may contain an amount of compounds of the invention which is effective at such a dose, or at a multiple of such a dose, for example units containing 5 mg to 500 mg, generally from about 10 mg to 200 mg.
- the invention also relates to a new composition containing, on the one hand at least one inhibitor of any molecule or group of molecules capable of participating in the synthesis of NO in the organism, in particular an inhibitor of nitric oxide synthase, and d on the other hand at least one inhibitor of peroxide ions such as glutathione. catalase or superoxide dismutase.
- the invention also relates to a therapeutic composition comprising, as active substance, at least one of the new compositions containing, on the one hand at least one inhibitor of any molecule or group of molecules capable of participating in the synthesis of NO in the body, in particular an inhibitor of nitric oxide synthase, and on the other hand at least one inhibitor of peroxide ions such as glutathione.
- the invention also relates to the use of a composition containing, on the one hand at least one inhibitor of any molecule or group of molecules capable of contributing to the synthesis of NO in the organism, in particular a nitric oxide inhibitor synthase, and on the other hand at least one inhibitor of peroxide ions such as glutathione, catalase or superoxide dismutase, for the preparation of a medicament intended for the treatment of pathologies involving either the inhibition by NO of the proliferation of cells T, or the destruction by NO, in particular by apoptosis, of T cells, in particular of thymocytes, in particular of T lymphocytes, the abovementioned pathologies being in particular viral pathologies, and involving the inhibition of peroxide ions.
- a composition containing, on the one hand at least one inhibitor of any molecule or group of molecules capable of contributing to the synthesis of NO in the organism, in particular a nitric oxide inhibitor synthase, and on the other hand at least one inhibitor of peroxid
- the invention also relates to a method for treating pathologies involving either inhibition by NO of the proliferation of T cells, or destruction by NO, in particular by apoptosis.
- T cells especially thymocytes, in particular T lymphocytes, the abovementioned pathologies being in particular viral pathologies, by appropriate administration of an inhibitor of any molecule or group of molecules capable of participating in the synthesis of NO in the organism, in particular a nitric inhibitor- oxide synthase.
- the invention relates to a method of treatment of pathologies resulting from the infection of an individual by a virus of the HIV type.
- Thymocytes and PBL-T cells are incubated (10 6 / ml) in medium alone or in the presence of L-NMMA (1 mM), or of SIN-1 (200 ⁇ M). Viable cells are counted every other day.
- the means of 5 separate thymocyte preparations are represented (standard error mean ⁇ 15%) and a data representative of a PBL-T experiment, on three separate experiments (standard deviation ⁇ 13%).
- thymocytes are also incubated with the Ca "l ⁇ ⁇ t" ionophore (1 ⁇ M) as a positive control of apoptosis.
- the cells are recovered 36 hours later, counted and an equal number of cells (4.10 6 ), analyzed to determine the presence of the DNA fragments.
- Representative data relate to one in four thymocyte preparations.
- - Figure 3 Rescue of thymocytes, altered by NO, of apoptosis and growth in the presence of IL-2.
- Thymocytes or PBL-T cells are incubated (10 4 cells / 200 ⁇ l / well) in the presence of L-NMMA, SIN-1 or buffer for 1 hour before addition of anti-CD3 monoclonal antibody.
- monoclonal anti-CD2 or recombinant human IL-2 antibody 100 U / ml.
- 3 Htdr tritiated thymidine
- 3 Htdr tritiated thymidine
- Peripheral blood leukocytes are obtained from normal volunteers. After centrifugation on a Ficoll gradient, the T cells are isolated from other mononuclear cells by direct sorting of CD2 + cells.
- the thymus fragments are obtained from children (under 3 years of age) who are undergoing corrective heart surgery.
- the thymocytes (> 95% CD2 +) are separated from the thymic stroma by gently loosening the tissues. Complete cells of the thymic stroma are then washed thoroughly to remove residual thymocytes. The tissue is then digested with collagenase (350 U / ml, Sigma) to obtain unique cell suspensions. Pure cultures of thymus epithelial cells are obtained as detailed in Dalloul, AH, Arock, M ..
- the CD23 ligation has recently been shown to be a specific activation process linked to NO (Bécherel, PA, Mossalayi, MD, Ouaaz, F., Le Goff, L., Dugas, B., Paul-Eu seemingly, N. , Frances, C, Chosidow, O., Kilchherr, E., Guillosson, JJ, Debré, P. and Arock, M. (1994)
- the total thymocytes are cultured in a DMEM medium supplemented with L-glutamine, penicillin, streptomycin, and 5% fetal calf serum (all from Gibco-BRL) with or without: co-mitogenic anti-CD2 ⁇ + m monoclonal antibody (clones D66 and XI 1, 1/400 of ascites each; gift from A. Bernard, Nice, France); anti-CD3 antibody (clone OKT3; 20 ⁇ g / ml); ionophore Ca + + (A23187, 1 ⁇ M, Sigma) and / or rIL-2 (lOOU / ml, Boehringer Manheim, Meylan.
- co-mitogenic anti-CD2 ⁇ + m monoclonal antibody clones D66 and XI 1, 1/400 of ascites each; gift from A. Bernard, Nice, France
- anti-CD3 antibody clone OKT3; 20 ⁇ g / ml
- Apoptosis The DNA fragmentation analysis is carried out as described in Albina, J.E. et al. (1993) mentioned above. Planned cell death is also analyzed using an in situ apoptosis detection kit
- thymocytes undergo rapid death due to apoptosis in comparison with T cells derived from PBL (Smith, C.A., et al.
- Figure 1B shows programmed cell death increased following ligation with CD3, and apoptosis after addition of L-NMMA.
- the cells are pretreated with L-NMMA or SIN-1 for 1 hour before the addition of the anti-CD2 antibody, of the antibody anti-CD3 or ionophore Ca "" " " • ".
- L-NMMA saves cells from death in a medium alone (Fig. 2), and decreases cell mortality in the presence of anti-CD2 antibodies or antibodies anti-CD3, but has no effect on cell death induced by ionophore
- IL-2 during thymocyte activation saves most thymocytes from apoptosis and induces their proliferation (Cohen, J.J. (1993) mentioned above).
- L-NMMA increases the proliferative responses of thymocytes (p ⁇ 0.001), while it does not induce cell growth of PBL-T (Fig. 3).
- SIN-1 hinders the growth of thymocytes in the presence of IL-2 (Fig. 3).
- SIN-1 shows an inhibitory effect on their proliferation induced both via CD3 or via CD2 with less intensity with respect to the CD3 pathway, than what is observed in thymocytes ( p ⁇ 0.01) (Fig. 3).
- thymocytes are involved in a NO process and may be able to produce nitrites, one of the end products of NO metabolism (Moncada, S. and Higgs, EA (1993) mentioned above. ; Nathan, C. (1992) above; Nussler, A. and Billiar, TR (1993) above; Stuehr, DJ and Griffith, OW (1992) above).
- DMEM medium for 4 days, significant amounts of nitrites are detected in culture supernatants derived from thymocytes in the absence of apparent additional activation.
- Thymocyte-free stroma cells of the same thymus have significantly higher (p ⁇ 0.001) levels of nitrites in their supernatants. These data demonstrate the possible exocrine and autocrine sources of NO for human thymocytes. In order to clarify the cellular origin of NO among cells of the human thymic stroma, we tested the possibility of purified epithelial cells derived from the thymus, to produce nitrites in vitro.
- Thymocytes were also treated with SIN-1 (NO and ONOO donor) or SNAP (NO donor) and their apoptosis was quantified.
- the cells are also incubated with CD3 antibody in the presence of L-NMMA, SOD or catalase. While L-NMMA inhibits the generation of
- NO, SOD and catalase are peroxide inhibitors. Similar inhibition of apoptosis is achieved with L-NMMA, SOD or both. The absence of synergy between these two agents militates in favor of the involvement of peroxy nitrites, but against a direct role of NO in this phenomenon. This correlates to the fact that higher thymocyte apoptosis is observed in the presence of SIN-1, compared to cells treated with SNAP.
- Thymocytes are incubated in the medium alone or in the presence of the anti-CD3 antibody (20 ⁇ g / ml). Some cultures are supplemented with SIN-1 (200 ⁇ M), SNAP-1 (1 mM), L-NMMA (1 ⁇ iM), superoxide dismutase (SOD, 120 units / ml), or catalase (240 units / ml), 1 hour before the addition of the anti-CD3 antibody. The cells are analyzed 48 hours later to determine the percentage of cells which undergo apoptosis by the Apoptag kit (peroxidase +), marketed Oncor (Gaithersburg, MD, USA). The results given in the table below correspond to the standard deviation, from three distinct thymuses.
- Peripheral blood mononuclear cells or spleen CD4 + cells from subjects suffering from AIDS, are incubated in the medium alone or in the presence of the anti-CD3 antibody (20 ⁇ g / ml). Some cultures are supplemented with SNAP (ImM) or L-NMMA (ImM), 1 hour before the addition of the anti-CD3 antibody. The cells are analyzed 48 hours later to determine the number of cells as well as the percentage of cells which undergo apoptosis by the Apoptag kit (peroxidase +), marketed by Oncor (Gaithersburg, MD, USA). The results given in the table below are the average of two ⁇ tests. the standard deviation. % apoptotic cells number of cells ⁇ l (/ ml
- the chemical donors of NO exert an apoptotic effect on the thymocytes as well as the T cells originating from HIV --- subjects.
- the in vitro inhibition of the NO pathway by L-NMMA allows prolonged survival of these cells. These latest data are in favor of an in vivo role of NO in the apoptosis of these cells.
- the inhibition of this pathway can thus constitute an appropriate treatment to increase the number of T lymphocytes (more cells produced by the thymus) and / or decrease their mortality in vivo (less apoptosis of peripheral lymphocytes).
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Epidemiology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Chemical & Material Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Gastroenterology & Hepatology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The use of at least one inhibitor of any molecule or group of molecules that may take part in NO synthesis in the body, particularly a nitric oxide synthase inhibitor such as L-N-monomethyl-arginine (L-NMMA), for preparing a drug for treating diseases involving the inhibition by NO of T cell proliferation or the destruction by NO, and particularly by apoptosis, of T cells, especially thymocytes, and more especially T lymphocytes, said diseases in particular being viral diseases.
Description
MEDICAMENTS CONTENANT DES INHIBITEURS DE LA NITRIQUE-OXYDE SYNTHASE POUR LE TRAITEMENT DES PATHOLOGIES D ' IMMUNODEFICIENCEDRUGS CONTAINING NITRIC OXIDE SYNTHASE INHIBITORS FOR THE TREATMENT OF IMMUNODEFICIENCY CONDITIONS
L'invention a pour objet des médicaments contenant, à titre de substance active, des inhibiteurs de la nitrique-oxyde synthase. et l'utilisation d'inhibiteurs de la nitrique-oxyde synthase pour la préparation de médicament destinés à inhiber la formation d'oxyde nitrique.The invention relates to medicaments containing, as active substance, inhibitors of nitric oxide synthase. and the use of nitric oxide synthase inhibitors for the preparation of medicaments for inhibiting the formation of nitric oxide.
La mort programmée des cellules par apoptose est une forme morphologiquement distincte de mortalité cellulaire qui est impliquée dans beaucoup de réactions immunitaires telles que la sélection thymique, la cytotoxicité induite par des cellules, la mort induite par activation et la pathogénèse du syndrome de déficience immunitaire acquise (Korsmeyer, S.J. (1992), Immunol. Today, 13: 285-288; Cohen, J.J. (1993) Immunol. Today 14: 126-130: Smith, C.A., Williams, G.T., Kingston. R.. Jenkinson. E.J. et Owen, J.J.T. (1989) Nature 337: 181-184; Krammer, P. H.. Behrmann. I.. Daniel. P..Programmed cell death by apoptosis is a morphologically distinct form of cell death that is involved in many immune responses such as thymic selection, cell-induced cytotoxicity, activation-induced death and the pathogenesis of acquired immune deficiency syndrome (Korsmeyer, SJ (1992), Immunol. Today, 13: 285-288; Cohen, JJ (1993) Immunol. Today 14: 126-130: Smith, CA, Williams, GT, Kingston. R .. Jenkinson. EJ and Owen, JJT (1989) Nature 337: 181-184; Krammer, PH. Behrmann. I .. Daniel. P ..
Dhein, J. et Debatin. K.M. (1994) Curr. Opin. Immunol. 6: 279-289; Gougeon, M.L. , Olivier, R., Garcia, S., Guetard, D., Dragic, T., Dauguet, C. et Montagnier, L. (1991) Comptes Rendu Acad. Sci. 312: 529-537; Terai, C, Kornbluth, R.S., Pauza, CD., Richman, D.D. et Carsen, D.A. (1991) J. Clin. Invest. 87: 1710-1715; Groux, H.G., Torpier. G. , Monte, D. , Mouton, Y. ,Dhein, J. and Debatin. K.M. (1994) Curr. Opin. Immunol. 6: 279-289; Gougeon, M.L., Olivier, R., Garcia, S., Guetard, D., Dragic, T., Dauguet, C. and Montagnier, L. (1991) Comptes Rendu Acad. Sci. 312: 529-537; Terai, C, Kornbluth, R.S., Pauza, CD., Richman, D.D. and Carsen, D.A. (1991) J. Clin. Invest. 87: 1710-1715; Groux, H.G., Torpier. G., Monte, D., Mouton, Y.,
Capron, A. et Amiesen, J.C. (1992) J. Exp. Med. 175: 331-340; Meyaard, L., Otto, S.A., Jonker, R.R., Mijnster, M.J., Keet. R.P.M. et Miedema. F. (1992) Science 257: 217-219).Capron, A. and Amiesen, J.C. (1992) J. Exp. Med. 175: 331-340; Meyaard, L., Otto, S.A., Jonker, R.R., Mijnster, M.J., Keet. R.P.M. and Miedema. F. (1992) Science 257: 217-219).
Des études récentes suggèrent un rôle pour les intermédiaires de l'oxygène dans l' apoptose de quelques lignées cellulaires T humaines et des lymphocytes T infectés par HIV (Sandstorm, P. A., Tebbey, P.W., Van Cleave. S. et Buttke, T.M. (1994) J. Biol. Che ., 269: 798-801 ; Buttke, T.M. et Sandstorm, P.A. (1994) Immunol. Today, 15: 7-10). L'addition d'intermédiaires de l'oxygène ou la diminution d' anti-oxydants cellulaires peut induire la mort cellulaire programmée, tandis que les enzymes antioxydants bloquent ce phénomèneRecent studies suggest a role for oxygen intermediates in the apoptosis of some human T cell lines and T cells infected with HIV (Sandstorm, PA, Tebbey, PW, Van Cleave. S. and Buttke, TM (1994) ) J. Biol. Che., 269: 798-801; Buttke, TM and Sandstorm, PA (1994) Immunol. Today, 15: 7-10). Addition of oxygen intermediates or reduction of cellular antioxidants can induce programmed cell death, while antioxidant enzymes block this phenomenon.
(Sandstorm, P.A. et al. (1994) susmentionné; Buttke, T.M. et Sandstorm, P.A. (1994) susmentionné; Lennon, S.V., Martin, S.J. et Cotter, T. G. (1991) Cell. Prolif. 24: 203-214; Larrick, J.W. et Wright, S.C. (1990) FASEB J. 4: 3215- 3223). En plus des intermédiaires réactifs de l'oxygène tels que H2O2 et OHβ, l'oxyde nitrique (NO) a été récemment impliqué comme un inducteur de l'apoptose dans les monocytes/macrophages (Albina. J.E., Lui, S., Mateo, R.B. et Reicher, J.S. (1993) J. Immunol. 150: 5080-5085). Ayant un électron unique non apparié, NO est lui-même considéré comme étant un radical libre avec des
. . . effets anti-microbiens puissants (Moncada, S. et Higgs, E.A. (1993) N. Engl. J. Med. 329: 2002-2012; Nathan, C. (1992) FASEB J. 6: 3051-3064; Nussler, A. et Billiar, T.R. (1993) J. Leuk. Biol. 54: 171-178). Cependant, il réagit aussi avec l'oxygène moléculaire pour former des péroxynitrites, un composant oxydant puissant (Moncada. S. et Higgs, E.A. (1993) susmentionné; Nathan. C.(Sandstorm, PA et al. (1994) above; Buttke, TM and Sandstorm, PA (1994) above; Lennon, SV, Martin, SJ and Cotter, TG (1991) Cell. Prolif. 24: 203-214; Larrick, JW and Wright, SC (1990) FASEB J. 4: 3215-3223). In addition to reactive oxygen intermediates such as H2O2 and OH β , nitric oxide (NO) has recently been implicated as an inducer of apoptosis in monocytes / macrophages (Albina. JE, Lui, S., Mateo , RB and Reicher, JS (1993) J. Immunol. 150: 5080-5085). Having a single unpaired electron, NO is itself considered to be a free radical with . . . powerful anti-microbial effects (Moncada, S. and Higgs, EA (1993) N. Engl. J. Med. 329: 2002-2012; Nathan, C. (1992) FASEB J. 6: 3051-3064; Nussler, A . and Billiar, TR (1993) J. Leuk. Biol. 54: 171-178). However, it also reacts with molecular oxygen to form peroxynitrites, a powerful oxidizing component (Moncada. S. and Higgs, EA (1993) above; Nathan. C.
(1992) susmentionné; Nussler, A. et Billiar, T.R. (1993) susmentionné; Stuehr, D.J. et Griffith, O.W. (1992) Adv. Enzymol. 65: 287-346).(1992) above; Nussler, A. and Billiar, T.R. (1993) above; Stuehr, D.J. and Griffith, O.W. (1992) Adv. Enzymol. 65: 287-346).
Une sélection négative intrathymique par apoptose est impliquée dans la suppression de thymocytes autoréactifs à la suite de la ligation de TCR/CD3 par les antigènes du soi (Smith, C.A., et al. (1989) susmentionné; Von Boehmer,Intrathymic negative selection by apoptosis is implicated in the suppression of self-reactive thymocytes following the ligation of TCR / CD3 by self antigens (Smith, C.A., et al. (1989) above; Von Boehmer,
H., (1992) Immunol. Today 13: 454-458). In vitro, la mort cellulaire programmée des thymocytes humains peut être également obtenue ou accélérée par le biais de la ligation des antigènes de surface CD3/TCR (Smith. C.A. et al. (1989) susmentionné), CD2 (Merkenschlager, M. et Fischer, A. G. (1991) Int. Immunol. 3: 1-7) ou Apo-1 (Krammer et al. (1994) susmentionné), par les anticorps appropriés. On pensait que l'influx de Ca+ + qui suit l'activation cellulaire par l'intermédiaire de ces récepteurs est le second messager principal impliqué dans la mort programmée cellulaire (King, L.B. et Ashwell. J.D.H., (1992) Immunol. Today 13: 454-458). In vitro, the programmed cell death of human thymocytes can also be obtained or accelerated by ligation of the surface antigens CD3 / TCR (Smith. CA et al. (1989) mentioned above), CD2 (Merkenschlager, M. and Fischer , AG (1991) Int. Immunol. 3: 1-7) or Apo-1 (Krammer et al. (1994) above), by appropriate antibodies. The influx of Ca ++ that follows cellular activation through these receptors was thought to be the second main messenger involved in programmed cell death (King, LB and Ashwell. JD
(1993) Curr. Opin. Immunol. 5: 368-373). Cependant, les données maintenant s'accumulent sur le rôle possible de la poussée oxydative comme médiateurs de l' apoptose cellulaire T (Gougeon, M.L. et al. (1991) susmentionné; Terai, C. et al. (1991) susmentionné; Groux, H.G. et al. (1992) susmentionné; Meyaard. L. et al. (1992) susmentionné; Sandstorm, P.A. et al. (1994) susmentionné; Buttke, T.M. et Sanstorm, P.A. (1994) susmentionné; Lennon, S.V. et al. (1991) susmentionné; Larrick, J.W. et Wright, S.C. (1990) susmentionné; Albina, J.E. et al. (1993) susmentionné). Cependant, les formes des radicaux oxygénés directement impliquées dans cette apoptose n'étaient pas caractérisées. Ces données conduisent à investiguer si le processus de transduction L-arginine/NO a une quelconque fonction dans la mort programmée cellulaire des thymocytes humains normaux et les lymphocytes T provenant de sujets HIV positifs. On démontre l'implication des oxydants, en particulier des radicaux d'azote, comme médiateurs de l' apoptose de ces cellules.(1993) Curr. Opin. Immunol. 5: 368-373). However, data are now accumulating on the possible role of oxidative surge as mediators of T cell apoptosis (Gougeon, ML et al. (1991) above; Terai, C. et al. (1991) above; Groux, HG et al. (1992) above; Meyaard. L. et al. (1992) above; Sandstorm, PA et al. (1994) above; Buttke, TM and Sanstorm, PA (1994) above; Lennon, SV et al. (1991) above; Larrick, JW and Wright, SC (1990) above; Albina, JE et al. (1993) above). However, the forms of oxygen radicals directly involved in this apoptosis were not characterized. These data lead us to investigate whether the L-arginine / NO transduction process has any function in the programmed cell death of normal human thymocytes and T lymphocytes originating from HIV positive subjects. The involvement of oxidants, in particular nitrogen radicals, as mediators of apoptosis of these cells is demonstrated.
On sait que NO est synthétisé à partir de la L-arginine par l'enzyme NO- synthase. On sait qu'il y a au moins trois types d'enzymes de NO-synthase:We know that NO is synthesized from L-arginine by the enzyme NO-synthase. We know that there are at least three types of NO-synthase enzymes:
- une enzyme constitutive, dépendante de Ca+ +/calmoduline, située dans l'endothélium, qui relâche NO en réponse à un récepteur ou une stimulation physique,
- une enzyme dépendante de Ca+ ' calmoduline. située dans le cerveau, qui relâche NO en réponse à un récepteur ou une stimulation physique,- a constitutive enzyme, dependent on Ca ++ / calmodulin, located in the endothelium, which releases NO in response to a receptor or physical stimulation, - an enzyme dependent on Ca + ' calmodulin. located in the brain, which releases NO in response to a receptor or physical stimulation,
- une enzyme indépendante de Ca+ + qui est induite après activation de muscle vasculaire lisse, de macrophages, de cellules endothéliales et un nombre d'autres cellules par l'endotoxine et les cytokines.- an enzyme independent of Ca ++ which is induced after activation of smooth vascular muscle, macrophages, endothelial cells and a number of other cells by endotoxin and cytokines.
Cependant, à ce jour, la relation entre l'apoptose des cellules T, normales ou infectées, et la voie NO n'a pas été établie.However, to date, the relationship between apoptosis of normal or infected T cells and the NO pathway has not been established.
L'un des aspects de l'invention est de proposer des médicaments pour la prévention ou le traitement de l'apoptose de cellules T normales et infectées. L'un des aspects de l'invention est de proposer des médicaments pour empêcher de façon préventive ou curative l'inhibition de la prolifération des cellules T.One aspect of the invention is to provide drugs for the prevention or treatment of apoptosis of normal and infected T cells. One aspect of the invention is to provide medicaments to prevent preventively or curatively the inhibition of the proliferation of T cells.
Ces différents aspects sont rendus possible par l'utilisation d'au moins un inhibiteur de toute molécule ou groupe de molécules susceptible de participer à la synthèse de NO dans l'organisme, notamment un inhibiteur de la nitrique- oxyde synthase, pour la préparation d'un médicament destiné au traitement de pathologies impliquant soit l'inhibition par NO de la prolifération de cellules T, soit la destruction par NO. notamment par apoptose, de cellules T, en particulier de thymocytes, notamment de lymphocytes T, les susdites pathologies étant notamment des pathologies virales.These various aspects are made possible by the use of at least one inhibitor of any molecule or group of molecules capable of participating in the synthesis of NO in the organism, in particular an inhibitor of nitric oxide synthase, for the preparation of a drug intended for the treatment of pathologies involving either the inhibition by NO of the proliferation of T cells, or the destruction by NO. in particular by apoptosis, of T cells, in particular of thymocytes, in particular of T lymphocytes, the abovementioned pathologies being in particular viral pathologies.
Selon un mode de réalisation avantageux, l'invention concerne l'utilisation d'au moins un inhibiteur de toute molécule ou groupe de molécules susceptible de participer à la synthèse de NO dans l'organisme, notamment un inhibiteur de la nitrique-oxyde synthase, pour la préparation d'un médicament destiné au traitement de pathologies impliquant soit l'inhibition par NO à l'exception duAccording to an advantageous embodiment, the invention relates to the use of at least one inhibitor of any molecule or group of molecules capable of participating in the synthesis of NO in the organism, in particular an inhibitor of nitric oxide synthase, for the preparation of a medicinal product intended for the treatment of pathologies involving either NO inhibition with the exception of
NO exogène provenant de macrophages, de la prolifération de cellules T, soit la destruction par NO, notamment par apoptose, de cellules T, en particulier de thymocytes, notamment de lymphocytes T, les susdites pathologies étant notamment des pathologies virales. Le but de l'invention est donc d'éviter la formation soit de NO endogèneExogenous NO originating from macrophages, from the proliferation of T cells, ie the destruction by NO, in particular by apoptosis, of T cells, in particular of thymocytes, in particular of T lymphocytes, the aforementioned pathologies being in particular viral pathologies. The object of the invention is therefore to avoid the formation of either endogenous NO
(dans les cellules T), soit de NO exogène, qui serait susceptible(in T cells), or exogenous NO, which would be susceptible
- de détruire, notamment par apoptose les cellules T, cet NO exogène pouvant provenir de cellules du thymus autres que les cellules T, ou de cellules autres que celles du thymus, par exemple des macrophages ou d'inhiber leur prolifération, cet NO exogène pouvant provenir de cellules du thymus autres que les cellules T, ou de cellules autres que celles du thymus, à l'exclusion des macrophages.
- J - S'agissant plus particulièrement de la prolifération des cellules T, le but est de l'invention est d'éviter leur inhibition soit par NO endogène, soit par NO exogène, étant entendu que par NO exogène, on exclut NO exogène provenant de macrophages. A titre d'exemple de NO exogène, on peut citer NO provenant des cellules épithéliales, des cellules endothéliales. des cellules de Langerhans, et des cellules dendritiques ou folliculaires dendritiques.- to destroy, in particular by apoptosis the T cells, this exogenous NO which can come from thymus cells other than T cells, or from cells other than those of the thymus, for example macrophages or to inhibit their proliferation, this exogenous NO can come from thymus cells other than T cells, or from cells other than thymus cells, excluding macrophages. - J - With regard more particularly to the proliferation of T cells, the aim is of the invention to avoid their inhibition either by endogenous NO or by exogenous NO, it being understood that by exogenous NO, exogenous NO originating from of macrophages. As an example of exogenous NO, mention may be made of NO originating from epithelial cells, from endothelial cells. Langerhans cells, and dendritic or follicular dendritic cells.
Selon un mode de réalisation avantageux, l'invention a pour objet l'utilisation d'au moins un inhibiteur tel que défini ci-dessus, pour la préparation d'un médicament destiné au traitement de pathologies non bactériennes.According to an advantageous embodiment, the invention relates to the use of at least one inhibitor as defined above, for the preparation of a medicament intended for the treatment of non-bacterial pathologies.
Selon un mode de réalisation avantageux, l'invention a pour objet l'utilisation d'au moins un inhibiteur tel que défini ci-dessus, pour la préparation d'un médicament destiné au traitement de pathologies humaines.According to an advantageous embodiment, the invention relates to the use of at least one inhibitor as defined above, for the preparation of a medicament intended for the treatment of human pathologies.
Selon un mode de réalisation avantageux, l'invention a pour objet l'utilisation d'au moins un inhibiteur, pour la préparation d'un médicament destiné au traitement de pathologies impliquant l'immunité T dépendante, telles que des pathologies d'immunodéficience d'origine virale, parasitaire ou fungique, ou des pathologies d'origine virale, parasitaire ou fungique impliquant l'immunité T dépendante, ou de troubles métaboliques, de cancers, notamment d'hé atopathies malignes.According to an advantageous embodiment, the invention relates to the use of at least one inhibitor, for the preparation of a medicament intended for the treatment of pathologies involving T-dependent immunity, such as pathologies of immunodeficiency d viral, parasitic or fungal origin, or pathologies of viral, parasitic or fungal origin involving T-dependent immunity, or metabolic disorders, cancers, in particular malignant hepatopathies.
Selon un mode de réalisation avantageux, l'invention a pour objet l'utilisation d'au moins un inhibiteur, pour la préparation d'un médicament destiné au traitement de pathologies impliquant l'immunité T dépendante, telles que des pathologies d'immunodéficience d'origine virale, ou fungique, ou des pathologies d'origine virale, ou fungique impliquant l'immunité T dépendante, ou de troubles métaboliques, de cancers, notamment d'hématopathies malignes. Comme pathologies, on peut citer les suivantes:According to an advantageous embodiment, the invention relates to the use of at least one inhibitor, for the preparation of a medicament intended for the treatment of pathologies involving T-dependent immunity, such as pathologies of immunodeficiency d 'viral or fungal origin, or pathologies of viral or fungal origin involving T-dependent immunity, or metabolic disorders, cancers, especially malignant hematopathies. As pathologies, the following can be cited:
- états inflammatoires relatifs à la réponse immune affectant les lymphocytes T; - syndrome d'immunodéficience acquise (SIDA) suite à l'infection par le- inflammatory states relating to the immune response affecting T lymphocytes; - acquired immunodeficiency syndrome (AIDS) following infection with
HIV;HIV;
- infections virales telles que celles provoquées par le cytomégalovirus ou l'herpès;- viral infections such as those caused by cytomegalovirus or herpes;
- infections fungiques telles que celles provoquées par Candida ou Aspergillus;- fungal infections such as those caused by Candida or Aspergillus;
- infections parasitaires telles que la malaria ou la leishmaniose;- parasitic infections such as malaria or leishmaniasis;
- post chimiothérapie dans les leucémies, les lymphomes et les cancers non hématopoïétiques ;
96/13256 PC-7FR95/01419- post chemotherapy in leukemias, lymphomas and non-hematopoietic cancers; 96/13256 PC-7FR95 / 01419
- 5 -- 5 -
- post radiothérapie dans les cancers désignés ci-dessus;- post radiotherapy in the cancers designated above;
- troubles métaboliques (malnutrition protéique(s), insuffisance rénale et hémodialyse).- metabolic disorders (protein malnutrition (s), renal failure and hemodialysis).
Selon un autre mode de réalisation avantageux, l'invention concerne l'utilisation d'au moins un inhibiteur de la nitrique-oxyde synthase, pour la préparation d'un médicament destiné au traitement de pathologies impliquant l'apoptose de thymocytes, notamment de lymphocytes T périphériques, en particulier de pathologies virales, notamment celles résultant de l'infection d'un individu par un virus du type HIV. Selon un mode de réalisation particulier de l'invention, l'inhibiteur de la nitrique-oxyde synthase est la L-N-monométhyl-arginine (L-NMMA).According to another advantageous embodiment, the invention relates to the use of at least one nitric oxide synthase inhibitor, for the preparation of a medicament intended for the treatment of pathologies involving the apoptosis of thymocytes, in particular of lymphocytes Peripheral T, in particular viral pathologies, in particular those resulting from the infection of an individual by a virus of the HIV type. According to a particular embodiment of the invention, the nitric oxide synthase inhibitor is L-N-monomethyl-arginine (L-NMMA).
Les médicaments selon l'invention se présentent sous forme administrable par voie orale, parentérale (incluant sous-cutanée. intradermique, intramusculaire, intraveineuse et intra-articulaire), et topique (incluant buccale, nasale), bien que la forme d'administration la plus appropriée dépende de l'état du patient et de la maladie à traiter.The medicaments according to the invention are presented in an oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous and intra-articular), and topical (including oral, nasal) form, although the administration form is more appropriate depends on the patient's condition and the disease to be treated.
Les formulations appropriées pour l'administration orale peuvent être présentées sous forme unitaire telles que capsules, comprimés ou cachets contenant chacun une quantité prédéterminée de substance active; sous forme de poudre ou de granulés; sous forme de solution ou de suspension dans un liquide aqueux ou un liquide non aqueux; ou sous forme d'émulsion liquide d'huile dans l'eau ou émulsion liquide d'eau dans l'huile.The formulations suitable for oral administration can be presented in unit form such as capsules, tablets or cachets each containing a predetermined quantity of active substance; in the form of powder or granules; in the form of a solution or suspension in an aqueous liquid or a non-aqueous liquid; or in the form of a liquid oil in water emulsion or a liquid water in oil emulsion.
Les formulations pour l' administration parentérale incluent les injections stériles aqueuses ou non aqueuses qui peuvent contenir des anti-oxydants, des tampons, des agents bactériostatiques et des solutés qui rendent la formulation isotonique avec le sang du malade; et les suspensions stériles aqueuses et non aqueuses qui peuvent inclure des agents de suspension et des agents de d'épaississement. Les formulations peuvent être présentées dans des conteneurs de doses unitaires ou multidoses, par exemples des ampoules scellées, et peuvent être stockées sous forme lyophilisée nécessitant seulement l'addition de liquide stérile, par exemple soluté salin, eau pour injection, immédiatement avant utilisation.Formulations for parenteral administration include sterile aqueous or non-aqueous injections which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the patient's blood; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations can be presented in unit dose or multi-dose containers, for example sealed ampoules, and can be stored in lyophilized form requiring only the addition of sterile liquid, for example saline, water for injection, immediately before use.
Dans un autre mode de réalisation, les formulations peuvent être présentées pour une injection continue. Des solutions et des suspensions extemporanées pour injection peuvent être préparées à partir de poudre stérile, de granulés et de cachets du genre décrit ci-dessus.
II doit aussi être compris qu'en plus des ingrédients mentionnés ci-dessus, les formulations de l'invention comprennent d'autres agents classiques dans les domaines vis à vis des formulations en question.In another embodiment, the formulations can be presented for continuous injection. Extemporaneous solutions and suspensions for injection can be prepared from sterile powder, granules and cachets of the kind described above. It should also be understood that in addition to the ingredients mentioned above, the formulations of the invention include other agents which are conventional in the fields with respect to the formulations in question.
Pour chacune des formulations mentionnées ci-dessus, les composés de l'invention peuvent être administrés oralement ou par injection à une dose deFor each of the formulations mentioned above, the compounds of the invention can be administered orally or by injection at a dose of
0,1 à 500 mg/kg/jour. La dose pour les adultes est généralement de 5 mg à 35 g/jour, de préférence 5 mg à 2 g/jour. Les comprimés ou autres formes de présentation sous forme unitaire peuvent contenir une quantité de composés de l'invention qui est efficace à une telle dose, ou à un multiple d'une telle dose, par exemple des unités contenant 5 mg à 500 mg, généralement d'environ 10 mg à 200 mg.0.1 to 500 mg / kg / day. The dose for adults is usually 5 mg to 35 g / day, preferably 5 mg to 2 g / day. Tablets or other forms of unit dosage form may contain an amount of compounds of the invention which is effective at such a dose, or at a multiple of such a dose, for example units containing 5 mg to 500 mg, generally from about 10 mg to 200 mg.
L'invention concerne également une nouvelle composition contenant, d'une part au moins un inhibiteur de toute molécule ou groupe de molécules susceptible de participer à la synthèse de NO dans l'organisme, notamment un inhibiteur de la nitrique-oxyde synthase, et d'autre part au moins un inhibiteur des ions peroxydes tel que le glutathion. la catalase ou la superoxyde dismutase. L'invention concerne également une composition thérapeutique comprenant, à titre de substance active, l'une au moins des nouvelles compositions contenant, d'une part au moins un inhibiteur de toute molécule ou groupe de molécules susceptible de participer à la synthèse de NO dans l'organisme, notamment un inhibiteur de la nitrique-oxyde synthase, et d'autre part au moins un inhibiteur des ions peroxydes tel que le glutathion. la catalase ou la superoxyde dismutase, en association avec un véhicule pharmaceutiquement acceptable. L'invention concerne également l'utilisation d'une composition contenant, d'une part au moins un inhibiteur de toute molécule ou groupe de molécules susceptible de paπiciper à la synthèse de NO dans l'organisme, notamment un inhibiteur de la nitrique-oxyde synthase, et d'autre part au moins un inhibiteur des ions peroxydes tel que le glutathion, la catalase ou la superoxyde dismutase, pour la préparation d'un médicament destiné au traitement de pathologies impliquant soit l'inhibition par NO de la prolifération de cellules T, soit la destruction par NO, notamment par apoptose, de cellules T, en particulier de thymocytes, notamment de lymphocytes T, les susdites pathologies étant notamment des pathologies virales, et impliquant l'inhibition des ions peroxydes.The invention also relates to a new composition containing, on the one hand at least one inhibitor of any molecule or group of molecules capable of participating in the synthesis of NO in the organism, in particular an inhibitor of nitric oxide synthase, and d on the other hand at least one inhibitor of peroxide ions such as glutathione. catalase or superoxide dismutase. The invention also relates to a therapeutic composition comprising, as active substance, at least one of the new compositions containing, on the one hand at least one inhibitor of any molecule or group of molecules capable of participating in the synthesis of NO in the body, in particular an inhibitor of nitric oxide synthase, and on the other hand at least one inhibitor of peroxide ions such as glutathione. catalase or superoxide dismutase, in combination with a pharmaceutically acceptable vehicle. The invention also relates to the use of a composition containing, on the one hand at least one inhibitor of any molecule or group of molecules capable of contributing to the synthesis of NO in the organism, in particular a nitric oxide inhibitor synthase, and on the other hand at least one inhibitor of peroxide ions such as glutathione, catalase or superoxide dismutase, for the preparation of a medicament intended for the treatment of pathologies involving either the inhibition by NO of the proliferation of cells T, or the destruction by NO, in particular by apoptosis, of T cells, in particular of thymocytes, in particular of T lymphocytes, the abovementioned pathologies being in particular viral pathologies, and involving the inhibition of peroxide ions.
L'invention concerne également un procédé de traitement de pathologies impliquant soit l'inhibition par NO de la prolifération de cellules T, soit la destruction par NO, notamment par apoptose. de cellules T, en particulier de
thymocytes, notamment de lymphocytes T, les susdites pathologies étant notamment des pathologies virales, par administration appropriée d'un inhibiteur de toute molécule ou groupe de molécules susceptible de participer à la synthèse de NO dans l'organisme, notamment un inhibiteur de la nitrique-oxyde synthase.The invention also relates to a method for treating pathologies involving either inhibition by NO of the proliferation of T cells, or destruction by NO, in particular by apoptosis. T cells, especially thymocytes, in particular T lymphocytes, the abovementioned pathologies being in particular viral pathologies, by appropriate administration of an inhibitor of any molecule or group of molecules capable of participating in the synthesis of NO in the organism, in particular a nitric inhibitor- oxide synthase.
Selon un mode de réalisation avantageux, l'invention concerne une méthode de traitement de pathologies résultant de l'infection d'un individu par un virus du type HIV.According to an advantageous embodiment, the invention relates to a method of treatment of pathologies resulting from the infection of an individual by a virus of the HIV type.
Légendes des figures.Legends of figures.
- Figures 1A et 1B: Rôle du processus dépendant de L-arginine dans la mort in vitro de thymocytes humains.- Figures 1A and 1B: Role of the L-arginine dependent process in the in vitro death of human thymocytes.
1A) Des thymocytes et des cellules PBL-T sont incubés (106/ml) dans du milieu seul ou en présence de L-NMMA (1 mM), ou de SIN-1 (200 μM). Les cellules viables sont comptées tous les deux jours. On a représenté les moyennes de 5 préparations de thymocytes distinctes (moyenne d'erreur standard < 15%) et une donnée représentative d'une expérience de PBL-T, sur trois expériences distinctes (déviation standard < 13%).1A) Thymocytes and PBL-T cells are incubated (10 6 / ml) in medium alone or in the presence of L-NMMA (1 mM), or of SIN-1 (200 μM). Viable cells are counted every other day. The means of 5 separate thymocyte preparations are represented (standard error mean <15%) and a data representative of a PBL-T experiment, on three separate experiments (standard deviation <13%).
1B) Effet inhibiteur de L-NMMA sur l'apoptose des thymocytes en milieu seul ou suite à la ligation de CD3. Les thymocytes sont incubés en milieu seul1B) Inhibitory effect of L-NMMA on the apoptosis of thymocytes in medium alone or following the ligation of CD3. Thymocytes are incubated in medium alone
(indiqué par "rien" sur la figure), avec L-NMMA, avec l'anticorps monoclonal anti-CD3 (20 μg/ml), ou avec les deux. Les thymocytes sont aussi incubés avec l'ionophore Ca"l~ ~t" (1 μM) comme contrôle positif de l'apoptose. Les cellules sont récupérées 36 heures plus tard, comptées et un nombre égal de cellules (4.106), analysées pour déterminer la présence des fragments d'ADN. Les données représentatives concernent une préparation de thymocytes sur quatre.(indicated by "nothing" in the figure), with L-NMMA, with the anti-CD3 monoclonal antibody (20 μg / ml), or with both. The thymocytes are also incubated with the Ca "l ~ ~ t" ionophore (1 μM) as a positive control of apoptosis. The cells are recovered 36 hours later, counted and an equal number of cells (4.10 6 ), analyzed to determine the presence of the DNA fragments. Representative data relate to one in four thymocyte preparations.
- Figure 2: Implication du processus L-arginine/NO dans la mort in vitro de thymocytes humains suivant la ligation de CD2 et de CD3. Les cellules sont traitées (5.10-Vml) avec des concentrations variées de L-NMMA ou de SIN-1, 1 heure avant l'addition de l'anticorps anti-CD3 (20 μg/ml), de l'anticorps anti-CD2ι + III (1/400 ascite de chaque) ou l'ionophore Ca+ + (1 μM). Les cellules viables sont comptées 4 jours plus tard. Les moyennes ± moyenne d'erreur standard résultent de 4 cultures distinctes.- Figure 2: Implication of the L-arginine / NO process in the in vitro death of human thymocytes following the ligation of CD2 and CD3. The cells are treated (5.10-Vml) with various concentrations of L-NMMA or SIN-1, 1 hour before the addition of the anti-CD3 antibody (20 μg / ml), of the anti-CD2ι antibody + III (1/400 ascites of each) or the Ca + + ionophore (1 μM). Viable cells are counted 4 days later. The means ± standard error mean result from 4 distinct cultures.
- Figure 3: Sauvetage de thymocytes, altérés par NO, de l'apoptose et la croissance en présence de IL-2. Les thymocytes ou cellules PBL-T sont incubés (104 cellules/200 μl/puits) en présence de L-NMMA, de SIN-1 ou du tampon
pendant 1 heure avant addition d'anticorps monoclonal anti-CD3. d'anticorps monoclonal anti-CD2 ou de IL-2 recombinant humain (100 U/ml). 3Htdr (thymidine tritiée) (1 μCi/puits) est ajouté au jour 4, et la mesure de la radioactivité est effectuée 10 heures plus tard. On a donné la moyenne ± déviation standard de trois préparations cellulaires différentes, chacune faite en triple.- Figure 3: Rescue of thymocytes, altered by NO, of apoptosis and growth in the presence of IL-2. Thymocytes or PBL-T cells are incubated (10 4 cells / 200 μl / well) in the presence of L-NMMA, SIN-1 or buffer for 1 hour before addition of anti-CD3 monoclonal antibody. monoclonal anti-CD2 or recombinant human IL-2 antibody (100 U / ml). 3 Htdr (tritiated thymidine) (1 μCi / well) is added on day 4, and the measurement of radioactivity is carried out 10 hours later. The mean ± standard deviation was given of three different cell preparations, each made in triplicate.
ExempleExample
Procédures expérimentales Réactifs. L'interleukine-4 humaine recombinante (IL-4, don de J.Reactive experimental procedures. Recombinant human interleukin-4 (IL-4, gift from J.
Banchereau, Schering Plough, Dardilly, France), la NG-monométhyl-L-arginine (L-NMMA), la L-arginine, la D-arginine, la superoxyde dismutase (SOD), et la catalase (tous provenant de Sigma, St. Louis MO); la S-nitroso-acétyl- pénicillamine (SNAP, Alexis Corporation, Làufelfmgen, Suisse); la 6- morpholino-sydnonimine (SIN-1 , Glaxo, Paris, France); l'anticorps monoclonal anti-CD2 conjugué à FITC (CD2-mAb, Immunotech, Lumigny, France); l'anticorps monoclonal anti-cytokératine (Immunotech, Marseille, France); et l'anticorps monoclonal anti-vimentine (Amersham, Les Ulis, France) sont obtenus comme indiqué. Dans certaines expériences, la réactivité des anticorps monoclonaux est également visualisée par la méthode à l' immunoperoxydaseBanchereau, Schering Plow, Dardilly, France), NG-monomethyl-L-arginine (L-NMMA), L-arginine, D-arginine, superoxide dismutase (SOD), and catalase (all from Sigma, St. Louis MO); S-nitroso-acetylpenicillamine (SNAP, Alexis Corporation, Làufelfmgen, Switzerland); 6-morpholino-sydnonimine (SIN-1, Glaxo, Paris, France); the anti-CD2 monoclonal antibody conjugated to FITC (CD2-mAb, Immunotech, Lumigny, France); monoclonal anti-cytokeratin antibody (Immunotech, Marseille, France); and the anti-vimentin monoclonal antibody (Amersham, Les Ulis, France) are obtained as indicated. In certain experiments, the reactivity of the monoclonal antibodies is also visualized by the immunoperoxidase method.
(Laboratoires Vector, Burlingame, CA) suivi d'une coloration subséquente à l'hémalun de Mayer.(Vector Laboratories, Burlingame, CA) followed by a subsequent staining with Mayer's hemalun.
Préparations cellulaires. Les leucocytes de sang périphérique (PBL) sont obtenus à partir de volontaires normaux. Après la centrifugation sur gradient de Ficoll, les cellules T sont isolées à partir d'autres cellules mononucléaires par triage direct de cellules CD2 + . Les fragments de thymus sont obtenus sur des enfants (d'âge inférieur à 3 ans) qui subissent une chirurgie cardiaque correctrice. Les thymocytes ( >95% CD2+) sont séparés du stroma thymique en délacérant doucement les tissus. Des cellules complètes du stroma thymique sont ensuite lavées intensément pour éliminer les thymocytes résiduels. Le tissu est alors digéré avec de la collagénase (350 U/ml, Sigma) pour obtenir des suspensions cellulaires uniques. Les cultures pures de cellules épithéliales de thymus sont obtenues comme détaillé dans Dalloul, A. H., Arock, M..Cell preparations. Peripheral blood leukocytes (PBL) are obtained from normal volunteers. After centrifugation on a Ficoll gradient, the T cells are isolated from other mononuclear cells by direct sorting of CD2 + cells. The thymus fragments are obtained from children (under 3 years of age) who are undergoing corrective heart surgery. The thymocytes (> 95% CD2 +) are separated from the thymic stroma by gently loosening the tissues. Complete cells of the thymic stroma are then washed thoroughly to remove residual thymocytes. The tissue is then digested with collagenase (350 U / ml, Sigma) to obtain unique cell suspensions. Pure cultures of thymus epithelial cells are obtained as detailed in Dalloul, AH, Arock, M ..
Fourcade, C. Hatzfeld, A., Bertho, J.M., Debré, P. et Mossalayi, M.D. (1991) Blood 77: 69-74. En bref, les fragments de thymus sont coupés en morceaux deFourcade, C. Hatzfeld, A., Bertho, J.M., Debré, P. and Mossalayi, M.D. (1991) Blood 77: 69-74. In short, the thymus fragments are cut into pieces of
0,02 cm2 et libérés des thymocytes par lavage important. Les explants sont ancrés dans des boîtes de culture et incubés pendant 3 à 5 semaines (Dalloul,0.02 cm 2 and released from the thymocytes by heavy washing. The explants are anchored in culture dishes and incubated for 3 to 5 weeks (Dalloul,
A. H. et al. (1991) susmentionné). En suivant cette procédure, seules les cellules
épithéliales avec un taux de cytokératine + supérieur à 95% et un taux inférieur à 2% de CD2 + ou les cellules vimentine+ sont utilisées.AH et al. (1991) mentioned above). By following this procedure, only cells epithelial cells with a cytokeratin + level higher than 95% and a level lower than 2% CD2 + or vimentin + cells are used.
Cultures cellulaires. L'induction du processus NO dans les cellules épithéliales demande des conditions de culture spéciales: avant l'activation, les cellules épithéliales thymiques sont cultivées à confluence (faible état proliférant) pendant 3 jours dans un milieu DMEM (Gibco, BRL, Grand Island, NY), en l'absence de facteur de croissance épidermique, à propos duquel il a été montré qu'il abaisse la génération de NO (Heck, D.E., Laskin, D.L., Gardner, C.R., et Laskin, J.D. (1992) J. Biol. Chem. 267: 21277-21281). Us cellules sont ensuite incubées avec IL-4 pendant 24 heures, lavées puis activées avec l'anticorps monoclonal CD23 (20 μg/ml, clone 135, don de E. Kilchherr, Ciba Geigy, Bâle, Suisse). La ligation de CD23 a été montrée récemment comme étant un processus d'activation spécifique liée à NO (Bécherel, P.A., Mossalayi, M.D., Ouaaz, F., Le Goff, L., Dugas, B., Paul-Eugène, N., Frances, C, Chosidow, O., Kilchherr, E., Guillosson, J.J., Debré, P. et Arock, M. (1994)Cell cultures. The induction of the NO process in epithelial cells requires special culture conditions: before activation, the thymic epithelial cells are cultured at confluence (low proliferative state) for 3 days in DMEM medium (Gibco, BRL, Grand Island, NY), in the absence of epidermal growth factor, about which it has been shown to lower the generation of NO (Heck, DE, Laskin, DL, Gardner, CR, and Laskin, JD (1992) J. Biol. Chem. 267: 21277-21281). The cells are then incubated with IL-4 for 24 hours, washed and then activated with the monoclonal antibody CD23 (20 μg / ml, clone 135, donation from E. Kilchherr, Ciba Geigy, Basel, Switzerland). The CD23 ligation has recently been shown to be a specific activation process linked to NO (Bécherel, PA, Mossalayi, MD, Ouaaz, F., Le Goff, L., Dugas, B., Paul-Eugène, N. , Frances, C, Chosidow, O., Kilchherr, E., Guillosson, JJ, Debré, P. and Arock, M. (1994)
J. Clin. Invest. 93: 2275-5579). Les thymocytes totaux sont cultivés dans un milieu DMEM additionné de L-glutamine, pénicilline, streptomycine, et 5 % de sérum foetal de veau (tous de Gibco-BRL) avec ou sans: anticorps monoclonal co-mitogénique anti-CD2ι+m (clones D66 et XI 1 , 1/400 d'ascite de chaque; don de A. Bernard, Nice, France); l'anticorps anti-CD3 (clone OKT3; 20 μg/ml); ionophore Ca+ + (A23187, 1 μM, Sigma) et/ou rIL-2 (lOOU/ml, Boehringer Manheim, Meylan. France). En suivant des périodes d'incubation variables, les cellules viables par ml sont comptées par exclusion au bleu de Trypan. La prolifération cellulaire est également évaluée par l'incorporation de thymidine tritiée (1 μCi/puits, CEA, France), pendant 10 h. Le rôle du processus NO est investigué par l'addition de L-NMMA aux cultures, un inhibiteur de la conversion de L-arginine en L-citrulline par la synthase de l'oxyde nitrique (Stuehr, D.J., et Griffith, O.W. (1992) susmentionné). Les cellules sont également traitées avec SOD ou la catalase afin d'inhiber les superoxydes. L'effet direct de NO est également testé par l'addition de donneurs de NO chimiques, SIN-1 et SNAP aux cultures de thymocytes. Les concentrations optimum des produits chimiques désignés ci-dessus sont déterminées d'après les études préliminaires. Les résultats sont analysés et comparés en utilisant le test t de Student pour les données associées . Détermination de Nθ2~- Les surnageants de culture à partir de sous- ensembles de différentes cellules thymiques sont testés pour le produit final stable de NO, NO2* en utilisant la réaction de Greiss modifiée comme détaillée
dans Kolb, J.P., Paul-Eugène, N., Damais, C, Yamaoka, K.. Drapier. J.C. et Dugas, B. (1994) J. Biol. Chem. 269: 9811-9816.J. Clin. Invest. 93: 2275-5579). The total thymocytes are cultured in a DMEM medium supplemented with L-glutamine, penicillin, streptomycin, and 5% fetal calf serum (all from Gibco-BRL) with or without: co-mitogenic anti-CD2ι + m monoclonal antibody (clones D66 and XI 1, 1/400 of ascites each; gift from A. Bernard, Nice, France); anti-CD3 antibody (clone OKT3; 20 μg / ml); ionophore Ca + + (A23187, 1 μM, Sigma) and / or rIL-2 (lOOU / ml, Boehringer Manheim, Meylan. France). Following variable incubation periods, viable cells per ml are counted by exclusion with Trypan blue. Cell proliferation is also evaluated by the incorporation of tritiated thymidine (1 μCi / well, CEA, France), for 10 h. The role of the NO process is investigated by the addition of L-NMMA to cultures, an inhibitor of the conversion of L-arginine to L-citrulline by nitric oxide synthase (Stuehr, DJ, and Griffith, OW (1992 ) above). The cells are also treated with SOD or catalase in order to inhibit the superoxides. The direct effect of NO is also tested by the addition of chemical NO donors, SIN-1 and SNAP to the thymocyte cultures. The optimum concentrations of the chemicals designated above are determined from the preliminary studies. The results are analyzed and compared using the Student's t-test for the associated data. Determination of Nθ2 ~ - Culture supernatants from subsets of different thymic cells are tested for the stable end product of NO, NO2 * using the modified Greiss reaction as detailed in Kolb, JP, Paul-Eugène, N., Damais, C, Yamaoka, K .. Drapier. JC and Dugas, B. (1994) J. Biol. Chem. 269: 9811-9816.
Apoptose. L'analyse de fragmentation d'ADN est effectuée comme décrite dans Albina, J.E. et al. (1993) susmentionné. La mort cellulaire programmée est également analysée en utilisant une trousse de détection d' apoptose in situApoptosis. The DNA fragmentation analysis is carried out as described in Albina, J.E. et al. (1993) mentioned above. Planned cell death is also analyzed using an in situ apoptosis detection kit
(Apoptag, Oncor, Gaithersburg, MD) en suivant les recommandations du fabricant, et les données sont exprimées en pourcentage de cellules (apoptotiques) positives en peroxydase.(Apoptag, Oncor, Gaithersburg, MD) following the manufacturer's recommendations, and the data are expressed as a percentage of peroxidase-positive (apoptotic) cells.
In vitro, les thymocytes subissent une mort rapide due à l'apoptose en comparaison avec des lymphocytes T dérivés de PBL (Smith, C.A., et al.In vitro, thymocytes undergo rapid death due to apoptosis in comparison with T cells derived from PBL (Smith, C.A., et al.
(1989) susmentionné (Fig. 1A)). Afin d'investiguer le rôle du processus NO dans la mort cellulaire programmée des thymocytes, des thymocytes fraîchement isolés sont traités avec des produits chimiques qui, soit inhibent la génération de(1989) mentioned above (Fig. 1A)). In order to investigate the role of the NO process in the programmed cell death of thymocytes, freshly isolated thymocytes are treated with chemicals which either inhibit the generation of
NO (L-NMMA), soit produisent NO (SIN-1) (Moncada, S. et Higgs, E.A. (1993) susmentionné). Le nombre de cellules viables par ml est ensuite évalué.NO (L-NMMA), or produce NO (SIN-1) (Moncada, S. and Higgs, E.A. (1993) above). The number of viable cells per ml is then evaluated.
L'addition de L-NMMA à des thymocytes humains fraîchement isolés décroît leur mortalité in vitro (p < 0.003), tandis qu'elle n'a pas d'effet sur la survie des cellules PBL-T. Cet effet de L-NMMA est renversé par l'addition, à ces cultures, de L-arginine mais pas de D-arginine. Lorsque les cellules sont traitées avec SIN-1, le nombre de cellules décroît dans les deux préparations de cellulesAddition of L-NMMA to freshly isolated human thymocytes decreases their mortality in vitro (p <0.003), while it has no effect on the survival of PBL-T cells. This effect of L-NMMA is reversed by the addition, to these cultures, of L-arginine but not of D-arginine. When cells are treated with SIN-1, the number of cells decreases in both cell preparations
T (p < 0.05), bien que les thymocytes soient plus sensibles que PBL-T à l'effet de SIN-1 (p < 0.001). Ces données suggèrent un rôle du processusT (p <0.05), although thymocytes are more sensitive than PBL-T to the effect of SIN-1 (p <0.001). These data suggest a role for the process
L-arginine/ oxyde nitrique dans la mort des thymocytes humains in vitro.L-arginine / nitric oxide in the death of human thymocytes in vitro.
L'addition de L-NMMA aux thymocytes décroît leur apoptose in vitro en milieu seul (Fig. 1B). L'apoptose des thymocytes peut être accélérée suite à la ligation in vitro des antigènes de surface CD3/TCR ou CD2 par des anticorps appropriés (Smith, C.A. et al. (1989) susmentionné; Murphy, K.M.,The addition of L-NMMA to the thymocytes decreases their apoptosis in vitro in medium alone (FIG. 1B). Thymocyte apoptosis can be accelerated following in vitro ligation of CD3 / TCR or CD2 surface antigens by appropriate antibodies (Smith, C.A. et al. (1989) above; Murphy, K.M.,
Heimberger, A.B. et Loh, D.Y. (1990) Science 250, 1720-1723; Li, J.,Heimberger, A.B. and Loh, D.Y. (1990) Science 250, 1720-1723; Li, J.,
Campell, D. et Howard, A.R. (1992) Immunology 75, 305-315). La figure 1B montre une mort programmée des cellules augmentée à la suite de la ligation avec CD3, et de l'apoptose après addition de L-NMMA. Afin d'investiguer le rôle de NO dans la mort cellulaire programmée des thymocytes activés, les cellules sont pré-traitées avec L-NMMA ou SIN-1 pendant 1 heure avant l'addition de l'anticorps anti-CD2, de l'anticorps anti-CD3 ou du ionophore Ca """ "•" . La L-NMMA sauve les cellules de la mort en milieu seul (Fig. 2), et décroît la mortalité cellulaire en présence d'anticorps anti-CD2 ou d'anticorps anti-CD3, mais n'a pas d'effet sur la mort cellulaire induite par l'ionophoreCampell, D. and Howard, AR (1992) Immunology 75, 305-315). Figure 1B shows programmed cell death increased following ligation with CD3, and apoptosis after addition of L-NMMA. In order to investigate the role of NO in the programmed cell death of activated thymocytes, the cells are pretreated with L-NMMA or SIN-1 for 1 hour before the addition of the anti-CD2 antibody, of the antibody anti-CD3 or ionophore Ca """" • ". L-NMMA saves cells from death in a medium alone (Fig. 2), and decreases cell mortality in the presence of anti-CD2 antibodies or antibodies anti-CD3, but has no effect on cell death induced by ionophore
Ca+ + (Fig. 2). L'addition de SIN-1 de plus augmente la mort des thymocytes
dans les cultures ci-dessus, même lorsqu'il y a des synergies avec l'ionophore Ca+ + .Ca ++ (Fig. 2). Addition of additional SIN-1 increases thymocyte death in the above cultures, even when there are synergies with the Ca + + ionophore.
L'addition de IL-2 pendant l'activation des thymocytes sauve la plupart des thymocytes de l'apoptose et induit leur prolifération (Cohen, J.J. (1993) susmentionné). Ici, la L-NMMA augmente les réponses proliférantes des thymocytes (p < 0.001), tandis qu'elle n'induit pas la croissance cellulaire de PBL-T (Fig. 3). Par contraste, l'addition de SIN-1 gêne la croissance des thymocytes en présence de IL-2 (Fig. 3). Sur les cellules PBL-T, SIN-1 montre un effet inhibiteur sur leur prolifération induite à la fois via le CD3 ou via le CD2 avec moins d'intensité vis à vis de la voie CD3, que ce qui est observé dans les thymocytes (p < 0.01) (Fig. 3). Ces données confirment de plus le rôle de NO sur la lignée cellulaire T.The addition of IL-2 during thymocyte activation saves most thymocytes from apoptosis and induces their proliferation (Cohen, J.J. (1993) mentioned above). Here, L-NMMA increases the proliferative responses of thymocytes (p <0.001), while it does not induce cell growth of PBL-T (Fig. 3). In contrast, the addition of SIN-1 hinders the growth of thymocytes in the presence of IL-2 (Fig. 3). On PBL-T cells, SIN-1 shows an inhibitory effect on their proliferation induced both via CD3 or via CD2 with less intensity with respect to the CD3 pathway, than what is observed in thymocytes ( p <0.01) (Fig. 3). These data further confirm the role of NO on the T cell line.
D'après ces données, on en déduit que les thymocytes sont impliqués dans un processus NO et peuvent être capables de produire des nitrites, l'un des produits finals du métabolisme de NO (Moncada, S. et Higgs, E.A. (1993) susmentionné; Nathan, C. (1992) susmentionné; Nussler, A. et Billiar, T.R. (1993) susmentionné; Stuehr, D.J. et Griffith, O.W. (1992) susmentionné). Suite à l'incubation dans du milieu DMEM pendant 4 jours, des quantités significatives de nitrites sont détectées dans des surnageants de culture dérivée de thymocytes en l'absence d'activation supplémentaire apparente. Les cellules du stroma libres de thymocytes des mêmes thymus ont des niveaux significativement plus élevés (p < 0.001) de nitrites dans leurs surnageants. Ces données démontrent les sources possibles exocrines et autocrines de NO pour les thymocytes humains. Afin de clarifier l'origine cellulaire de NO parmi les cellules du stroma thymique humain, on a testé la possibilité des cellules purifiées épithéliales dérivées du thymus, à produire des nitrites in vitro.From these data, it can be deduced that thymocytes are involved in a NO process and may be able to produce nitrites, one of the end products of NO metabolism (Moncada, S. and Higgs, EA (1993) mentioned above. ; Nathan, C. (1992) above; Nussler, A. and Billiar, TR (1993) above; Stuehr, DJ and Griffith, OW (1992) above). Following incubation in DMEM medium for 4 days, significant amounts of nitrites are detected in culture supernatants derived from thymocytes in the absence of apparent additional activation. Thymocyte-free stroma cells of the same thymus have significantly higher (p <0.001) levels of nitrites in their supernatants. These data demonstrate the possible exocrine and autocrine sources of NO for human thymocytes. In order to clarify the cellular origin of NO among cells of the human thymic stroma, we tested the possibility of purified epithelial cells derived from the thymus, to produce nitrites in vitro.
Des thymocytes ont également été traités avec SIN-1 , (donneur de NO et de ONOO) ou SNAP (donneur de NO) et leur apoptose a été quantifiée. Les cellules sont également incubées avec de l'anticorps CD3 en présence de L- NMMA, de SOD ou de catalase. Tandis que L-NMMA inhibe la génération deThymocytes were also treated with SIN-1 (NO and ONOO donor) or SNAP (NO donor) and their apoptosis was quantified. The cells are also incubated with CD3 antibody in the presence of L-NMMA, SOD or catalase. While L-NMMA inhibits the generation of
NO, la SOD et la catalase sont des inhibiteurs de peroxydes. Une inhibition similaire d'apoptose est obtenue avec L-NMMA, SOD ou les deux. L'absence de synergie entre ces deux agents milite en faveur de l'implication de peroxy nitrites, mais contre un rôle direct de NO dans ce phénomène. Ceci corrèle le fait qu'une apoptose plus élevée des thymocytes est observée en présence de SIN-1, comparé aux cellules traitées avec SNAP.
Exemple :NO, SOD and catalase are peroxide inhibitors. Similar inhibition of apoptosis is achieved with L-NMMA, SOD or both. The absence of synergy between these two agents militates in favor of the involvement of peroxy nitrites, but against a direct role of NO in this phenomenon. This correlates to the fact that higher thymocyte apoptosis is observed in the presence of SIN-1, compared to cells treated with SNAP. Example:
Des thymocytes sont incubés en milieu seul ou en présence de l'anticorps anti-CD3 (20μg/ml). Certaines cultures sont additionnées de SIN-1 (200 μM), de SNAP-1 (1 mM), de L-NMMA (1 πiM), de superoxyde dismutase (SOD, 120 unités/ml), ou de catalase (240 unités/ml), 1 heure avant l'addition de l'anticorps anti-CD3. Les cellules sont analysées 48 heures plus tard pour déterminer le pourcentage de cellules qui subissent l'apoptose par le kit Apoptag (peroxydase +), commercialisé Oncor (Gaithersburg, MD, USA). Les résultats donnés dans le tableau ci-après correspondent ± la déviation standard, à partir de trois thymus distincts.Thymocytes are incubated in the medium alone or in the presence of the anti-CD3 antibody (20 μg / ml). Some cultures are supplemented with SIN-1 (200 μM), SNAP-1 (1 mM), L-NMMA (1 πiM), superoxide dismutase (SOD, 120 units / ml), or catalase (240 units / ml), 1 hour before the addition of the anti-CD3 antibody. The cells are analyzed 48 hours later to determine the percentage of cells which undergo apoptosis by the Apoptag kit (peroxidase +), marketed Oncor (Gaithersburg, MD, USA). The results given in the table below correspond to the standard deviation, from three distinct thymuses.
% cellules apoptotiques% apoptotic cells
L-NMMA (1 mM) + - - +L-NMMA (1 mM) + - - +
SNAP (1 mM) • - - + - -SNAP (1 mM) • - - + - -
SOD + Catalase - - - + +SOD + Catalase - - - + +
Thymus 1 62 14 84 18 12Thymus 1 62 14 84 18 12
Thymus 2 35 12 92 14 10Thymus 2 35 12 92 14 10
Thymus 3 41 24 67 22 20Thymus 3 41 24 67 22 20
Rôle de NO sur l'apoptose des cellules T des sujets HIV + :Role of NO on apoptosis of T cells in HIV + subjects:
Des cellules mononuclées du sang périphériques (PBL), ou des cellules CD4+ de rate provenant des sujets atteints par le SIDA, sont incubées en milieu seul ou en présence de l'anticorps anti-CD3 (20μg/ml). Certaines cultures sont additionnées de SNAP (ImM) ou de L-NMMA (ImM), 1 heure avant l'addition de l'anticorps anti-CD3. Les cellules sont analysées 48 heures plus tard pour déterminer le nombre des cellules ainsi que le pourcentage de cellules qui subissent l'apoptose par le kit Apoptag (peroxydase +), commercialisé par Oncor (Gaithersburg, MD, USA). Les résultats donnés dans le tableau ci-après sont la moyenne de deux essais ±. la déviation standard .
% cellules apoptotiques nombre de cellules χ l( /mlPeripheral blood mononuclear cells (PBL), or spleen CD4 + cells from subjects suffering from AIDS, are incubated in the medium alone or in the presence of the anti-CD3 antibody (20 μg / ml). Some cultures are supplemented with SNAP (ImM) or L-NMMA (ImM), 1 hour before the addition of the anti-CD3 antibody. The cells are analyzed 48 hours later to determine the number of cells as well as the percentage of cells which undergo apoptosis by the Apoptag kit (peroxidase +), marketed by Oncor (Gaithersburg, MD, USA). The results given in the table below are the average of two ± tests. the standard deviation. % apoptotic cells number of cells χ l (/ ml
L-NMMA ( ImM) + - +L-NMMA (ImM) + - +
SNAP (ImM) - + +SNAP (ImM) - + +
PBL-SIDA-1 38 h6 14±4 67±14 4,0±0,2 5,3±0,4 2,1±0,5PBL-SIDA-1 38 h6 14 ± 4 67 ± 14 4.0 ± 0.2 5.3 ± 0.4 2.1 ± 0.5
PBL-SIDA-2 43_±14 23±7 76±20 3,2+0,5 5,0+_0,6 1 ,8±0,4PBL-SIDA-2 43_ ± 14 23 ± 7 76 ± 20 3.2 + 0.5 5.0 + _0.6 1.8 ± 0.4
CD4 + -SIDA 64±3 25±6 92±10 2,2+_0,2 5, 1±0, 1 0,9+_0,2CD4 + -SIDA 64 ± 3 25 ± 6 92 ± 10 2.2 + _0.2 5, 1 ± 0, 1 0.9 + _0.2
Les donneurs chimiques de NO exercent un effet apoptotique sur les thymocytes ainsi que les cellules T provenant des sujets HIV--- . De plus, l'inhibition in vitro de la voie NO par la L-NMMA permet une survie prolongée de ces cellules. Ces dernières données sont en faveur d'un rôle in vivo de NO dans l'apoptose de ces cellules. L'inhibition de cette voie peut ainsi constituer un traitement approprié pour augmenter le nombre des lymphocytes T (plus des cellules produites par le thymus) et/ou diminuer leur mortalité in vivo (moins d' apoptose des lymphocytes périphériques).
The chemical donors of NO exert an apoptotic effect on the thymocytes as well as the T cells originating from HIV --- subjects. In addition, the in vitro inhibition of the NO pathway by L-NMMA allows prolonged survival of these cells. These latest data are in favor of an in vivo role of NO in the apoptosis of these cells. The inhibition of this pathway can thus constitute an appropriate treatment to increase the number of T lymphocytes (more cells produced by the thymus) and / or decrease their mortality in vivo (less apoptosis of peripheral lymphocytes).
Claims
1. Utilisation d'au moins un inhibiteur de toute molécule ou groupe de molécules susceptible de participer à la synthèse de NO dans l'organisme, notamment un inhibiteur de la nitrique-oxyde synthase, pour la préparation d'un médicament destiné au traitement de pathologies impliquant soit l'inhibition par NO (à l'exception de NO provenant de macrophages) de la prolifération de cellules T, soit la destruction par NO, notamment par apoptose, de cellules T, en particulier de thymocytes, notamment de lymphocytes T, les susdites pathologies étant notamment des pathologies virales.1. Use of at least one inhibitor of any molecule or group of molecules capable of participating in the synthesis of NO in the organism, in particular an inhibitor of nitric oxide synthase, for the preparation of a medicament intended for the treatment of pathologies involving either inhibition by NO (with the exception of NO from macrophages) of the proliferation of T cells, or destruction by NO, in particular by apoptosis, of T cells, in particular of thymocytes, in particular T lymphocytes, the aforementioned pathologies being in particular viral pathologies.
2. Utilisation d'au moins un inhibiteur selon la revendication 1, pour la préparation d'un médicament destiné au traitement de pathologies impliquant l'immunité T dépendante, telles que des pathologies d'immunodéficience d'origine virale ou fungique ou des pathologies d'origine virale, ou fungique impliquant l'immunité T dépendante, ou de troubles métaboliques, de cancer, notamment d'hématopathies malignes.2. Use of at least one inhibitor according to claim 1, for the preparation of a medicament intended for the treatment of pathologies involving T dependent immunity, such as immunodeficiency pathologies of viral or fungal origin or pathologies d 'viral or fungal origin involving T-dependent immunity, or metabolic disorders, cancer, in particular malignant hematopathies.
3. Utilisation d'au moins un inhibiteur de la nitrique-oxyde synthase selon la revendication 1 , pour la préparation d'un médicament destiné au traitement de pathologies impliquant l'apoptose de thymocytes, notamment de lymphocytes T périphériques, en particulier de pathologies virales, notamment celles résultant de l'infection d'un individu par un virus du type HIV.3. Use of at least one nitric oxide synthase inhibitor according to claim 1, for the preparation of a medicament intended for the treatment of pathologies involving the apoptosis of thymocytes, in particular peripheral T lymphocytes, in particular viral pathologies , especially those resulting from the infection of an individual with a virus of the HIV type.
4. Utilisation selon l'une des revendications 1 à 3, dans laquelle l'inhibiteur de la nitrique-oxyde synthase est la L-N-monométhyl-arginine (L-NMMA).4. Use according to one of claims 1 to 3, in which the nitric oxide synthase inhibitor is L-N-monomethyl-arginine (L-NMMA).
5. Utilisation selon l'une des revendications 1 à 4, dans laquelle l'inhibiteur de la nitrique-oxyde synthase est administrable à une dose de 0,1 à5. Use according to one of claims 1 to 4, in which the nitric oxide synthase inhibitor can be administered at a dose of 0.1 to
500 mg/kg/jour, et notamment pour un adulte une dose de 5 mg à 35 g/jour, de préférence 5 mg à 2 g/jour.500 mg / kg / day, and in particular for an adult a dose of 5 mg to 35 g / day, preferably 5 mg to 2 g / day.
6. Utilisation selon l'une des revendications 1 à 5, caractérisée en ce que le médicament se présente sous forme administrable par voie orale, parentérale ou nasale. 6. Use according to one of claims 1 to 5, characterized in that the medicament is in a form which can be administered orally, parenterally or nasally.
7. Composition contenant, d'une part au moins un inhibiteur de toute molécule ou groupe de molécules susceptible de participer à la synthèse de NO dans l'organisme, notamment un inhibiteur de la nitrique-oxyde synthase. et d'autre part au moins un inhibiteur des ions peroxydes tel que le glutathion. la catalase ou la superoxyde dismutase.7. Composition containing, on the one hand at least one inhibitor of any molecule or group of molecules capable of participating in the synthesis of NO in the organism, in particular an inhibitor of nitric oxide synthase. and on the other hand at least one inhibitor of peroxide ions such as glutathione. catalase or superoxide dismutase.
8. Composition pharmaceutique comprenant, à titre de substance active, l'une au moins des compositions selon la revendication 7, en association avec un véhicule pharmaceutiquement acceptable.8. Pharmaceutical composition comprising, as active substance, at least one of the compositions according to claim 7, in combination with a pharmaceutically acceptable vehicle.
9. Utilisation d'une au moins des compositions selon la revendication 7, pour la préparation d'un médicament destiné au traitement de pathologies impliquant soit l'inhibition par NO de la prolifération de cellules T, soit la destruction par NO, notamment par apoptose, de cellules T, en particulier de thymocytes, notamment de lymphocytes T. les susdites pathologies étant notamment des pathologies virales, et impliquant l'inhibition des ions peroxydes. 9. Use of at least one of the compositions according to claim 7, for the preparation of a medicament intended for the treatment of pathologies involving either the inhibition by NO of the proliferation of T cells, or the destruction by NO, in particular by apoptosis , T cells, in particular thymocytes, in particular T lymphocytes, the abovementioned pathologies being in particular viral pathologies, and involving the inhibition of peroxide ions.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR9412946A FR2726186B1 (en) | 1994-10-28 | 1994-10-28 | DRUGS CONTAINING NITRIC OXIDE SYNTHASE INHIBITORS |
| FR94/12946 | 1994-10-28 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO1996013256A1 true WO1996013256A1 (en) | 1996-05-09 |
Family
ID=9468332
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/FR1995/001419 WO1996013256A1 (en) | 1994-10-28 | 1995-10-26 | Drugs containing nitric oxide synthase inhibitors for treating immunodeficiency diseases |
Country Status (2)
| Country | Link |
|---|---|
| FR (1) | FR2726186B1 (en) |
| WO (1) | WO1996013256A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998035667A1 (en) * | 1997-02-13 | 1998-08-20 | Smithkline Beecham Plc | Use of nitric oxid synthase inhibitors for the treatment of diabetes |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994016729A1 (en) * | 1993-01-28 | 1994-08-04 | Neorx Corporation | Targeted nitric oxide pathway or nitric oxide synthase modulation |
-
1994
- 1994-10-28 FR FR9412946A patent/FR2726186B1/en not_active Expired - Fee Related
-
1995
- 1995-10-26 WO PCT/FR1995/001419 patent/WO1996013256A1/en active Application Filing
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994016729A1 (en) * | 1993-01-28 | 1994-08-04 | Neorx Corporation | Targeted nitric oxide pathway or nitric oxide synthase modulation |
Non-Patent Citations (11)
| Title |
|---|
| AL-RAMADI BK ET AL: "Immunosuppression induced by nitric oxide and its inhibition by interleukin-4.", EUR J IMMUNOL (GERMANY), SEP 1992, VOL. 22, NO. 9, PAGE(S) 2249-54, * |
| ANKARCRONA M ET AL: "in pancreatic RINm5F cells.", EXP CELL RES (UNITED STATES), JUL 1994, VOL. 213, NO. 1, PAGE(S) 172-7, * |
| DAWSON VL ET AL: "Human immunodeficiency virus type 1 coat protein neurotoxicity mediated by nitric oxide in primary cortical cultures.", PROC NATL ACAD SCI U S A (UNITED STATES), APR 15 1993, VOL. 90, NO. 8, PAGE(S) 3256-9, * |
| J.E. ALBINA: "Nitric oxide-mediated apoptosis in murine peritoneal macrophages", J. IMMUNOL., vol. 150, no. 11, pages 5080 - 85 * |
| KITAJIMA I ET AL: "Nitric oxide-mediated apoptosis in murine mastocytoma.", BIOCHEM BIOPHYS RES COMMUN (UNITED STATES), OCT 14 1994, VOL. 204, NO. 1, PAGE(S) 244-51, * |
| OYANAGUI Y: "Nitric oxide- and hydrogen peroxide-mediated gene expression by glucocorticoids and FK506 in histamine paw edema of mice.", LIFE SCI (ENGLAND), 1994, VOL. 55, NO. 9, PAGE(S) PL177-85, * |
| OYANAGUI Y: "Nitric oxide and superoxide radical are involved in both initiation and development of adjuvant arthritis in rats.", LIFE SCI (ENGLAND), 1994, VOL. 54, NO. 17, PAGE(S) PL285-9, * |
| ROCKETT KA ET AL: "Possible role of nitric oxide in malarial immunosuppression.", PARASITE IMMUNOL (ENGLAND), MAY 1994, VOL. 16, NO. 5, PAGE(S) 243-9, * |
| STERNBERG J ET AL: "Inhibition of nitric oxide synthesis leads to reduced parasitemia in murine Trypanosoma brucei infection.", INFECT IMMUN (UNITED STATES), MAY 1994, VOL. 62, NO. 5, PAGE(S) 2135-7, * |
| STERNBERG J ET AL: "Nitric oxide mediates suppression of T cell responses in murine Trypanosoma brucei infection.", EUR J IMMUNOL (GERMANY), OCT 1992, VOL. 22, NO. 10, PAGE(S) 2741-4, * |
| XIE K ET AL: "Induction of apoptosis in transformed fibroblasts can be mediated via endogenous and exogenous nitric oxide (Meeting abstract).", PROC ANNU MEET AM ASSOC CANCER RES, VOL. 34, A 564,, 1993 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998035667A1 (en) * | 1997-02-13 | 1998-08-20 | Smithkline Beecham Plc | Use of nitric oxid synthase inhibitors for the treatment of diabetes |
Also Published As
| Publication number | Publication date |
|---|---|
| FR2726186A1 (en) | 1996-05-03 |
| FR2726186B1 (en) | 1996-12-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Müller | Reactive oxygen intermediates and human immunodeficiency virus (HIV) infection | |
| Malley et al. | Synergistic anti-human immunodeficiency virus type 1 effect of hydroxamate compounds with 2', 3'-dideoxyinosine in infected resting human lymphocytes. | |
| EP0147283B1 (en) | Immunomodulating medicine based on fc fragments of human igg | |
| Zoschke et al. | Suppression of human lymphocyte mitogenesis mediated by phagocyte-released reactive oxygen species: comparative activities in normals and in chronic granulomatous disease | |
| US20020049157A1 (en) | Use of proteasome inhibitors for treating cancer, inflammation, autoimmune disease, graft rejection and septic shock | |
| WO2011021833A2 (en) | Composition for preventing or treating inflammation | |
| Waegell et al. | A420983, a novel, small molecule inhibitor of LCK prevents allograft rejection | |
| CA2334990A1 (en) | Novel use of hiv protease inhibiting compounds | |
| EP0542630A2 (en) | Use of derivatives of amphotericin B as protease inhibitors | |
| Buhl | Imbalance between oxidants and antioxidants in the lungs of HIV-seropositive individuals | |
| Kouoh et al. | Antioxidant effects and anti-elastase activity of the calcium antagonist nicardipine on activated human and rabbit neutrophils—a potential antiatherosclerotic property of calcium antagonists? | |
| WO1996013256A1 (en) | Drugs containing nitric oxide synthase inhibitors for treating immunodeficiency diseases | |
| Munch-Petersen | Azidothymidine inhibits mitogen stimulated growth and DNA-repair in human peripheral lymphocytes | |
| CA2085399A1 (en) | Method for inhibiting nitric oxide formation | |
| EP1303296B1 (en) | Composition containing an iron complexing protein and a precursor of nitrogen monoxide metabolism and/or a chemical donor of nitrogen monoxide and uses thereof | |
| US6429208B1 (en) | Methods and compositions for restoring impaired cellular immune function | |
| JP2003519088A (en) | Use of GSSG reductase for treatment and prevention of HIV infected patients | |
| WO1999043308A2 (en) | Treating pulmonary hypertension through tenascin suppression and elastase inhibition | |
| US10092595B2 (en) | Compositions and methods for treating inflammatory arthritis | |
| EP1032401B1 (en) | Method for inhibiting cytokine production by cells | |
| EP0185584A1 (en) | Utilization of the heteropolyanion (NaSb9W21086)18- for the manufacture of a pharmaceutical composition for the treatment of AIDS and analogous syndromes | |
| Ni et al. | Inhibition of aldosterone turn-off phenomenon following chronic adrenocorticotropin treatment with in vivo administration of antiglucocorticoid and antioxidants in rats | |
| US5780513A (en) | Method of inhibiting the release of bioactive IL-1 | |
| Miesel et al. | Activity of Cu2Zn2 superoxide dismutase against the human immunodeficiency virus type 1 | |
| EP1079852B1 (en) | Composition based on pentoxifylline and anticytokin |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| AK | Designated states |
Kind code of ref document: A1 Designated state(s): JP US |
|
| AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE |
|
| DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
| 122 | Ep: pct application non-entry in european phase | ||
| 122 | Ep: pct application non-entry in european phase |