CA2303591C - Method and compositions for preventing or reducing hiv infection - Google Patents
Method and compositions for preventing or reducing hiv infection Download PDFInfo
- Publication number
- CA2303591C CA2303591C CA 2303591 CA2303591A CA2303591C CA 2303591 C CA2303591 C CA 2303591C CA 2303591 CA2303591 CA 2303591 CA 2303591 A CA2303591 A CA 2303591A CA 2303591 C CA2303591 C CA 2303591C
- Authority
- CA
- Canada
- Prior art keywords
- lapase
- estrogen
- use according
- inhibitor
- antibody
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 239000000203 mixture Substances 0.000 title claims abstract description 32
- 208000031886 HIV Infections Diseases 0.000 title claims description 34
- 208000037357 HIV infectious disease Diseases 0.000 title claims description 30
- 208000033519 human immunodeficiency virus infectious disease Diseases 0.000 title claims description 30
- 238000000034 method Methods 0.000 title abstract description 49
- 239000003112 inhibitor Substances 0.000 claims abstract description 68
- 150000001875 compounds Chemical class 0.000 claims abstract description 39
- 230000001833 anti-estrogenic effect Effects 0.000 claims abstract description 33
- 239000000328 estrogen antagonist Substances 0.000 claims abstract description 33
- 229940046836 anti-estrogen Drugs 0.000 claims abstract description 32
- 239000000262 estrogen Substances 0.000 claims abstract description 25
- 229940011871 estrogen Drugs 0.000 claims abstract description 25
- 102000002704 Leucyl aminopeptidase Human genes 0.000 claims abstract description 8
- 108010004098 Leucyl aminopeptidase Proteins 0.000 claims abstract description 8
- 102000002933 Thioredoxin Human genes 0.000 claims description 33
- 108060008226 thioredoxin Proteins 0.000 claims description 33
- 229940094937 thioredoxin Drugs 0.000 claims description 33
- 210000001744 T-lymphocyte Anatomy 0.000 claims description 29
- 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 21
- VGGGPCQERPFHOB-MCIONIFRSA-N Bestatin Chemical group CC(C)C[C@H](C(O)=O)NC(=O)[C@@H](O)[C@H](N)CC1=CC=CC=C1 VGGGPCQERPFHOB-MCIONIFRSA-N 0.000 claims description 18
- VGGGPCQERPFHOB-UHFFFAOYSA-N Bestatin Natural products CC(C)CC(C(O)=O)NC(=O)C(O)C(N)CC1=CC=CC=C1 VGGGPCQERPFHOB-UHFFFAOYSA-N 0.000 claims description 18
- 229950009811 ubenimex Drugs 0.000 claims description 18
- 210000004408 hybridoma Anatomy 0.000 claims description 17
- 230000036967 uncompetitive effect Effects 0.000 claims description 17
- 150000003573 thiols Chemical group 0.000 claims description 13
- NKANXQFJJICGDU-QPLCGJKRSA-N Tamoxifen Chemical compound C=1C=CC=CC=1C(/CC)=C(C=1C=CC(OCCN(C)C)=CC=1)/C1=CC=CC=C1 NKANXQFJJICGDU-QPLCGJKRSA-N 0.000 claims description 12
- 239000003814 drug Substances 0.000 claims description 12
- 108010024636 Glutathione Proteins 0.000 claims description 11
- 229960003180 glutathione Drugs 0.000 claims description 10
- 239000003937 drug carrier Substances 0.000 claims description 9
- ZQZFYGIXNQKOAV-OCEACIFDSA-N Droloxifene Chemical compound C=1C=CC=CC=1C(/CC)=C(C=1C=C(O)C=CC=1)\C1=CC=C(OCCN(C)C)C=C1 ZQZFYGIXNQKOAV-OCEACIFDSA-N 0.000 claims description 6
- JJKOTMDDZAJTGQ-DQSJHHFOSA-N Idoxifene Chemical compound C=1C=CC=CC=1C(/CC)=C(C=1C=CC(OCCN2CCCC2)=CC=1)/C1=CC=C(I)C=C1 JJKOTMDDZAJTGQ-DQSJHHFOSA-N 0.000 claims description 6
- 230000002860 competitive effect Effects 0.000 claims description 6
- 229950004203 droloxifene Drugs 0.000 claims description 6
- 229950002248 idoxifene Drugs 0.000 claims description 6
- 238000004519 manufacturing process Methods 0.000 claims description 6
- VIKNJXKGJWUCNN-XGXHKTLJSA-N norethisterone Chemical compound O=C1CC[C@@H]2[C@H]3CC[C@](C)([C@](CC4)(O)C#C)[C@@H]4[C@@H]3CCC2=C1 VIKNJXKGJWUCNN-XGXHKTLJSA-N 0.000 claims description 6
- 229960001603 tamoxifen Drugs 0.000 claims description 6
- OSVMTWJCGUFAOD-KZQROQTASA-N formestane Chemical compound O=C1CC[C@]2(C)[C@H]3CC[C@](C)(C(CC4)=O)[C@@H]4[C@@H]3CCC2=C1O OSVMTWJCGUFAOD-KZQROQTASA-N 0.000 claims description 5
- 229940053934 norethindrone Drugs 0.000 claims description 5
- GZUITABIAKMVPG-UHFFFAOYSA-N raloxifene Chemical compound C1=CC(O)=CC=C1C1=C(C(=O)C=2C=CC(OCCN3CCCCC3)=CC=2)C2=CC=C(O)C=C2S1 GZUITABIAKMVPG-UHFFFAOYSA-N 0.000 claims description 5
- 229960004622 raloxifene Drugs 0.000 claims description 5
- 238000004890 malting Methods 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 53
- 241000725303 Human immunodeficiency virus Species 0.000 abstract description 39
- 208000030507 AIDS Diseases 0.000 abstract description 10
- 230000002265 prevention Effects 0.000 abstract description 6
- 238000011282 treatment Methods 0.000 abstract description 6
- 230000002068 genetic effect Effects 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 88
- 208000015181 infectious disease Diseases 0.000 description 23
- 239000011159 matrix material Substances 0.000 description 19
- 230000005764 inhibitory process Effects 0.000 description 14
- 229960005309 estradiol Drugs 0.000 description 12
- 239000006228 supernatant Substances 0.000 description 12
- 239000008055 phosphate buffer solution Substances 0.000 description 10
- 230000002829 reductive effect Effects 0.000 description 10
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 9
- 230000001566 pro-viral effect Effects 0.000 description 9
- 230000000840 anti-viral effect Effects 0.000 description 8
- 239000000427 antigen Substances 0.000 description 8
- 102000036639 antigens Human genes 0.000 description 8
- 108091007433 antigens Proteins 0.000 description 8
- 230000027455 binding Effects 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 8
- 238000011534 incubation Methods 0.000 description 8
- 239000008194 pharmaceutical composition Substances 0.000 description 8
- 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 8
- 208000026310 Breast neoplasm Diseases 0.000 description 7
- 241000713772 Human immunodeficiency virus 1 Species 0.000 description 7
- 206010006187 Breast cancer Diseases 0.000 description 6
- 206010035226 Plasma cell myeloma Diseases 0.000 description 6
- 239000000872 buffer Substances 0.000 description 6
- 230000004927 fusion Effects 0.000 description 6
- 230000002401 inhibitory effect Effects 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 201000000050 myeloid neoplasm Diseases 0.000 description 6
- 238000000746 purification Methods 0.000 description 6
- 239000007787 solid Substances 0.000 description 6
- 238000010367 cloning Methods 0.000 description 5
- 230000000670 limiting effect Effects 0.000 description 5
- 239000012528 membrane Substances 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 230000003612 virological effect Effects 0.000 description 5
- 241000283707 Capra Species 0.000 description 4
- 101710205625 Capsid protein p24 Proteins 0.000 description 4
- 238000002965 ELISA Methods 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 102100034343 Integrase Human genes 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 102100030856 Myoglobin Human genes 0.000 description 4
- 108010062374 Myoglobin Proteins 0.000 description 4
- 239000000020 Nitrocellulose Substances 0.000 description 4
- 229930182555 Penicillin Natural products 0.000 description 4
- 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 4
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 241000700605 Viruses Species 0.000 description 4
- 210000000628 antibody-producing cell Anatomy 0.000 description 4
- 230000037396 body weight Effects 0.000 description 4
- 201000008275 breast carcinoma Diseases 0.000 description 4
- 230000008878 coupling Effects 0.000 description 4
- 238000010168 coupling process Methods 0.000 description 4
- 238000005859 coupling reaction Methods 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 230000003053 immunization Effects 0.000 description 4
- 230000001404 mediated effect Effects 0.000 description 4
- 229920001220 nitrocellulos Polymers 0.000 description 4
- 229940049954 penicillin Drugs 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 235000018102 proteins Nutrition 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 229960005322 streptomycin Drugs 0.000 description 4
- 230000007502 viral entry Effects 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 3
- 101710177166 Phosphoprotein Proteins 0.000 description 3
- 239000012980 RPMI-1640 medium Substances 0.000 description 3
- 101710149279 Small delta antigen Proteins 0.000 description 3
- 102100022563 Tubulin polymerization-promoting protein Human genes 0.000 description 3
- 239000004480 active ingredient Substances 0.000 description 3
- 238000007792 addition Methods 0.000 description 3
- 239000012148 binding buffer Substances 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 239000001913 cellulose Substances 0.000 description 3
- 229920002678 cellulose Polymers 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 210000000349 chromosome Anatomy 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 238000012744 immunostaining Methods 0.000 description 3
- 230000036963 noncompetitive effect Effects 0.000 description 3
- 108090000765 processed proteins & peptides Proteins 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 210000004989 spleen cell Anatomy 0.000 description 3
- 230000000638 stimulation Effects 0.000 description 3
- 239000003981 vehicle Substances 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- 102100035875 C-C chemokine receptor type 5 Human genes 0.000 description 2
- 102100031650 C-X-C chemokine receptor type 4 Human genes 0.000 description 2
- 101710082513 C-X-C chemokine receptor type 4 Proteins 0.000 description 2
- 241000283073 Equus caballus Species 0.000 description 2
- 101000946926 Homo sapiens C-C chemokine receptor type 5 Proteins 0.000 description 2
- 108010044467 Isoenzymes Proteins 0.000 description 2
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- 101710120037 Toxin CcdB Proteins 0.000 description 2
- 208000036142 Viral infection Diseases 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 238000001042 affinity chromatography Methods 0.000 description 2
- 230000000890 antigenic effect Effects 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 150000001718 carbodiimides Chemical class 0.000 description 2
- 230000003833 cell viability Effects 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 2
- 230000016784 immunoglobulin production Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000003367 kinetic assay Methods 0.000 description 2
- 239000002502 liposome Substances 0.000 description 2
- 239000008176 lyophilized powder Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000007911 parenteral administration Methods 0.000 description 2
- 102000013415 peroxidase activity proteins Human genes 0.000 description 2
- 108040007629 peroxidase activity proteins Proteins 0.000 description 2
- 238000007747 plating Methods 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- ZCCUUQDIBDJBTK-UHFFFAOYSA-N psoralen Chemical compound C1=C2OC(=O)C=CC2=CC2=C1OC=C2 ZCCUUQDIBDJBTK-UHFFFAOYSA-N 0.000 description 2
- 230000009257 reactivity Effects 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 239000006152 selective media Substances 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 210000004988 splenocyte Anatomy 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 230000002195 synergetic effect Effects 0.000 description 2
- 229940124597 therapeutic agent Drugs 0.000 description 2
- 229940104230 thymidine Drugs 0.000 description 2
- 230000009385 viral infection Effects 0.000 description 2
- SQKUFYLUXROIFM-UHFFFAOYSA-N 2-[2-[carboxymethyl-[[3-hydroxy-2-methyl-5-(phosphonooxymethyl)pyridin-4-yl]methyl]amino]ethyl-[[3-hydroxy-2-methyl-5-(phosphonooxymethyl)pyridin-4-yl]methyl]amino]acetic acid Chemical compound CC1=NC=C(COP(O)(O)=O)C(CN(CCN(CC(O)=O)CC=2C(=C(C)N=CC=2COP(O)(O)=O)O)CC(O)=O)=C1O SQKUFYLUXROIFM-UHFFFAOYSA-N 0.000 description 1
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 1
- JBIJLHTVPXGSAM-UHFFFAOYSA-N 2-naphthylamine Chemical compound C1=CC=CC2=CC(N)=CC=C21 JBIJLHTVPXGSAM-UHFFFAOYSA-N 0.000 description 1
- TVZRAEYQIKYCPH-UHFFFAOYSA-N 3-(trimethylsilyl)propane-1-sulfonic acid Chemical compound C[Si](C)(C)CCCS(O)(=O)=O TVZRAEYQIKYCPH-UHFFFAOYSA-N 0.000 description 1
- VXGRJERITKFWPL-UHFFFAOYSA-N 4',5'-Dihydropsoralen Natural products C1=C2OC(=O)C=CC2=CC2=C1OCC2 VXGRJERITKFWPL-UHFFFAOYSA-N 0.000 description 1
- TVZGACDUOSZQKY-LBPRGKRZSA-N 4-aminofolic acid Chemical compound C1=NC2=NC(N)=NC(N)=C2N=C1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 TVZGACDUOSZQKY-LBPRGKRZSA-N 0.000 description 1
- LPXQRXLUHJKZIE-UHFFFAOYSA-N 8-azaguanine Chemical compound NC1=NC(O)=C2NN=NC2=N1 LPXQRXLUHJKZIE-UHFFFAOYSA-N 0.000 description 1
- 229960005508 8-azaguanine Drugs 0.000 description 1
- 241000238876 Acari Species 0.000 description 1
- 206010003445 Ascites Diseases 0.000 description 1
- 238000011725 BALB/c mouse Methods 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 102000009410 Chemokine receptor Human genes 0.000 description 1
- 108050000299 Chemokine receptor Proteins 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- GUBGYTABKSRVRQ-WFVLMXAXSA-N DEAE-cellulose Chemical compound OC1C(O)C(O)C(CO)O[C@H]1O[C@@H]1C(CO)OC(O)C(O)C1O GUBGYTABKSRVRQ-WFVLMXAXSA-N 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 239000012981 Hank's balanced salt solution Substances 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000852559 Homo sapiens Thioredoxin Proteins 0.000 description 1
- 108091006905 Human Serum Albumin Proteins 0.000 description 1
- 102000008100 Human Serum Albumin Human genes 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 241000091754 Hyposoter fugitivus ichnovirus Species 0.000 description 1
- 108010091358 Hypoxanthine Phosphoribosyltransferase Proteins 0.000 description 1
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 102000017727 Immunoglobulin Variable Region Human genes 0.000 description 1
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 description 1
- 235000019454 L-leucine Nutrition 0.000 description 1
- 239000004395 L-leucine Substances 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108010058846 Ovalbumin Proteins 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 239000004372 Polyvinyl alcohol Substances 0.000 description 1
- 102000009843 Thyroglobulin Human genes 0.000 description 1
- 108010034949 Thyroglobulin Proteins 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 108010067390 Viral Proteins Proteins 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 238000005904 alkaline hydrolysis reaction Methods 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- QFAADIRHLBXJJS-ZAZJUGBXSA-N amastatin Chemical compound CC(C)C[C@@H](N)[C@H](O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CC(O)=O QFAADIRHLBXJJS-ZAZJUGBXSA-N 0.000 description 1
- 108010052590 amastatin Proteins 0.000 description 1
- 229960003896 aminopterin Drugs 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000002259 anti human immunodeficiency virus agent Substances 0.000 description 1
- 230000036436 anti-hiv Effects 0.000 description 1
- 239000003443 antiviral agent Substances 0.000 description 1
- 229940121357 antivirals Drugs 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000036765 blood level Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 238000010370 cell cloning Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000011210 chromatographic step Methods 0.000 description 1
- 238000002648 combination therapy Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 238000011461 current therapy Methods 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- 230000000120 cytopathologic effect Effects 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- IJKVHSBPTUYDLN-UHFFFAOYSA-N dihydroxy(oxo)silane Chemical compound O[Si](O)=O IJKVHSBPTUYDLN-UHFFFAOYSA-N 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 1
- 231100000676 disease causative agent Toxicity 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 230000005684 electric field Effects 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 239000003687 estradiol congener Substances 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 108010074605 gamma-Globulins Proteins 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000003054 hormonal effect Effects 0.000 description 1
- 102000056461 human TXN Human genes 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 230000009610 hypersensitivity Effects 0.000 description 1
- 230000000984 immunochemical effect Effects 0.000 description 1
- 238000003125 immunofluorescent labeling Methods 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 230000001524 infective effect Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 238000005511 kinetic theory Methods 0.000 description 1
- 238000011005 laboratory method Methods 0.000 description 1
- 229960003136 leucine Drugs 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000002483 medication Methods 0.000 description 1
- 239000012913 medium supplement Substances 0.000 description 1
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 229940126619 mouse monoclonal antibody Drugs 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 1
- 239000002687 nonaqueous vehicle Substances 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 239000000346 nonvolatile oil Substances 0.000 description 1
- 229940092253 ovalbumin Drugs 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 210000005105 peripheral blood lymphocyte Anatomy 0.000 description 1
- 230000002572 peristaltic effect Effects 0.000 description 1
- 230000003285 pharmacodynamic effect Effects 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 235000019422 polyvinyl alcohol Nutrition 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 239000003419 rna directed dna polymerase inhibitor Substances 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000012064 sodium phosphate buffer Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000012730 sustained-release form Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000017960 syncytium formation Effects 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 229960002175 thyroglobulin Drugs 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 238000003211 trypan blue cell staining Methods 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 210000002845 virion Anatomy 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
- 239000011592 zinc chloride Substances 0.000 description 1
- 235000005074 zinc chloride Nutrition 0.000 description 1
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 description 1
Classifications
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
- G01N33/56988—HIV or HTLV
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Virology (AREA)
- General Engineering & Computer Science (AREA)
- Tropical Medicine & Parasitology (AREA)
- Cell Biology (AREA)
- AIDS & HIV (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Food Science & Technology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Steroid Compounds (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
In spite of having achieved an in depth and comprehensive genetic structure of the human immunodeficiency virus (HIV) and of that of its several structural and geographical clusters, a very limited success in the prevention and treatment of AIDS
has been attained. Described herein is a method for preventing or reducing HIV
infectivity by using an antibody specific for estrogen-stimulated leucine aminopeptidase (es-LAPase), or one or more inhibitors of LAPase activity, or a combination thereof.
Also described is a method for preventing or reducing HIV infectivity by using an antibody specific for es-LAPase, one or more inhibitors of es-LAPase activity and an anti-estrogen compound. The novel compositions are also defined.
has been attained. Described herein is a method for preventing or reducing HIV
infectivity by using an antibody specific for estrogen-stimulated leucine aminopeptidase (es-LAPase), or one or more inhibitors of LAPase activity, or a combination thereof.
Also described is a method for preventing or reducing HIV infectivity by using an antibody specific for es-LAPase, one or more inhibitors of es-LAPase activity and an anti-estrogen compound. The novel compositions are also defined.
Description
METHOD AND COMPOSITIONS FOR PREVENTING OR REDUCING HIV
INFECTION
The present invention relates to a method to inhibit HIV infection by using an antibody to estrogen-stimulated leucine aminopeptidase (es-LAPase) or one ore more inhibitors of es-LAPase activity, or a combination thereof. The invention further relates to a method to inhibit 1~IV infection by using an antibody to es-LAPase, one ore more inhibitors of es-LAPase activity and an anti-estrogen compound. Novel compositions which inhibit HIV infection are also part of the present invention.
BACKGROUND OF THE INVENTION
In spite of having .achieved an in depth and comprehensive genetic structure of the human immunodeficiency virus (HIV) and of that of its several structural and geographical clusters, a very limited success in the prevention and treatment of AIDS
has been attained. Although HIV and its variants are well characterized as the prime causative agents leading to the human acquired immunodeficiency syndrome (AIDS), a less comprehensive identification of key cellular factors involved in the early molecular mechanisms leading to viral entry have been accomplished.
CD4 T-cells are the principal target of HIV infection as shown from both in vitro and in vivo studies. The CD4 glycoprotein at the surface of T-cells exhibits a high affinity for HIV virions. Other molecules, particularly from the chemokine-receptor family, also participate to promote viral entry into target cells.
Therapeutics modalities to thwart the progress of the disease in HIV infected patients relies principally on two types of medications: Reverse transcriptase inhibitors and protease inhibitors. Although successful at slowing the replication of the virus, they are limited by the high rate of mutagenesis in the virus which results in modification of the drugs' binding site and their concomitant lost of efficacy. One approach to resolve the problem has been to administer several drugs simultaneously to lower the probability of the development of resistant strains of the virus. However, these multidrug regimen create serious side effects. As a result compliance may be low.
Furthermore, current therapies for HIV infections are not specific for particular groups of individuals such, as women. In a recent report it was found that the number of women infected with HIV during 1991 and 1995 increased by 63 % , more than any other group of people that had contracted AIDS, regardless of race or mode of exposure to HIV virus [Centers for Disease Control and Prevention. (1996).
HIVlAIDSSurveill. Rep. Centers for Disease Control and Prevention; 8. Atlanta, Ga.].
The increased incidence of AIDS epidemic on morbidity and mortality among women (ages of 25-44) has been recently confirmed by other groups [Wortley, P.M., and Flemming, P.L. (1097). AIDS in Women in the United States: Recent Trends.
JAMA, 278: 911-916.]. The reasons for the increased incidence of AIDS in women are largely unknown. Not only women are more prone to infection than men (supra) but studies have shown that anti-HIV drugs work differently in women and men. For example, toxicities, side effE:cts and blood levels have been shown to be gender specific.
Hormonal differences between men and women are cited to explain the differences but no clear link has been established. In view of the above data, there is a need to tailor therapeutic modalities to exploit the differences in the infection process between men and women to improve the efficiency of treatments.
Independently of the role of CD4, the putative receptor for HIV and that of the (3-chemokine co-receptors (CKR-5, CCRS and CXCR-4), we have described the important role of both membrane-bound and extracellular Leucine Aminopeptidase in HIV viral entry. (F'ulido-Cejudo, G. et al. (1997) Antiviral Research 36, 167-177) The present invention overcomes the limitation of the prior art by providing a link between LAPase activity and estrogen. This link has been exploited herein to discover new HIV viral inhibitors which can be used alone or in combination with improved efficacy.
INFECTION
The present invention relates to a method to inhibit HIV infection by using an antibody to estrogen-stimulated leucine aminopeptidase (es-LAPase) or one ore more inhibitors of es-LAPase activity, or a combination thereof. The invention further relates to a method to inhibit 1~IV infection by using an antibody to es-LAPase, one ore more inhibitors of es-LAPase activity and an anti-estrogen compound. Novel compositions which inhibit HIV infection are also part of the present invention.
BACKGROUND OF THE INVENTION
In spite of having .achieved an in depth and comprehensive genetic structure of the human immunodeficiency virus (HIV) and of that of its several structural and geographical clusters, a very limited success in the prevention and treatment of AIDS
has been attained. Although HIV and its variants are well characterized as the prime causative agents leading to the human acquired immunodeficiency syndrome (AIDS), a less comprehensive identification of key cellular factors involved in the early molecular mechanisms leading to viral entry have been accomplished.
CD4 T-cells are the principal target of HIV infection as shown from both in vitro and in vivo studies. The CD4 glycoprotein at the surface of T-cells exhibits a high affinity for HIV virions. Other molecules, particularly from the chemokine-receptor family, also participate to promote viral entry into target cells.
Therapeutics modalities to thwart the progress of the disease in HIV infected patients relies principally on two types of medications: Reverse transcriptase inhibitors and protease inhibitors. Although successful at slowing the replication of the virus, they are limited by the high rate of mutagenesis in the virus which results in modification of the drugs' binding site and their concomitant lost of efficacy. One approach to resolve the problem has been to administer several drugs simultaneously to lower the probability of the development of resistant strains of the virus. However, these multidrug regimen create serious side effects. As a result compliance may be low.
Furthermore, current therapies for HIV infections are not specific for particular groups of individuals such, as women. In a recent report it was found that the number of women infected with HIV during 1991 and 1995 increased by 63 % , more than any other group of people that had contracted AIDS, regardless of race or mode of exposure to HIV virus [Centers for Disease Control and Prevention. (1996).
HIVlAIDSSurveill. Rep. Centers for Disease Control and Prevention; 8. Atlanta, Ga.].
The increased incidence of AIDS epidemic on morbidity and mortality among women (ages of 25-44) has been recently confirmed by other groups [Wortley, P.M., and Flemming, P.L. (1097). AIDS in Women in the United States: Recent Trends.
JAMA, 278: 911-916.]. The reasons for the increased incidence of AIDS in women are largely unknown. Not only women are more prone to infection than men (supra) but studies have shown that anti-HIV drugs work differently in women and men. For example, toxicities, side effE:cts and blood levels have been shown to be gender specific.
Hormonal differences between men and women are cited to explain the differences but no clear link has been established. In view of the above data, there is a need to tailor therapeutic modalities to exploit the differences in the infection process between men and women to improve the efficiency of treatments.
Independently of the role of CD4, the putative receptor for HIV and that of the (3-chemokine co-receptors (CKR-5, CCRS and CXCR-4), we have described the important role of both membrane-bound and extracellular Leucine Aminopeptidase in HIV viral entry. (F'ulido-Cejudo, G. et al. (1997) Antiviral Research 36, 167-177) The present invention overcomes the limitation of the prior art by providing a link between LAPase activity and estrogen. This link has been exploited herein to discover new HIV viral inhibitors which can be used alone or in combination with improved efficacy.
SUMMARY OF THE INVENTION
The present invention relates to a method to inhibit HIV infection by using an antibody to es-LAPase or an inhibitor of es-LAPase activity, or a combination thereof.
The invention further relates to a method to inhibit HIV infection by using an antibody to es-LAPase, an inhibitor of es-LAPase activity and an anti-estrogen compound.
Novel compositions which inhibit HIV infection are also part of the present invention.
Thus according to the present invention there is provided a method for reducing HIV
infectivity towards T lymphocytes comprising contacting said T lymphocytes with a sufficient amount of an inhibitor of LAPase.
In a further embodiment of the present invention there is provided a method for reducing HIV infc:ctivity towards T lymphocytes comprising contacting said T
lymphocytes with a sufficient amount of an antibody specific for LAPase, more specifically a monoclonal antibody specific for es-LAPase.
This invention also provides a method for reducing HIV infectivity towards T
lymphocytes comprising contacting said T lymphocytes with a sufficient amount of an antibody specific for es-LAPase and an inhibitor of LAPase.
This invention also provides a method for reducing HIV infectivity towards T
lymphocytes comprising contacting said T lymphocytes with a sufficient amount of an antibody specific for es-LAPase, an inhibitor of LAPase and an anti-estrogen compound.' Further according to the present invention there is provided a method for preventing or reducing HIV infection comprising administering to a patient in need thereof, a pharmaceutically effective amount of a composition which comprises an antibody specific to es-LAPase and a pharmaceutically acceptable carrier.
Also provided in this invention is a method for preventing or reducing HIV
infection comprising administering to a patient in need thereof, a pharmaceutically effective amount of a composition which comprises an inhibitor of LAPase and a pharmaceutically acceptable carrier.
S This invention is also directed to a method for preventing or reducing HIV
infection comprising administering to a patient in need thereof, a pharmaceutically effective amount of a composition which comprises an antibody specific to es-LAPase and an inhibitor of LAPasf; together with a pharmaceutically acceptable carrier.
This invention is also directed to a method for preventing or reducing HIV
infection comprising admini<,;tering to a patient in need thereof, a pharmaceutically effective amount of a composition which comprises an antibody specific to es-LAPase, an inhibitor of LAPase: and an anti-estrogen compound together with a pharmaceutically acceptable carrier.
Also according to the present invention there is provided a composition comprising an antibody specific to es-LAPase and an inhibitor of LAPase.
Also according to tree present invention there is provided a composition comprising an antibody specific ~:o es-LAPase, an inhibitor of LAPase and an anti-estrogen compound.
BRIEF DESCRIPTION OF THE DRAWINGS
These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings wherein:
FIGURE 1 shows ~:he effect of estrogen on LAPase activity in cell supernatents of primary parental breast cancer cell lines.
FIGURE 2 is a SDS PAGE of crude (lane 1) and purified LAPase (lane 2) followed by western blot with MAb 7B6.
FIGURE 3 shows the kinetics of inhibition of LAPase by Bestatin, thioredoxin and glutathione.
DETAILED DESCRIPTIOiV OF PREFERRED EMBODIMENT
The present invention relates to a method to inhibit HIV infection by using an antibody to es-LAPase or an inhibitor of es-LAPase activity, or a combination thereof.
The invention further relates to a method to inhibit HIV infection by using an antibody to es-LAPase, an inhibitor of es-IJAPase activity and an anti-estrogen compound.
Novel compositions which inhibit HfIV infection are also part of the present invention.
It has been observed that, independently of the role of CD4, the putative receptor for HIV and that of the 13-chemokine co-receptor.s (CKR-5/CCRS;CXCR-4), both membrane-bound and extracellular leucine aminopeptidase (LAPase;EC 3.4.11.1) can promote HIV entry in T lymphocytes (Pulido-Cejudo et al. Antiviral Res. 36:167-177, 1997).
In the present invention it is disclosed that LAPase activity in T lymphocytes and in breast cancer parental cells can be stimulated by estrogens, or estrogen analogues. One aspect of this invention is directed to the identification and purification of an estrogen-stimulated LAPase isoenzyrne, referred to herein as es-LAPase. Details of the estrogen-stimulated LAPase isoenzyme are described in Applicants copending Canadian patent application No. 2,303,505 entitled "A Monoclonal Antibody against Estrogen Stimulated Leucine Aminopeptidase".
A further aspect of the present invention is the preparation of antibodies against the es-LAPase isoenzyme. Antibodies can be produced by methods well known in the art.
In general, the antibody producing cells are prepared by immunizing an animal, for example, mouse, rat, rabbit, sheep, horse, or bovine, with an antigen. The immunization schedule and the concentration of the antigen in suspension is such as to provide useful quantities of suitably primed antibody producing cells. These antibody producing cells can be either spleen cells, thymocytes, lymph node cells and/or peripheral blood lymphocytes.
The antibody producing cells are then fused with myeloma cells, cell lines originating from various animals such <is mice, rats, rabbits. and humans can be used, using a suitable fusion promoter. N(any mouse myeloma cell lines are known and available generally from members of the academic community and various depositories, such as the American Type Culture Collection, Rockville, Maryland. The myeloma cell line used should preferably be medium sensitive so that unfused myeloma cells will not survive in a selective media, while hybrids will survive. The cell line most conunonly used is an 8-azaguanine resistant cell line, which lacks the enzyme hypoxanthine-guanine- phosphoribosyl-transferase and therefore will not be supported by HAT
(hypoxanthine-aminopterin-thymidine) medium. In general, the cell line is also preferably a "non-secretor" type, in that it does not produce any antibody.
The preferred fusion promoter is polyethyleneglycol having an average molecular weight from about 1000 to about 4000. Other fusion promoters such as polyvinylalcohol, a virus or an electrical field can also be used.
The immortalized cells (hybridoma) must then be screened for those which secrete antibody of the correct specificity. The initial screening is generally carried out using an enzyme-linked immunosorbent assay (ELISA). Specifically, the hybridoma culture supernatants are added to mic:rotitre plates which have been previously coated with the antigen, in this case the es-LAPase. A bound specific antibody from the culture supernatants can be detected using a labelled second antibody, for example, goat antimouse IgG labelled with peroxidase, which is commercially available.
Cultures that are positive against the es-LAPase are then subjected to cloning by the limiting dilution method. Secondary hybridoma cultures are re-screened as described above, and further positive cultures acre then examined using the BIAcore ~M system (Pharmacia Biosensor AB, Uppsala, Sweden). The cultures are then evaluated as to determine whether or not the antibody binds the antigen and to determine the kinetic profile of antigen binding. Selected cultures based on these results are subject to further cloning until culture stability and clo:nality are obtained. Immediately after hybridization, the fusion products will have approximately 80 chromosomes, and as these cells proceed to divide they will randomly lose some of these chromosomes. The cloning process is to select those cells which still have the chromosomes coding for antibody production. The cloning process is repeated until 100% of the sub-population exhibits the production of a specific antibody, which is indicative of the "stability"
of the hybridoma. In addition, hybridoma culture wells often have multiple colonies some of which may be antibody non-producers. The cloning process allows the selection of a positive hybrid which is derived from a single cell.
In one embodiment of the present invention there is provided a monoclonal antibody, specific against the es-LAPase, which has been designated Mab 7B6. This monoclonal antibody does not inhibit the enzymatic activity of LAPase. For this reason it will be referred to hereinafter as a non-inhibitory monoclonal antibody. A hybridoma cell line producing this monoclonal antibody has been deposited with the International Depository Authority of Canada, Winnipeg, Manitoba on March 23, 2000 (Accession #
IDAC 230300-1 ) in compliance with the Budapest Treaty.
The present invention also encompasses MAb 7B6 and any fragments thereof containing the active binding region of the antibody such as Fab, F(ab)2 and Fv fragments. These fragments can be obtained from the 7B6 antibody by using techniques well known to those of skills in the art (Rousseaux et al. Methods Enzymology, 121:663-69, Academic Press, 1986).
A further embodiment of the present invention encompasses antibodies or fragments thereof capable of binding the same antigenic determinant as the 7B6 antibody.
Including, but not limited to, antibodies possessing the same antigenic specificity as the 7B6 antibody but originating from a different species or having a different isotype or exhibiting different binding affinities. It is envisioned that class and isotype variants of the antibody of the present invention can be prepared using recombinant class switching and fusion techniques that are well known to those skilled in the art (see for example: Thammana et al. Eur. J. Immunol, 13:614, 1983; Oi et al., Biotechnologies, _g_ 4(3):214-221, Liu et al. Proc. Nat. Acid. Sci. (USA), 84:3439-43, 1987;
Neuberger et al., Nature 31:2:604-608, 1984 and Spira et al. J. Immunol. Meth., 74:307-15, 1984).
The monoclonal ;antibody of the present invention can be produced either using a bioreactar or from ascites, both procedures of which are well known in the art.
In one embodiment of the present invention there is provided a method of reducing HIV infection wherein Mab 7B6 is incubated with T lymphocytes prior to and during infection with HI'J-1. This incubation prevents or reduces the infectivity of towards T lymph~~cytes. Even though not described in the examples (infra), this embodiment also encompasses incubating T lymphocytes with Mab 7B6 after they have been infected with HIV-1 to reduce further infection as would be obvious to one skilled in the art.
The monoclonal antibody can be added directly to the incubation medium or it can be coupled to.a solid :matrix. The solid matrix can be any matrix, as would be known to someone skilled in the art, onto which the antibody can be coupled as long as this coupling does not interfere with the binding of the antibody to its epitope on es-LAPase. The solid matrix can be, but is not limited to, a protein G matrix.
Without wishing to be bound by any theory, the coupling of the antibody to the matrix may reduce the level of non-specific binding of the anti-body to its epitope.
HIV infection of T lymphocytes can be measured using a number of techniques.
The three most commonly used are the quantification of viral capsid protein p24 production, cyopathycity, and the determination of proviral load by polymerise chain reaction (PCR)(Conway et al., Clin. Diag. Virol. 3:95-104, 1995). These methods are well known in the a.rt. Other methods as would be known to one skilled in the art may also be used to determine the levels of HIV infectivity.
According to one aspect of the present invention anti-es-LAPase antibodies are used to reduce HIV infection. According to this embodiment of the present invention cells can be treated at a concentration of from about Song to about 200ng of antibody per ml of cells, wherein the cells are at a density of about 1.0 x 105 cells/ml.
The preferred amount of antibody can be determined empirically, by a person of ordinary 5 skill in the art. In one example of this embodiment the concentration of antibody used is about 100ng per 10 ml of cells.
The present invention also discloses that thiol containing compounds unexpectedly inhibit the activity of es-LAPase. By thiol containing compounds it is meant any 10 compound having a SH group such as, but not limited to, peptides having reduced cysteine residues. lfn one embodiment of this aspect of the invention, thioredoxin and glutathione have been found to inhibit es-LAPase.
Thus, in a further embodiment of the present invention, there is provided a method wherein an inhibitor of es-LAPase is incubated with T lymphocytes prior to and during infection with HIV. According to this aspect of the invention, the inhibitor of es-LAPase activity cam be a competitive, an uncompetitive or non-competitive inhibitor.
The meaning of these terms is taken in the context of the enzyme kinetic theory, as would be known to persons skilled in the art. In this aspect of the invention, an example of an uncompetitive inhibitor is thioredoxin and an example of a non-competitive inhibitor is glutathione.
The inhibitor of LAPase can be added directly to the incubation medium or it can be coupled to a solid matrix. The solid matrix can be any matrix, as would be known to someone skilled in the art, onto which the inhibitor can be coupled as long as this coupling does not interfere with the binding of the inhibitor to es-LAPase.
The solid matrix can be, but is not limited to, DPDP, an amide bonding matrix (Pierce, Rockford,IL, USAj.
In this aspect of the present invention the inhibitors of es-LAPase activity are used to reduce HIV infection. According to this embodiment of the present invention cells can be treated with the inhibitor at a concentration of from about 10-~M to about 10-5M, wherein the cells are at a density of about 1.0 x 10' cells/ml. The preferred amount of inhibitor can be determined empirically, by a person of ordinary skill in the art. In one example of this embodirr'ent the concentration of inhibitor used is from about 4ng to about 8ng per ml of cells.
In another aspect of the preaent invention there is provided a method wherein an antibody to es-LAPase and am inhibitor of LAPase can be combined and incubated with T lymphocytes prior, during or after infection with HIV to reduce HIV
infectivity. In this aspect of the present invention, the antibody and inhibitor are used together according to the methods already disclosed for their individual use.
In a previous publication (Mesange et al. Mol. Pharmacol. , 50:75-79, 1996) it has been shown that TamoxifenT"', an anti-estrogen compound can reduce HIV infectivity.
However, incomplete inhibition of HIV infection were observed at TamoxifenT~'' concentrations of 10-~M or higher. In a further aspect of the present invention there is provided a method wherein not only TamoxifenT~' but other anti-estrogens can be used together with an antibody to es-LAPase and an inhibitor of LAPase to reduce HIV
infectivity.
In this example of the present invention the anti-estrogen compound is selected from the following, used alone car in combination: 4-hydroxyandrostenedione, RaloxifeneT'~', 3~3,Sa-tetrahydro Norethisterone, Tamoxifen, Droloxifene"'' and Idoxifene'M.
In this aspect of the present invention the anti-estrogen compound is used at a final concentration ranging from about 106 M to about 10-~ M. The concentrations of the other components in this method are used as describe above.
In a further aspect of this invention a competitive inhibitor of es-LAPase activity can also be used together with thf: above described compounds. An example of a suitable competitive inhibitor is BestatinTM, but the invention also encompasses other comptetive inhibitors such as Amastatin rM.
The present invention also encompasses a method for treating patients infected with HIV, or at risk of being infected, with an antibody to es-LAPase or one or more inhibitors of es-LAPase activity or a combination thereof. The present invention also encompasses a method for treating patients infected with HIV, or at risk of being infected, with an antibody to es-LAPase, one or more inhibitors of es-LAPase activity and an anti-estrogen. In this aspect of the invention there is provided a pharmaceutical composition comprising an antibody to es-LAPase. In a further aspect of this embodiment the pharmaceutical composition comprises one or more inhibitors of es-LAPase activity. In a still further aspect of this embodiment the pharmaceutical composition comprises an antibody to es-LAPase and one or more inhibitors of es-LAPase activity. In a still further aspect of this embodiment the pharmaceutical composition comprises an antibody to es-LAPase and one or more inhibitors of es-LAPase activity and an anti-estrogen compound.
Pharmaceutical compositions of the present invention generally will include a therapeutically effective amount of at least one of the compounds referred to above together with a pharmaceutically effective carrier. The antibody of the present invention may be administered by any means that enables the active agent to reach the agent's site of action in the body of a mammal. In the case of the antibodies of this invention, the primary focus is the ability to reach and bind with both membrane-bound and extra cellular es-LAPase. Because proteins are subject to being digested when administered orally, parenteral administration, i.e., intravenous, subcutaneous, intramuscular, would ordinarily be used to optimize absorption.
The antibodies of this invention may be administered either as individual therapeutic agents or in combination with other therapeutic agents, as discussed above.
They can be administered alone, but are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.
The dosage administered will, of course, vary depending upon known factors such as the pharmacodynamic characteristics of the particular agent, and its mode and route of administration; ag;e, health, and weight of the recipient; nature and extent of symptoms, kind of concurrent treatment, frequency of treatment, and the effect desired. Usually a daily dosage of active ingredient can be about 0.1 to 100 milligrams per kilogram of body weight. Ordinarily 0.5 to 50, and preferably 1 to 10 milligrams per kilogram per day given in divided doses 1 to 6 times a day or in sustained release form is effective to obtain desired results.
Dosage forms (composition) suitable for internal administration generally contain from about 1 milligram to about 500 milligrams of active ingredient per unit. In these pharmaceutical compositions the active ingredient will ordinarily be present in an amount of about 0.5 - 95 % by weight based on the total weight of the composition.
For parenteral administration, the antibody can be formulated as a solution, suspension, emulsion or lyophilized powder in association with a pharmaceutically acceptable parenteral vehicle. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 5 % human serum albumin. Liposomes and nonaqueous vehicles such as fixed oils may also be used. The vehicle or lyophilized powder may contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and cherr~ical stability (e.g., buffers and preservatives). The formulation is sterilized by commonly used techniques.
In further embodiments of the present invention, the antibody can be administered together with one or more LAPase inhibitors, as discussed in detail above. In this embodiment, the L.APase inhibitors can be added at a dose of about 0.1 to about 100 milligrams per kilogram of body weight.
In yet a further embodiment of the present invention, the antibody can be administered together with one or more anti-estrogen compound, as discussed in detail above. In this embodiment, the anti-estrogen compound can be added at a dose of about 0.1 to about 100 milligrams per kilogram of body weight.
In another embodiment of the present invention, the antibody can be administered together with one or more LAPase inhibitors and one or more anti-estrogen compounds as discussed in detail above. In this embodiment, both the LAPase inhibitors and anti-estrogen compounds can be added at doses of about 0.1 to about 100 milligrams per kilogram of body weight.
The antibody and the inhibitors to be included in the pharmaceutical composition may be coupled to a matrix as described above, as long as the matrix is non-toxic to mammals. Coupling of the antibody and the inhibitors to an appropriate matrix may also facilitate the incorporation of these compounds into liposomes.
In a further embodiment, the inhibitor of LAPase in the pharmaceutical composition is the uncompetitive inhibitor thioredoxin, which may be maintained in its reduced state by including reducing compounds in the composition. Such compounds may include but are limited to g:lutathione and cysteine.
In order that the invention described herein may be more fully understood, the following examples are set forth. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting the scope of this invention in any manner.
EXAMPLES
Example 1: Estrogen mediated LAPase release from HUT78 cells CD4+ T-HUT78 cells were incubated in the presence of various concentrations of 17-~3-Estradiol and I~APase levels were determined as described by G. Pulido-Cejudo et al. (Antiviral Research, 36:167-177, 1997).
Table 1 show that estrogen promoted the release of LAPase in HUT78 cells in a dose response manner, with a maximum stimulation observed at 10-'M.
Table 1 Extracellular LAPase activity of HUT78 cells incubated with 17-~-Estradiol LAPase activity (U* x 10-S/ml) [~3-Estradiol] Control 17-[3-Estradiol Stimulation 10-SM 4.2 10.8 10-6M 5 . 6 20.4 10-'M 4.8 148.6 10-gM 5.7 110.7 * One unit of LAPase is defined as the amount of enzyme required to hydrolyze 1 ~mol of L-leucine-(~-naphthylamide to L-leucine and (3-naphthylamide per min at pEl 7.5 and 37°C.
Following estrogen stimulation, maximum release of LAPase from breast carcinoma parental cells was observed at an estrogen ( 17(3-Estradiol) concentration of 100 nM
following 24 hours of incubation (see Fig. 1 where LAPase activity was determined in supernatants of primary parental cell lines immunoprecipitated with anti-LAPase-SepharoseT'''' bound Mab 7B6 antibodies. Cells were incubated in the presence of 17-~3-Estradiol and following a :?4 hr incubation, supernatants were removed from the primary culture, immunoprecipitated and assayed tluorometrically for LAPasf:
activity by the method of Kuramochi et al. [1987, J. Antibiot., 40: 1605:1611]
Control cells were cultured in the absence of 17-~3-Estradiol.). During the same period, LAPase in cell supernatants of control cells remain unchanged. In addition, LAPase activity was determined in supernatants of primary parental cell lines immunoprecipitated with anti-LAP-Protein G MAb 7B6 bound antibodies. These results show that the extracellular LAPase activity of estrogen stimulated cells (100 nM) was 7.7 x 10-5 U/ml in comparison to 6.4 x 10-~ U/ml detected in the supernatant of parental cells incubated for 24 hours with cell media alone as control. In addition, there was no effect of estrogen incubation on LAPase activity in purified preparations of this enzyme alone. Collectively, these results suggest that estrogen effect on LAPase activity encompasses a cellular mediated process.
Example 2: Purification ot_es-LAPase Primary parental breast carcinoma cells obtained from human tumour biopsies.
were stimulated with 100nM 17-~3--Iatradiol for 24 hours or cell media alone as a control.
The cell media was RPMI 1640 medium + 10 % FCS + 100U/ml Penicillin + 100 ~,g/ml Streptomycin. Cell supernatants were collected and then dialyzed against PBS in seamless cellulose tubing (MW 12,400) for 12 hours at 4°C. Es-LAPase was subsequently purified from the dialyzed cell supernatants using HPLC-gel permeation followed by DIRE-Cellulose and Bestatin-Sepharose affinity Chromatography. Briefly, the cell supernatant was applied to a Bio-Sil SEC-2:50 column (600x7.5 mm) previously equilibrated in a buffer containing 100 mM
Sodium Phosphate buffer pH fi.8, 100 mM Na~SO,, 1~,M ZnCh and 10% glycerol.
The column was washed with 300 ml of the same buffer at a flow rate of 0.5 ml/min. Protein was concentrated to 10 ml by ultrafiltration using YM5 membrane (5000 M.W. cutoff, Amicon I)iv., Danvers, MA., USA). The concentrate was applied to a DEAE cellulose column (2.6 cm X 28.5 cm) equilibrated and washed with 50 mM Tris-HC1 buffer pH 7.5; 1~,M ZnCl2; and 10% (v/v) glycerol. Es-LAP was eluted using a linear gradient (0 to 1M NaCI in Tris buffer) at a flow rate of 0.50 ml/min. A Bestatin-affinity column was prepared using UltralinkTM
EDC/DADPA Amide bonding matrix (Pierce, Rockford, IL U.S.A.) by reacting 100mg of pure Bestatin with the carbodiimide EDC/DADPA matrix following the procedure provided by the manufacturer. Prior to loading the LAPase containing eluent, the Bestatin-affinity column was equilibrated with 10 mM Tris-HC1 pH
8.0 containing 1~M ZnClz and washed with 300 ml of this binding buffer. Es-LAl?ase was recirculated through the system using a peristaltic pump at a t7ow rate of O.lOml/min, for 2 hours. Following this recirculation, the column was washed with eight column volumes of binding buffer.
Bestatin-bound es-LAPase was eluted with a linear gradient (0-0.5 M NaCI) prepared in binding buffer 10 mM Tris-HCl pH 8.0 containing 1~M ZnClz .
Elution of bound es-LAPase was monitored by absorbance at 280 nm. Purified es-LAPase fractions were aliquoted in 500 ~1 and stored until further use in 50 mM Tris-HCl pH 7.8 and SO~cM ZnClz . Es-LAPase Protein concentration was estimated by the method of Lowry et al. [J Biol Chem 193:265 (1951)] using bovine serum albumin as a standard. A summary of the purification fold of the 17-~3-Estradiol-stimulated LAPase from parental cells of human breast carcinomas is set out in Table 2.
Es-LAPase was purified to an approximate 7000 fold after the last Bestatin-affinity chromatographic step.
Es-LAPase activities were determined fluorometrically using leucine-b-naphthylamide as the substrate [Kuramochi, H., et al. (1987) J. Antibiot. , 40,1605-1611] .
The reaction was stopped by boiling the samples at 100°C for 10 minutes, followed by centrifugation at 780 x g at 4°C for 10 minutes. Values obtained represent the average of LAPase activity determined in triplicate.
Table 2 Summary of Purification of LAP from Human Breast Carcinoma Parental Cells Step P:roteinlTotal Activity2Specific Activity2Fold Yield (rng) (nmole/min)(nmole/min/mg) Supernatant1 '.i.4 98.21 6.37 1 100 Gel Permeation4.2 82.13 19.55 3.07 83.63 Cellulose 0.72 48.25 67.01 10.51 49.13 DEAE
Bestatin- 0.005 37.11 7422 1165 37.79 Sepharose 1-the amount of protein was determined after alkaline hydrolysis and quantitative ninhydrin detection of hydrolyzed material as described by Pulido-Cejudo et al. [J.
Chromatogr. B 660 (1994) 37-47)].
2-determined fluorometrically using leucine-~3-naphtylamide as a subtrate as described by [Kuramochi, H., et al. (1987) J. Antibiot. , 40,1605-1611].
Example 3: Monoclonal Antibody Production and Purification In producing the hvbridoma cell line 7B6 secreting the mouse monoclonal antibody to 17-~i-Estradiol-stimulated LAPase, protocols for antigen preparation for immunization, preparation of spleen cells from immune animals, fusion of spleen cells with myeloma. cells and plating of fused cells in selective medium was conducted following detailed guidelines described by Campbell [Burdon RH, Knippenberg PHV (eds): Laboratory Techniques in Biochemistry and Molecular Biology, Amsterdam, Elsevier, p219 (1984)] and by Lietzke and Unsicker [Leitzke R. Unsicker K: A :statistical Approach to Determine Monoclonality After Limiting Cell Plating of a Hybridoma Clone, J Immunol Methods 76:223 (1985)].
Briefly, the primary immunization was performed with purified es-LAPase following desalting. Boosts with purified es-LAPase were performed at days 14, & 56. BALB/c mice were screened at days 24 & 45. The mice were sacrificed at day 59 and the splenocytes from the best responder were fused with myeloma cells.
Screening was performed by dot blot immunostaining on nitrocellulose.
The hybridoma clone 7B6 was obtained by single cell cloning using limiting dilution. Four dilution tubes in series containing hybridoma cells with medium supplement with 20% FBS -~ 2X OPI were prepared. 100 ~,1 of each dilution was plated in a 96-well plate with 50 ~.l of splenocyte feeder cells in each well and placed inside a 37°C 5 % C.""0, incubator. At day 7, supernatants from each well were removed and screened by dot blot immunostaining on nitrocellulose.
Hybridoma clone 7B6 cells were transferred from the 96 well plate to 0.5 nil medium supplemented with :?0% FBS + 1X OPI + 1X HAT in a 24 well plate.
Once the cells were dense, they were transferred into 5 mls in a 60 mm dish and then transferred to 10 mls in a 100 mm dish. Once in the 60 mm dish, the cells were weened off hypoxanthine, thymidine and aminopterin. 7B6 hybridoma cells were continued to be grown until in a log phase of growth. Anti-es-LAPase, Nlab 7B6 was isolated from collected hybridoma 7B6 cell supernatant by affinity chromatography using Immunopure~~'' IgG as per described by manufacturer.
Screening was performed by dot blot immunostaining on nitrocellulose.
The isotype of Mab 7B6 was determined using Sigma's Immunotype Kit~M. Briefly, the assay involves binding of Mab 7B6 to a precoated isotyping nitrocellulose membrane strip followed by immunodetection using a sensitive biotin-avidin-enzyme detection system. I'he immunoglobulin isotype is revealed by self description.
ELISA analysis of Human Breast Carcinoma parental cell lines was conducted to demonstrate the reactivity of Mab 7B6 against human es-LAPase. Briefly, 50 x parental cells were plated per well in a 96 well plate in RPMI 1640 medium -I-10%
FCS + 100 U/ml Penicillin -+- 100 ~.g/ml Streptomycin. The plated cells were cultivated at 37°C 5 % CO, for 24 hours. The cell supernatants were removed, the cells were washed with PBS and subsequently fixed with 1 % gluteraldehyde in PBS
for 1 hour at room temperaW re. Washing with PBS occurred prior to blocking with casein for 1 hour at 37°C S ~~ CO,. Following another wash with PBS, serial dilutions of Mab 7B6 were added to the wells and allowed to incubate for 2 hours at 37°C 5% CO~, Demonstration of the reactivity of Mab 7B6 was revealed upon the addition of a secondary antibody, anti-(IgG + IgM) peroxidase conjugated goat anti-mouse IgG + IgM (H+L) followed by the substate, OPD.
Following purification of the Mab 7B6, the corresponding IgGla isotype was subsequently immobilized to a DSS cross-linking system obtained from Pierce (Rockford, IL, U.S.A) according to the procedures described by the manufacturer.
As shown in Figure 2, Mab '7B6 specifically reacts with es-LAPase in both crude cell supernatants as well as purified es-LAPase after SDS PAGE followed by immunoelectrotransfer. The estimated Molecular Mass of 17-13-Estradiol-stimulated LAPase was 315 kDa as estimated by Gel Permeation on a Bio-SiITM SEC-250 column (600x7.5 mm) using Thyroglobulin (MW 670kDa); Bovine Gamma Globulin (MW 158 kDa); Chiken ovalbumin (MW 44 kDa) and Equine Myoglobin (l7kDa).
Example 4: Effect of Estrosten on increased infectivitv of HUT78 cells.
Estrogen-mediated HIV-induced cytopathic effect was determined as described by G. Pulido-Cejudo et al. [Pulido-Cejudo, G., Conway, B., Proulx, P., Brown, R., and Izaguirre, C.A. (1997). Anti viral Research, 36: 167-177]. The sensitivity of CD4+ T-HUT78 cells to HIV viral infection was determined by measuring the activity of reverse transcriptase in cells infected in the presence/absence of 17-~3-Estradiol.
In light of both the; clinical observations indicating the hypersensitivity of women to HIV infection [Centers for Disease Control and Prevention. (1996). HIVlAIDS
Surveill. Rep. 8. Atlanta, Ga., Wortley, P.M., and Flemming, P.L. (1997).
JAMA, 278: 911-916.] as well as the role of LAPase in viral entry [Pulido-Cejudo, G., Conway, B., Proul!x, P., Brown, R., and Izaguirre, C.A. (1997). Antiviral Research, 36: 167-177], together with the direct effect of estrogen on LAPase reported supra, the sensitivity of HUT78 cells to HIV viral infection was determined. As shown in Table 3, at 3 days and 5 days post infection, in the presence of estrogen, HIV viral infectivity as determined by the activity of reverse transcriptase (RT) was approximately 4 times higher than control cells infected in the absence of estrogen.
Table 3 Effect of Estrogen on increased infectivity RT Activity (cpm x 102/10,1) Day 0 Day 3 Day 5 (Infection) (Post Infection) (Post Infection) Control 0.82 19.82 32.44 Estrogen 0.68 72.42 124.44 ( 10-'M) Example 5: Inhibitors of es-LAPase Human Thioredoxin was purified from CD4+ T MP6 cells as described by Rosen et al. [Rosen, A. et al. (1995) lnt. Immunol. 7, 625-633 ]and compared to purified Thioredoxin activity from E.C'.oli. purchased from SIGMA-ALDRICH Canada (Oakville,Ontario Canada).
Thioredoxin was covalently :linked through an amide bond to Ultralink EDC/DADPA bonding matrix (Pierce, Rockford, IL U.S.A.) by reacting 5 mg of purified Thioredoxin with the carbodiimide EDC/DADPA matrix following the procedure provided by the manufacturer.
Measurements of LAPase were performed spectrophotometrically at 330nm to measure product formation of 13-naphthylamine, using 182~eM 1-leucine-13-naphthylamide as the substrate. The kinetic assays were performed using a Spectronic Genesys~M ~ spec~.rophotometer from Milton Roy (Rochester, NY;).
Absorbance was recorded every sixty seconds, over a period of 30 min. Enzyme kinetic assays were performed using 8.0 x 10-2 U of es-LAPase in a final reaction volume of 600.1 and triplicate samples. The reaction mixture contained the following materials added in order and all kept on ice prior to use: es-LAPase, Calcium-free Hank's solution making up a 600,1 final reaction volume and 182~,M
1-leucine-li-naphthylamide. A corresponding blank without es-LAPase was used as baseline. Both of the cuvettes were transferred to the spectrophotometer where the 1-leucine-13-naphthylmide solution was added last, marking time zero of the reaction. The inhibition studies were carried out in the presence of 167~M
Bestatin, 167~,M reduced Gluthathione and 17 ~M reduced thioredoxin.
Figure 3 shows the inhibitory effect of both Bestatin and two thiol-containing peptides, reduced thioredoxin and reduced glutathione respectively. Es-LAPase showed significant slower mites of reaction both in the presence of thioredoxin and glutathione in comparison to that observed in the control es-LAPase. In addition, es-LAPase reaction rates were slower in the presence of 17~M thioredoxin when compared to those observed in samples incubated with 167~,M reduced Gluthathione. In addition, 16~7~,M Bestatin clearly inhibits es-LAPase activity. As shown in Table 4., reduced thioredoxin is an effective inhibitor of es-LAPase activity. The analysis of the maximum reaction rates (Vmax), Michaelis-Menten (Km) constants and the inhibition constants (Ki) for each inhibitor as presented in Table 4 reveals significant differences in the inhibitory properties of each peptide.
S In this regard, es-LAPase incubated with Bestatin shows a Vmax value very similar to that observed to es-LAPase alone and a significantly higher Km value. This data confirms the competitive nature of es-LAPase inhibition by Bestatin. In contrast, es-LAPase incubated with reduced thioredoxin leads to a significantly lower Vmax and Km with an intermediate Ki value when compared to those obtained for Bestatin and glutathione. Collectively these data strongly suggest that reduce thioredoxin inhibits es-LAPase in an uncompetitive fashion while with reduced glutahthione es-LAPase inhibition is noncompetitive.
Table 4 ~ Bestatin, reduced thioredoxin and glutathione es-LAPase inhibition Constants Vmax Km Ki (10-6 M/min) (10-4 M) (10-6M) LAPase alone 36.81.9 1.670.19 N/A
Bestatin 167uM 39.03.9 48.78.4 6.221.61 Thioredoxin l7uM 5.310.02 0.2340.006 3.900.58 Glutathione 167uM 17.21.5 1.780.25 1.530.40 Example 6: Inhibition of HIV
Twelve hours prior to infection with HTLVIIIb~ HUT78 cells were incubated in viral culture medium alone or in the presence of Anti-es-LAPase; thioredoxin;
Bestatin and Anti-estrogens according to the combinations described below. In the continued presencc: of each of the factors aforementioned, the cells were subsequently infected at a multiplicity of two infective viral particles per cell for two hours at 25°C with constant agitation in a final volume of 1.0 ml.
Cells were subsequently washed with saline phosphate buffer solution (PBS) and seeded at a density of 1.0 x 105 cells/ml in RPMI 1640, supplemented withl0% FBS, 100 U/ml penicillin, 1(10 mg/ml streptomycin in 96-well plates. Syncytium formation was evaluated by light microscopy on days 3, 5 and 7. At appropriate time points, the cells in selected wells were subjected to psoralen/ultraviolet light inactivation (Watson et al., 1990) air-dried and fixed on glass plates with cold acetone.
Indirect immunofluorescent staining was performed using a modification of standard procedures (Aldovini, A. and Walker, B.D. (1990) Techniques in HIV Research.
Stockton Press, New York; Johnson, G.D. and Nogueira Araujo, G.M.(1981) J.
Immunol. Methods , 43, 349-350. Plates were incubated for 10 minutes in blocking buffer (PBS, 5 % goat serum) and reacted with either control mouse serum or anti-HIV-1 gag (p24, p~~s) monoclonal antibodies (Olympus Immunochemicals), which were diluted 1:100 in blocking buffer, for 30 minutes at room temperature.
Cells were washed three rimes with PBS and incubated for 30 minutes with goat anti-mouse-FITC antibo~3ies (Beckton and Dickinson). Slides were mounted in paraphenylene-diam.ine/glycerol [Johnson, G.D. and Nogueira Araujo, G.M.(1981) J. Immunol. Methods , 43, 349-350] and fluorescent cells were counted.
Following HIV-1 infection of HUT-78 cells, the proviral load in each experimental condition was determned by PCR [Conway, B., Shui-Wah Ko, D. and Cameron, W. ( 1995) Clin. Diag.Virol. 3, 95-104].
Prior to infection, H UT78 cells were washed three times with PBS and the cell viability was determined. Infection was performed as described above. At indicated time intervals, 1.0 ml aliquots of were taken and p24 antigen quantified using a commercial ;ELISA kit (p24 Antigen Capture ELISA, ABBOTT Diagnostics Division), following the instructions of the manufacturer.
The Effect of Mab 7:B6, thioredoxin, Bestatin and anti-estrogens on the viability of HUT78 cells was as:;essed as follows. HUT78 cells were seeded at a density of 1.0 x 105 cells/ml in RP:MI 1640, 10 % FBS, 100 U/ml penicillin, 100 mg/ml streptomycin in the presence or absence of each of the compounds aforementioned.
The medium was changed every third day and cell viability was determined by trypan blue dye exclusion. Thymidine incorporation was also measured [Noma, T.
, Klein, B., Cupissol, D., Yata, J. and Serrou, B.(1984). Int. J. Immunopharmac.
, 6, 87-92.] in dupli~rate cultures on day 7. Values obtained represent the mean ~
s.d. (n=10).
Incubation of HUT 78 cells with Mab 7B6-Protein G Matrices 7 hours prior to Infection with HIV'-1 significantly reduces the number of HIV-1 proviral copies when compared to control cells pre-incubated with mouse IgGI-Protein G
matrices (see Table 5).
Table 5 Inhibition of HIV-a proviral copies by Anti-es-LAPase Mab 7B6 bound to Protein G.
Days Post-infection Mab 7B6 0 86~5 189~2 220~24 (100ng/lOml) Control IgGla 0 2019~89 4002~46 5551~92 2$ (100ng/lOml) Incubation of HUT 78 cells with thioredoxin-EDC/DADPA Matrices 7 hours prior to Infection with HIV-1 significantly reduces the number of HIV-1 proviral copies when compared to control cells pre-incubated with control myoglobin IgGI-Protein G matrices (see Table 6).
Table 6 Inhibition of HIV-1 Proviral copies by thioredoxin-EDC/DADPA Matrix compared to Bestatin-HIV inlhibition Days Post-infection Thioredoxin 0 4~ 1 123 1228 (40ng/Sml) Bestatin 0 7 2 29 ~ 3 457 12 (120~g/ml Control/Myoglobin 0 24141178 4122 121 5851 ~
(40ng/Sml) The independent inhibition mediated by thioredoxin and anti-LAPase 7B6 monoclonal antibodies can be synergized by their simultaneous addition to HUT-cells prior to HIV-1 infection (Table 7). In this context, without wishing to be bound by any theory, the synergistic effect of thioredoxin and of non-inhibitory LAPase 7B6 monoclonal antibodies imply that both LAPase activity and Viral-protein interaction;; may be required during HIV-1 infectivity. It equally suggests that the elevation o~f plasma thioredoxin levels detected in late stages of HIV
infection as reported by Nakamura et al. (1996, Int. Immunol. 8, 603-611) may not only compensate for intracellular glutathione depletion but it may also be directly implicated in reducing HIV viral load.
Table 7 Synergistic Inhibition of HIV-1 Proviral copies by thioredoxin-EDC/DADPA and Anti-LAPase 7B6 combined Matrices Days Post-infection Mab 7B6/Thioredoxin 0 12~3 69~5 88~12 (100ng/40ng/lOml) Control IgGla/Myoglobin 0 1914~55 4330~26 5110~12 (100ng/40m1) As shown in Example 4, exposure to 17-13-estradiol increased LAPase activity, which in turn resulted in an increase in HIV infectivity. Thus, the selective increase in HIV infectivity of women between 1991-1995 regardless of race or mode of exposure to HIV virus could be explained in terms of the effect of estrogen on LAPase.
These observations, together with the uncompetitive inhibitory effect of thioredoxin on LAPase raise the possibility of the use of combined therapy for the prevention and treatment of AIDS.
The combined anti-HIV effect of LAPase inhibition by Mab 7B6, thioredoxin and Bestatin together 'with the administration of anti-estrogens to hamper the effect of estrogen on LAPase activity and hence of HIV infection can lead to a promising therapy against AIDS. Therefore, Tamoxifen, Droloxifene, Raloxifene, Idoxifene, 4-hydroxyandrostenedione and the 313,Sa-tetrahydro derivative of Norethisterone, which are all anti-estrogen compounds, were tested for their ability to senergize the antiviral properties of thioredoxin, Bestatin and of Mab 7B6.
Based on the effect of anti-astrogens on the antiviral properties of combined MAb 7B6/thioredoxin matrices, the most effective combination for the complete in vitro inhibition of HIV-1 infection. was evaluated according to the overall effect on the reduction of cytopathicity, p:?4 levels and proviral load. For simplicity in these experiments determinations on the various parameters herein above aforementioned were determined on day 5 post-infection. Prior to infection cells were simultaneouly incubated with thioredoxin-E;DC/DADPA and Anti-es-LAPase 7B6 Matrices. The inhibitory effect of this anti-fatrogens is sununarized in Table 8. All anti-estrogenic compounds were used at a final concentration of 10-'M.
Table 8 Combined antiviral effect of Mab 7B6 and thioredoxin matrices in combination with anti-estrogens 1S Day 5 Post-infection Agent P24 Cytopathicity Proviral (nmoles/ml)(Syncytia/well)Load(copies) Control L . 82 117 6 3777 82 (No additions) .003 4-hydroxyandrostenedione0.02.001 4.00.4 12 Raloxifene 0.01 .002 3.3 0.1 8 313,5x-tetrahydro 0 0 2 Norethisterone Tamoxifen, 0 0 0 Droloxifene, 0.04.003 5.20.6 14 Idoxifene, 0 0 0 -2g-The present invention has been defined in terms of certain examples, which are not to be construed as limiting. The full scope of the present invention is defined in the following claims.
The present invention relates to a method to inhibit HIV infection by using an antibody to es-LAPase or an inhibitor of es-LAPase activity, or a combination thereof.
The invention further relates to a method to inhibit HIV infection by using an antibody to es-LAPase, an inhibitor of es-LAPase activity and an anti-estrogen compound.
Novel compositions which inhibit HIV infection are also part of the present invention.
Thus according to the present invention there is provided a method for reducing HIV
infectivity towards T lymphocytes comprising contacting said T lymphocytes with a sufficient amount of an inhibitor of LAPase.
In a further embodiment of the present invention there is provided a method for reducing HIV infc:ctivity towards T lymphocytes comprising contacting said T
lymphocytes with a sufficient amount of an antibody specific for LAPase, more specifically a monoclonal antibody specific for es-LAPase.
This invention also provides a method for reducing HIV infectivity towards T
lymphocytes comprising contacting said T lymphocytes with a sufficient amount of an antibody specific for es-LAPase and an inhibitor of LAPase.
This invention also provides a method for reducing HIV infectivity towards T
lymphocytes comprising contacting said T lymphocytes with a sufficient amount of an antibody specific for es-LAPase, an inhibitor of LAPase and an anti-estrogen compound.' Further according to the present invention there is provided a method for preventing or reducing HIV infection comprising administering to a patient in need thereof, a pharmaceutically effective amount of a composition which comprises an antibody specific to es-LAPase and a pharmaceutically acceptable carrier.
Also provided in this invention is a method for preventing or reducing HIV
infection comprising administering to a patient in need thereof, a pharmaceutically effective amount of a composition which comprises an inhibitor of LAPase and a pharmaceutically acceptable carrier.
S This invention is also directed to a method for preventing or reducing HIV
infection comprising administering to a patient in need thereof, a pharmaceutically effective amount of a composition which comprises an antibody specific to es-LAPase and an inhibitor of LAPasf; together with a pharmaceutically acceptable carrier.
This invention is also directed to a method for preventing or reducing HIV
infection comprising admini<,;tering to a patient in need thereof, a pharmaceutically effective amount of a composition which comprises an antibody specific to es-LAPase, an inhibitor of LAPase: and an anti-estrogen compound together with a pharmaceutically acceptable carrier.
Also according to the present invention there is provided a composition comprising an antibody specific to es-LAPase and an inhibitor of LAPase.
Also according to tree present invention there is provided a composition comprising an antibody specific ~:o es-LAPase, an inhibitor of LAPase and an anti-estrogen compound.
BRIEF DESCRIPTION OF THE DRAWINGS
These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings wherein:
FIGURE 1 shows ~:he effect of estrogen on LAPase activity in cell supernatents of primary parental breast cancer cell lines.
FIGURE 2 is a SDS PAGE of crude (lane 1) and purified LAPase (lane 2) followed by western blot with MAb 7B6.
FIGURE 3 shows the kinetics of inhibition of LAPase by Bestatin, thioredoxin and glutathione.
DETAILED DESCRIPTIOiV OF PREFERRED EMBODIMENT
The present invention relates to a method to inhibit HIV infection by using an antibody to es-LAPase or an inhibitor of es-LAPase activity, or a combination thereof.
The invention further relates to a method to inhibit HIV infection by using an antibody to es-LAPase, an inhibitor of es-IJAPase activity and an anti-estrogen compound.
Novel compositions which inhibit HfIV infection are also part of the present invention.
It has been observed that, independently of the role of CD4, the putative receptor for HIV and that of the 13-chemokine co-receptor.s (CKR-5/CCRS;CXCR-4), both membrane-bound and extracellular leucine aminopeptidase (LAPase;EC 3.4.11.1) can promote HIV entry in T lymphocytes (Pulido-Cejudo et al. Antiviral Res. 36:167-177, 1997).
In the present invention it is disclosed that LAPase activity in T lymphocytes and in breast cancer parental cells can be stimulated by estrogens, or estrogen analogues. One aspect of this invention is directed to the identification and purification of an estrogen-stimulated LAPase isoenzyrne, referred to herein as es-LAPase. Details of the estrogen-stimulated LAPase isoenzyme are described in Applicants copending Canadian patent application No. 2,303,505 entitled "A Monoclonal Antibody against Estrogen Stimulated Leucine Aminopeptidase".
A further aspect of the present invention is the preparation of antibodies against the es-LAPase isoenzyme. Antibodies can be produced by methods well known in the art.
In general, the antibody producing cells are prepared by immunizing an animal, for example, mouse, rat, rabbit, sheep, horse, or bovine, with an antigen. The immunization schedule and the concentration of the antigen in suspension is such as to provide useful quantities of suitably primed antibody producing cells. These antibody producing cells can be either spleen cells, thymocytes, lymph node cells and/or peripheral blood lymphocytes.
The antibody producing cells are then fused with myeloma cells, cell lines originating from various animals such <is mice, rats, rabbits. and humans can be used, using a suitable fusion promoter. N(any mouse myeloma cell lines are known and available generally from members of the academic community and various depositories, such as the American Type Culture Collection, Rockville, Maryland. The myeloma cell line used should preferably be medium sensitive so that unfused myeloma cells will not survive in a selective media, while hybrids will survive. The cell line most conunonly used is an 8-azaguanine resistant cell line, which lacks the enzyme hypoxanthine-guanine- phosphoribosyl-transferase and therefore will not be supported by HAT
(hypoxanthine-aminopterin-thymidine) medium. In general, the cell line is also preferably a "non-secretor" type, in that it does not produce any antibody.
The preferred fusion promoter is polyethyleneglycol having an average molecular weight from about 1000 to about 4000. Other fusion promoters such as polyvinylalcohol, a virus or an electrical field can also be used.
The immortalized cells (hybridoma) must then be screened for those which secrete antibody of the correct specificity. The initial screening is generally carried out using an enzyme-linked immunosorbent assay (ELISA). Specifically, the hybridoma culture supernatants are added to mic:rotitre plates which have been previously coated with the antigen, in this case the es-LAPase. A bound specific antibody from the culture supernatants can be detected using a labelled second antibody, for example, goat antimouse IgG labelled with peroxidase, which is commercially available.
Cultures that are positive against the es-LAPase are then subjected to cloning by the limiting dilution method. Secondary hybridoma cultures are re-screened as described above, and further positive cultures acre then examined using the BIAcore ~M system (Pharmacia Biosensor AB, Uppsala, Sweden). The cultures are then evaluated as to determine whether or not the antibody binds the antigen and to determine the kinetic profile of antigen binding. Selected cultures based on these results are subject to further cloning until culture stability and clo:nality are obtained. Immediately after hybridization, the fusion products will have approximately 80 chromosomes, and as these cells proceed to divide they will randomly lose some of these chromosomes. The cloning process is to select those cells which still have the chromosomes coding for antibody production. The cloning process is repeated until 100% of the sub-population exhibits the production of a specific antibody, which is indicative of the "stability"
of the hybridoma. In addition, hybridoma culture wells often have multiple colonies some of which may be antibody non-producers. The cloning process allows the selection of a positive hybrid which is derived from a single cell.
In one embodiment of the present invention there is provided a monoclonal antibody, specific against the es-LAPase, which has been designated Mab 7B6. This monoclonal antibody does not inhibit the enzymatic activity of LAPase. For this reason it will be referred to hereinafter as a non-inhibitory monoclonal antibody. A hybridoma cell line producing this monoclonal antibody has been deposited with the International Depository Authority of Canada, Winnipeg, Manitoba on March 23, 2000 (Accession #
IDAC 230300-1 ) in compliance with the Budapest Treaty.
The present invention also encompasses MAb 7B6 and any fragments thereof containing the active binding region of the antibody such as Fab, F(ab)2 and Fv fragments. These fragments can be obtained from the 7B6 antibody by using techniques well known to those of skills in the art (Rousseaux et al. Methods Enzymology, 121:663-69, Academic Press, 1986).
A further embodiment of the present invention encompasses antibodies or fragments thereof capable of binding the same antigenic determinant as the 7B6 antibody.
Including, but not limited to, antibodies possessing the same antigenic specificity as the 7B6 antibody but originating from a different species or having a different isotype or exhibiting different binding affinities. It is envisioned that class and isotype variants of the antibody of the present invention can be prepared using recombinant class switching and fusion techniques that are well known to those skilled in the art (see for example: Thammana et al. Eur. J. Immunol, 13:614, 1983; Oi et al., Biotechnologies, _g_ 4(3):214-221, Liu et al. Proc. Nat. Acid. Sci. (USA), 84:3439-43, 1987;
Neuberger et al., Nature 31:2:604-608, 1984 and Spira et al. J. Immunol. Meth., 74:307-15, 1984).
The monoclonal ;antibody of the present invention can be produced either using a bioreactar or from ascites, both procedures of which are well known in the art.
In one embodiment of the present invention there is provided a method of reducing HIV infection wherein Mab 7B6 is incubated with T lymphocytes prior to and during infection with HI'J-1. This incubation prevents or reduces the infectivity of towards T lymph~~cytes. Even though not described in the examples (infra), this embodiment also encompasses incubating T lymphocytes with Mab 7B6 after they have been infected with HIV-1 to reduce further infection as would be obvious to one skilled in the art.
The monoclonal antibody can be added directly to the incubation medium or it can be coupled to.a solid :matrix. The solid matrix can be any matrix, as would be known to someone skilled in the art, onto which the antibody can be coupled as long as this coupling does not interfere with the binding of the antibody to its epitope on es-LAPase. The solid matrix can be, but is not limited to, a protein G matrix.
Without wishing to be bound by any theory, the coupling of the antibody to the matrix may reduce the level of non-specific binding of the anti-body to its epitope.
HIV infection of T lymphocytes can be measured using a number of techniques.
The three most commonly used are the quantification of viral capsid protein p24 production, cyopathycity, and the determination of proviral load by polymerise chain reaction (PCR)(Conway et al., Clin. Diag. Virol. 3:95-104, 1995). These methods are well known in the a.rt. Other methods as would be known to one skilled in the art may also be used to determine the levels of HIV infectivity.
According to one aspect of the present invention anti-es-LAPase antibodies are used to reduce HIV infection. According to this embodiment of the present invention cells can be treated at a concentration of from about Song to about 200ng of antibody per ml of cells, wherein the cells are at a density of about 1.0 x 105 cells/ml.
The preferred amount of antibody can be determined empirically, by a person of ordinary 5 skill in the art. In one example of this embodiment the concentration of antibody used is about 100ng per 10 ml of cells.
The present invention also discloses that thiol containing compounds unexpectedly inhibit the activity of es-LAPase. By thiol containing compounds it is meant any 10 compound having a SH group such as, but not limited to, peptides having reduced cysteine residues. lfn one embodiment of this aspect of the invention, thioredoxin and glutathione have been found to inhibit es-LAPase.
Thus, in a further embodiment of the present invention, there is provided a method wherein an inhibitor of es-LAPase is incubated with T lymphocytes prior to and during infection with HIV. According to this aspect of the invention, the inhibitor of es-LAPase activity cam be a competitive, an uncompetitive or non-competitive inhibitor.
The meaning of these terms is taken in the context of the enzyme kinetic theory, as would be known to persons skilled in the art. In this aspect of the invention, an example of an uncompetitive inhibitor is thioredoxin and an example of a non-competitive inhibitor is glutathione.
The inhibitor of LAPase can be added directly to the incubation medium or it can be coupled to a solid matrix. The solid matrix can be any matrix, as would be known to someone skilled in the art, onto which the inhibitor can be coupled as long as this coupling does not interfere with the binding of the inhibitor to es-LAPase.
The solid matrix can be, but is not limited to, DPDP, an amide bonding matrix (Pierce, Rockford,IL, USAj.
In this aspect of the present invention the inhibitors of es-LAPase activity are used to reduce HIV infection. According to this embodiment of the present invention cells can be treated with the inhibitor at a concentration of from about 10-~M to about 10-5M, wherein the cells are at a density of about 1.0 x 10' cells/ml. The preferred amount of inhibitor can be determined empirically, by a person of ordinary skill in the art. In one example of this embodirr'ent the concentration of inhibitor used is from about 4ng to about 8ng per ml of cells.
In another aspect of the preaent invention there is provided a method wherein an antibody to es-LAPase and am inhibitor of LAPase can be combined and incubated with T lymphocytes prior, during or after infection with HIV to reduce HIV
infectivity. In this aspect of the present invention, the antibody and inhibitor are used together according to the methods already disclosed for their individual use.
In a previous publication (Mesange et al. Mol. Pharmacol. , 50:75-79, 1996) it has been shown that TamoxifenT"', an anti-estrogen compound can reduce HIV infectivity.
However, incomplete inhibition of HIV infection were observed at TamoxifenT~'' concentrations of 10-~M or higher. In a further aspect of the present invention there is provided a method wherein not only TamoxifenT~' but other anti-estrogens can be used together with an antibody to es-LAPase and an inhibitor of LAPase to reduce HIV
infectivity.
In this example of the present invention the anti-estrogen compound is selected from the following, used alone car in combination: 4-hydroxyandrostenedione, RaloxifeneT'~', 3~3,Sa-tetrahydro Norethisterone, Tamoxifen, Droloxifene"'' and Idoxifene'M.
In this aspect of the present invention the anti-estrogen compound is used at a final concentration ranging from about 106 M to about 10-~ M. The concentrations of the other components in this method are used as describe above.
In a further aspect of this invention a competitive inhibitor of es-LAPase activity can also be used together with thf: above described compounds. An example of a suitable competitive inhibitor is BestatinTM, but the invention also encompasses other comptetive inhibitors such as Amastatin rM.
The present invention also encompasses a method for treating patients infected with HIV, or at risk of being infected, with an antibody to es-LAPase or one or more inhibitors of es-LAPase activity or a combination thereof. The present invention also encompasses a method for treating patients infected with HIV, or at risk of being infected, with an antibody to es-LAPase, one or more inhibitors of es-LAPase activity and an anti-estrogen. In this aspect of the invention there is provided a pharmaceutical composition comprising an antibody to es-LAPase. In a further aspect of this embodiment the pharmaceutical composition comprises one or more inhibitors of es-LAPase activity. In a still further aspect of this embodiment the pharmaceutical composition comprises an antibody to es-LAPase and one or more inhibitors of es-LAPase activity. In a still further aspect of this embodiment the pharmaceutical composition comprises an antibody to es-LAPase and one or more inhibitors of es-LAPase activity and an anti-estrogen compound.
Pharmaceutical compositions of the present invention generally will include a therapeutically effective amount of at least one of the compounds referred to above together with a pharmaceutically effective carrier. The antibody of the present invention may be administered by any means that enables the active agent to reach the agent's site of action in the body of a mammal. In the case of the antibodies of this invention, the primary focus is the ability to reach and bind with both membrane-bound and extra cellular es-LAPase. Because proteins are subject to being digested when administered orally, parenteral administration, i.e., intravenous, subcutaneous, intramuscular, would ordinarily be used to optimize absorption.
The antibodies of this invention may be administered either as individual therapeutic agents or in combination with other therapeutic agents, as discussed above.
They can be administered alone, but are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.
The dosage administered will, of course, vary depending upon known factors such as the pharmacodynamic characteristics of the particular agent, and its mode and route of administration; ag;e, health, and weight of the recipient; nature and extent of symptoms, kind of concurrent treatment, frequency of treatment, and the effect desired. Usually a daily dosage of active ingredient can be about 0.1 to 100 milligrams per kilogram of body weight. Ordinarily 0.5 to 50, and preferably 1 to 10 milligrams per kilogram per day given in divided doses 1 to 6 times a day or in sustained release form is effective to obtain desired results.
Dosage forms (composition) suitable for internal administration generally contain from about 1 milligram to about 500 milligrams of active ingredient per unit. In these pharmaceutical compositions the active ingredient will ordinarily be present in an amount of about 0.5 - 95 % by weight based on the total weight of the composition.
For parenteral administration, the antibody can be formulated as a solution, suspension, emulsion or lyophilized powder in association with a pharmaceutically acceptable parenteral vehicle. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 5 % human serum albumin. Liposomes and nonaqueous vehicles such as fixed oils may also be used. The vehicle or lyophilized powder may contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and cherr~ical stability (e.g., buffers and preservatives). The formulation is sterilized by commonly used techniques.
In further embodiments of the present invention, the antibody can be administered together with one or more LAPase inhibitors, as discussed in detail above. In this embodiment, the L.APase inhibitors can be added at a dose of about 0.1 to about 100 milligrams per kilogram of body weight.
In yet a further embodiment of the present invention, the antibody can be administered together with one or more anti-estrogen compound, as discussed in detail above. In this embodiment, the anti-estrogen compound can be added at a dose of about 0.1 to about 100 milligrams per kilogram of body weight.
In another embodiment of the present invention, the antibody can be administered together with one or more LAPase inhibitors and one or more anti-estrogen compounds as discussed in detail above. In this embodiment, both the LAPase inhibitors and anti-estrogen compounds can be added at doses of about 0.1 to about 100 milligrams per kilogram of body weight.
The antibody and the inhibitors to be included in the pharmaceutical composition may be coupled to a matrix as described above, as long as the matrix is non-toxic to mammals. Coupling of the antibody and the inhibitors to an appropriate matrix may also facilitate the incorporation of these compounds into liposomes.
In a further embodiment, the inhibitor of LAPase in the pharmaceutical composition is the uncompetitive inhibitor thioredoxin, which may be maintained in its reduced state by including reducing compounds in the composition. Such compounds may include but are limited to g:lutathione and cysteine.
In order that the invention described herein may be more fully understood, the following examples are set forth. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting the scope of this invention in any manner.
EXAMPLES
Example 1: Estrogen mediated LAPase release from HUT78 cells CD4+ T-HUT78 cells were incubated in the presence of various concentrations of 17-~3-Estradiol and I~APase levels were determined as described by G. Pulido-Cejudo et al. (Antiviral Research, 36:167-177, 1997).
Table 1 show that estrogen promoted the release of LAPase in HUT78 cells in a dose response manner, with a maximum stimulation observed at 10-'M.
Table 1 Extracellular LAPase activity of HUT78 cells incubated with 17-~-Estradiol LAPase activity (U* x 10-S/ml) [~3-Estradiol] Control 17-[3-Estradiol Stimulation 10-SM 4.2 10.8 10-6M 5 . 6 20.4 10-'M 4.8 148.6 10-gM 5.7 110.7 * One unit of LAPase is defined as the amount of enzyme required to hydrolyze 1 ~mol of L-leucine-(~-naphthylamide to L-leucine and (3-naphthylamide per min at pEl 7.5 and 37°C.
Following estrogen stimulation, maximum release of LAPase from breast carcinoma parental cells was observed at an estrogen ( 17(3-Estradiol) concentration of 100 nM
following 24 hours of incubation (see Fig. 1 where LAPase activity was determined in supernatants of primary parental cell lines immunoprecipitated with anti-LAPase-SepharoseT'''' bound Mab 7B6 antibodies. Cells were incubated in the presence of 17-~3-Estradiol and following a :?4 hr incubation, supernatants were removed from the primary culture, immunoprecipitated and assayed tluorometrically for LAPasf:
activity by the method of Kuramochi et al. [1987, J. Antibiot., 40: 1605:1611]
Control cells were cultured in the absence of 17-~3-Estradiol.). During the same period, LAPase in cell supernatants of control cells remain unchanged. In addition, LAPase activity was determined in supernatants of primary parental cell lines immunoprecipitated with anti-LAP-Protein G MAb 7B6 bound antibodies. These results show that the extracellular LAPase activity of estrogen stimulated cells (100 nM) was 7.7 x 10-5 U/ml in comparison to 6.4 x 10-~ U/ml detected in the supernatant of parental cells incubated for 24 hours with cell media alone as control. In addition, there was no effect of estrogen incubation on LAPase activity in purified preparations of this enzyme alone. Collectively, these results suggest that estrogen effect on LAPase activity encompasses a cellular mediated process.
Example 2: Purification ot_es-LAPase Primary parental breast carcinoma cells obtained from human tumour biopsies.
were stimulated with 100nM 17-~3--Iatradiol for 24 hours or cell media alone as a control.
The cell media was RPMI 1640 medium + 10 % FCS + 100U/ml Penicillin + 100 ~,g/ml Streptomycin. Cell supernatants were collected and then dialyzed against PBS in seamless cellulose tubing (MW 12,400) for 12 hours at 4°C. Es-LAPase was subsequently purified from the dialyzed cell supernatants using HPLC-gel permeation followed by DIRE-Cellulose and Bestatin-Sepharose affinity Chromatography. Briefly, the cell supernatant was applied to a Bio-Sil SEC-2:50 column (600x7.5 mm) previously equilibrated in a buffer containing 100 mM
Sodium Phosphate buffer pH fi.8, 100 mM Na~SO,, 1~,M ZnCh and 10% glycerol.
The column was washed with 300 ml of the same buffer at a flow rate of 0.5 ml/min. Protein was concentrated to 10 ml by ultrafiltration using YM5 membrane (5000 M.W. cutoff, Amicon I)iv., Danvers, MA., USA). The concentrate was applied to a DEAE cellulose column (2.6 cm X 28.5 cm) equilibrated and washed with 50 mM Tris-HC1 buffer pH 7.5; 1~,M ZnCl2; and 10% (v/v) glycerol. Es-LAP was eluted using a linear gradient (0 to 1M NaCI in Tris buffer) at a flow rate of 0.50 ml/min. A Bestatin-affinity column was prepared using UltralinkTM
EDC/DADPA Amide bonding matrix (Pierce, Rockford, IL U.S.A.) by reacting 100mg of pure Bestatin with the carbodiimide EDC/DADPA matrix following the procedure provided by the manufacturer. Prior to loading the LAPase containing eluent, the Bestatin-affinity column was equilibrated with 10 mM Tris-HC1 pH
8.0 containing 1~M ZnClz and washed with 300 ml of this binding buffer. Es-LAl?ase was recirculated through the system using a peristaltic pump at a t7ow rate of O.lOml/min, for 2 hours. Following this recirculation, the column was washed with eight column volumes of binding buffer.
Bestatin-bound es-LAPase was eluted with a linear gradient (0-0.5 M NaCI) prepared in binding buffer 10 mM Tris-HCl pH 8.0 containing 1~M ZnClz .
Elution of bound es-LAPase was monitored by absorbance at 280 nm. Purified es-LAPase fractions were aliquoted in 500 ~1 and stored until further use in 50 mM Tris-HCl pH 7.8 and SO~cM ZnClz . Es-LAPase Protein concentration was estimated by the method of Lowry et al. [J Biol Chem 193:265 (1951)] using bovine serum albumin as a standard. A summary of the purification fold of the 17-~3-Estradiol-stimulated LAPase from parental cells of human breast carcinomas is set out in Table 2.
Es-LAPase was purified to an approximate 7000 fold after the last Bestatin-affinity chromatographic step.
Es-LAPase activities were determined fluorometrically using leucine-b-naphthylamide as the substrate [Kuramochi, H., et al. (1987) J. Antibiot. , 40,1605-1611] .
The reaction was stopped by boiling the samples at 100°C for 10 minutes, followed by centrifugation at 780 x g at 4°C for 10 minutes. Values obtained represent the average of LAPase activity determined in triplicate.
Table 2 Summary of Purification of LAP from Human Breast Carcinoma Parental Cells Step P:roteinlTotal Activity2Specific Activity2Fold Yield (rng) (nmole/min)(nmole/min/mg) Supernatant1 '.i.4 98.21 6.37 1 100 Gel Permeation4.2 82.13 19.55 3.07 83.63 Cellulose 0.72 48.25 67.01 10.51 49.13 DEAE
Bestatin- 0.005 37.11 7422 1165 37.79 Sepharose 1-the amount of protein was determined after alkaline hydrolysis and quantitative ninhydrin detection of hydrolyzed material as described by Pulido-Cejudo et al. [J.
Chromatogr. B 660 (1994) 37-47)].
2-determined fluorometrically using leucine-~3-naphtylamide as a subtrate as described by [Kuramochi, H., et al. (1987) J. Antibiot. , 40,1605-1611].
Example 3: Monoclonal Antibody Production and Purification In producing the hvbridoma cell line 7B6 secreting the mouse monoclonal antibody to 17-~i-Estradiol-stimulated LAPase, protocols for antigen preparation for immunization, preparation of spleen cells from immune animals, fusion of spleen cells with myeloma. cells and plating of fused cells in selective medium was conducted following detailed guidelines described by Campbell [Burdon RH, Knippenberg PHV (eds): Laboratory Techniques in Biochemistry and Molecular Biology, Amsterdam, Elsevier, p219 (1984)] and by Lietzke and Unsicker [Leitzke R. Unsicker K: A :statistical Approach to Determine Monoclonality After Limiting Cell Plating of a Hybridoma Clone, J Immunol Methods 76:223 (1985)].
Briefly, the primary immunization was performed with purified es-LAPase following desalting. Boosts with purified es-LAPase were performed at days 14, & 56. BALB/c mice were screened at days 24 & 45. The mice were sacrificed at day 59 and the splenocytes from the best responder were fused with myeloma cells.
Screening was performed by dot blot immunostaining on nitrocellulose.
The hybridoma clone 7B6 was obtained by single cell cloning using limiting dilution. Four dilution tubes in series containing hybridoma cells with medium supplement with 20% FBS -~ 2X OPI were prepared. 100 ~,1 of each dilution was plated in a 96-well plate with 50 ~.l of splenocyte feeder cells in each well and placed inside a 37°C 5 % C.""0, incubator. At day 7, supernatants from each well were removed and screened by dot blot immunostaining on nitrocellulose.
Hybridoma clone 7B6 cells were transferred from the 96 well plate to 0.5 nil medium supplemented with :?0% FBS + 1X OPI + 1X HAT in a 24 well plate.
Once the cells were dense, they were transferred into 5 mls in a 60 mm dish and then transferred to 10 mls in a 100 mm dish. Once in the 60 mm dish, the cells were weened off hypoxanthine, thymidine and aminopterin. 7B6 hybridoma cells were continued to be grown until in a log phase of growth. Anti-es-LAPase, Nlab 7B6 was isolated from collected hybridoma 7B6 cell supernatant by affinity chromatography using Immunopure~~'' IgG as per described by manufacturer.
Screening was performed by dot blot immunostaining on nitrocellulose.
The isotype of Mab 7B6 was determined using Sigma's Immunotype Kit~M. Briefly, the assay involves binding of Mab 7B6 to a precoated isotyping nitrocellulose membrane strip followed by immunodetection using a sensitive biotin-avidin-enzyme detection system. I'he immunoglobulin isotype is revealed by self description.
ELISA analysis of Human Breast Carcinoma parental cell lines was conducted to demonstrate the reactivity of Mab 7B6 against human es-LAPase. Briefly, 50 x parental cells were plated per well in a 96 well plate in RPMI 1640 medium -I-10%
FCS + 100 U/ml Penicillin -+- 100 ~.g/ml Streptomycin. The plated cells were cultivated at 37°C 5 % CO, for 24 hours. The cell supernatants were removed, the cells were washed with PBS and subsequently fixed with 1 % gluteraldehyde in PBS
for 1 hour at room temperaW re. Washing with PBS occurred prior to blocking with casein for 1 hour at 37°C S ~~ CO,. Following another wash with PBS, serial dilutions of Mab 7B6 were added to the wells and allowed to incubate for 2 hours at 37°C 5% CO~, Demonstration of the reactivity of Mab 7B6 was revealed upon the addition of a secondary antibody, anti-(IgG + IgM) peroxidase conjugated goat anti-mouse IgG + IgM (H+L) followed by the substate, OPD.
Following purification of the Mab 7B6, the corresponding IgGla isotype was subsequently immobilized to a DSS cross-linking system obtained from Pierce (Rockford, IL, U.S.A) according to the procedures described by the manufacturer.
As shown in Figure 2, Mab '7B6 specifically reacts with es-LAPase in both crude cell supernatants as well as purified es-LAPase after SDS PAGE followed by immunoelectrotransfer. The estimated Molecular Mass of 17-13-Estradiol-stimulated LAPase was 315 kDa as estimated by Gel Permeation on a Bio-SiITM SEC-250 column (600x7.5 mm) using Thyroglobulin (MW 670kDa); Bovine Gamma Globulin (MW 158 kDa); Chiken ovalbumin (MW 44 kDa) and Equine Myoglobin (l7kDa).
Example 4: Effect of Estrosten on increased infectivitv of HUT78 cells.
Estrogen-mediated HIV-induced cytopathic effect was determined as described by G. Pulido-Cejudo et al. [Pulido-Cejudo, G., Conway, B., Proulx, P., Brown, R., and Izaguirre, C.A. (1997). Anti viral Research, 36: 167-177]. The sensitivity of CD4+ T-HUT78 cells to HIV viral infection was determined by measuring the activity of reverse transcriptase in cells infected in the presence/absence of 17-~3-Estradiol.
In light of both the; clinical observations indicating the hypersensitivity of women to HIV infection [Centers for Disease Control and Prevention. (1996). HIVlAIDS
Surveill. Rep. 8. Atlanta, Ga., Wortley, P.M., and Flemming, P.L. (1997).
JAMA, 278: 911-916.] as well as the role of LAPase in viral entry [Pulido-Cejudo, G., Conway, B., Proul!x, P., Brown, R., and Izaguirre, C.A. (1997). Antiviral Research, 36: 167-177], together with the direct effect of estrogen on LAPase reported supra, the sensitivity of HUT78 cells to HIV viral infection was determined. As shown in Table 3, at 3 days and 5 days post infection, in the presence of estrogen, HIV viral infectivity as determined by the activity of reverse transcriptase (RT) was approximately 4 times higher than control cells infected in the absence of estrogen.
Table 3 Effect of Estrogen on increased infectivity RT Activity (cpm x 102/10,1) Day 0 Day 3 Day 5 (Infection) (Post Infection) (Post Infection) Control 0.82 19.82 32.44 Estrogen 0.68 72.42 124.44 ( 10-'M) Example 5: Inhibitors of es-LAPase Human Thioredoxin was purified from CD4+ T MP6 cells as described by Rosen et al. [Rosen, A. et al. (1995) lnt. Immunol. 7, 625-633 ]and compared to purified Thioredoxin activity from E.C'.oli. purchased from SIGMA-ALDRICH Canada (Oakville,Ontario Canada).
Thioredoxin was covalently :linked through an amide bond to Ultralink EDC/DADPA bonding matrix (Pierce, Rockford, IL U.S.A.) by reacting 5 mg of purified Thioredoxin with the carbodiimide EDC/DADPA matrix following the procedure provided by the manufacturer.
Measurements of LAPase were performed spectrophotometrically at 330nm to measure product formation of 13-naphthylamine, using 182~eM 1-leucine-13-naphthylamide as the substrate. The kinetic assays were performed using a Spectronic Genesys~M ~ spec~.rophotometer from Milton Roy (Rochester, NY;).
Absorbance was recorded every sixty seconds, over a period of 30 min. Enzyme kinetic assays were performed using 8.0 x 10-2 U of es-LAPase in a final reaction volume of 600.1 and triplicate samples. The reaction mixture contained the following materials added in order and all kept on ice prior to use: es-LAPase, Calcium-free Hank's solution making up a 600,1 final reaction volume and 182~,M
1-leucine-li-naphthylamide. A corresponding blank without es-LAPase was used as baseline. Both of the cuvettes were transferred to the spectrophotometer where the 1-leucine-13-naphthylmide solution was added last, marking time zero of the reaction. The inhibition studies were carried out in the presence of 167~M
Bestatin, 167~,M reduced Gluthathione and 17 ~M reduced thioredoxin.
Figure 3 shows the inhibitory effect of both Bestatin and two thiol-containing peptides, reduced thioredoxin and reduced glutathione respectively. Es-LAPase showed significant slower mites of reaction both in the presence of thioredoxin and glutathione in comparison to that observed in the control es-LAPase. In addition, es-LAPase reaction rates were slower in the presence of 17~M thioredoxin when compared to those observed in samples incubated with 167~,M reduced Gluthathione. In addition, 16~7~,M Bestatin clearly inhibits es-LAPase activity. As shown in Table 4., reduced thioredoxin is an effective inhibitor of es-LAPase activity. The analysis of the maximum reaction rates (Vmax), Michaelis-Menten (Km) constants and the inhibition constants (Ki) for each inhibitor as presented in Table 4 reveals significant differences in the inhibitory properties of each peptide.
S In this regard, es-LAPase incubated with Bestatin shows a Vmax value very similar to that observed to es-LAPase alone and a significantly higher Km value. This data confirms the competitive nature of es-LAPase inhibition by Bestatin. In contrast, es-LAPase incubated with reduced thioredoxin leads to a significantly lower Vmax and Km with an intermediate Ki value when compared to those obtained for Bestatin and glutathione. Collectively these data strongly suggest that reduce thioredoxin inhibits es-LAPase in an uncompetitive fashion while with reduced glutahthione es-LAPase inhibition is noncompetitive.
Table 4 ~ Bestatin, reduced thioredoxin and glutathione es-LAPase inhibition Constants Vmax Km Ki (10-6 M/min) (10-4 M) (10-6M) LAPase alone 36.81.9 1.670.19 N/A
Bestatin 167uM 39.03.9 48.78.4 6.221.61 Thioredoxin l7uM 5.310.02 0.2340.006 3.900.58 Glutathione 167uM 17.21.5 1.780.25 1.530.40 Example 6: Inhibition of HIV
Twelve hours prior to infection with HTLVIIIb~ HUT78 cells were incubated in viral culture medium alone or in the presence of Anti-es-LAPase; thioredoxin;
Bestatin and Anti-estrogens according to the combinations described below. In the continued presencc: of each of the factors aforementioned, the cells were subsequently infected at a multiplicity of two infective viral particles per cell for two hours at 25°C with constant agitation in a final volume of 1.0 ml.
Cells were subsequently washed with saline phosphate buffer solution (PBS) and seeded at a density of 1.0 x 105 cells/ml in RPMI 1640, supplemented withl0% FBS, 100 U/ml penicillin, 1(10 mg/ml streptomycin in 96-well plates. Syncytium formation was evaluated by light microscopy on days 3, 5 and 7. At appropriate time points, the cells in selected wells were subjected to psoralen/ultraviolet light inactivation (Watson et al., 1990) air-dried and fixed on glass plates with cold acetone.
Indirect immunofluorescent staining was performed using a modification of standard procedures (Aldovini, A. and Walker, B.D. (1990) Techniques in HIV Research.
Stockton Press, New York; Johnson, G.D. and Nogueira Araujo, G.M.(1981) J.
Immunol. Methods , 43, 349-350. Plates were incubated for 10 minutes in blocking buffer (PBS, 5 % goat serum) and reacted with either control mouse serum or anti-HIV-1 gag (p24, p~~s) monoclonal antibodies (Olympus Immunochemicals), which were diluted 1:100 in blocking buffer, for 30 minutes at room temperature.
Cells were washed three rimes with PBS and incubated for 30 minutes with goat anti-mouse-FITC antibo~3ies (Beckton and Dickinson). Slides were mounted in paraphenylene-diam.ine/glycerol [Johnson, G.D. and Nogueira Araujo, G.M.(1981) J. Immunol. Methods , 43, 349-350] and fluorescent cells were counted.
Following HIV-1 infection of HUT-78 cells, the proviral load in each experimental condition was determned by PCR [Conway, B., Shui-Wah Ko, D. and Cameron, W. ( 1995) Clin. Diag.Virol. 3, 95-104].
Prior to infection, H UT78 cells were washed three times with PBS and the cell viability was determined. Infection was performed as described above. At indicated time intervals, 1.0 ml aliquots of were taken and p24 antigen quantified using a commercial ;ELISA kit (p24 Antigen Capture ELISA, ABBOTT Diagnostics Division), following the instructions of the manufacturer.
The Effect of Mab 7:B6, thioredoxin, Bestatin and anti-estrogens on the viability of HUT78 cells was as:;essed as follows. HUT78 cells were seeded at a density of 1.0 x 105 cells/ml in RP:MI 1640, 10 % FBS, 100 U/ml penicillin, 100 mg/ml streptomycin in the presence or absence of each of the compounds aforementioned.
The medium was changed every third day and cell viability was determined by trypan blue dye exclusion. Thymidine incorporation was also measured [Noma, T.
, Klein, B., Cupissol, D., Yata, J. and Serrou, B.(1984). Int. J. Immunopharmac.
, 6, 87-92.] in dupli~rate cultures on day 7. Values obtained represent the mean ~
s.d. (n=10).
Incubation of HUT 78 cells with Mab 7B6-Protein G Matrices 7 hours prior to Infection with HIV'-1 significantly reduces the number of HIV-1 proviral copies when compared to control cells pre-incubated with mouse IgGI-Protein G
matrices (see Table 5).
Table 5 Inhibition of HIV-a proviral copies by Anti-es-LAPase Mab 7B6 bound to Protein G.
Days Post-infection Mab 7B6 0 86~5 189~2 220~24 (100ng/lOml) Control IgGla 0 2019~89 4002~46 5551~92 2$ (100ng/lOml) Incubation of HUT 78 cells with thioredoxin-EDC/DADPA Matrices 7 hours prior to Infection with HIV-1 significantly reduces the number of HIV-1 proviral copies when compared to control cells pre-incubated with control myoglobin IgGI-Protein G matrices (see Table 6).
Table 6 Inhibition of HIV-1 Proviral copies by thioredoxin-EDC/DADPA Matrix compared to Bestatin-HIV inlhibition Days Post-infection Thioredoxin 0 4~ 1 123 1228 (40ng/Sml) Bestatin 0 7 2 29 ~ 3 457 12 (120~g/ml Control/Myoglobin 0 24141178 4122 121 5851 ~
(40ng/Sml) The independent inhibition mediated by thioredoxin and anti-LAPase 7B6 monoclonal antibodies can be synergized by their simultaneous addition to HUT-cells prior to HIV-1 infection (Table 7). In this context, without wishing to be bound by any theory, the synergistic effect of thioredoxin and of non-inhibitory LAPase 7B6 monoclonal antibodies imply that both LAPase activity and Viral-protein interaction;; may be required during HIV-1 infectivity. It equally suggests that the elevation o~f plasma thioredoxin levels detected in late stages of HIV
infection as reported by Nakamura et al. (1996, Int. Immunol. 8, 603-611) may not only compensate for intracellular glutathione depletion but it may also be directly implicated in reducing HIV viral load.
Table 7 Synergistic Inhibition of HIV-1 Proviral copies by thioredoxin-EDC/DADPA and Anti-LAPase 7B6 combined Matrices Days Post-infection Mab 7B6/Thioredoxin 0 12~3 69~5 88~12 (100ng/40ng/lOml) Control IgGla/Myoglobin 0 1914~55 4330~26 5110~12 (100ng/40m1) As shown in Example 4, exposure to 17-13-estradiol increased LAPase activity, which in turn resulted in an increase in HIV infectivity. Thus, the selective increase in HIV infectivity of women between 1991-1995 regardless of race or mode of exposure to HIV virus could be explained in terms of the effect of estrogen on LAPase.
These observations, together with the uncompetitive inhibitory effect of thioredoxin on LAPase raise the possibility of the use of combined therapy for the prevention and treatment of AIDS.
The combined anti-HIV effect of LAPase inhibition by Mab 7B6, thioredoxin and Bestatin together 'with the administration of anti-estrogens to hamper the effect of estrogen on LAPase activity and hence of HIV infection can lead to a promising therapy against AIDS. Therefore, Tamoxifen, Droloxifene, Raloxifene, Idoxifene, 4-hydroxyandrostenedione and the 313,Sa-tetrahydro derivative of Norethisterone, which are all anti-estrogen compounds, were tested for their ability to senergize the antiviral properties of thioredoxin, Bestatin and of Mab 7B6.
Based on the effect of anti-astrogens on the antiviral properties of combined MAb 7B6/thioredoxin matrices, the most effective combination for the complete in vitro inhibition of HIV-1 infection. was evaluated according to the overall effect on the reduction of cytopathicity, p:?4 levels and proviral load. For simplicity in these experiments determinations on the various parameters herein above aforementioned were determined on day 5 post-infection. Prior to infection cells were simultaneouly incubated with thioredoxin-E;DC/DADPA and Anti-es-LAPase 7B6 Matrices. The inhibitory effect of this anti-fatrogens is sununarized in Table 8. All anti-estrogenic compounds were used at a final concentration of 10-'M.
Table 8 Combined antiviral effect of Mab 7B6 and thioredoxin matrices in combination with anti-estrogens 1S Day 5 Post-infection Agent P24 Cytopathicity Proviral (nmoles/ml)(Syncytia/well)Load(copies) Control L . 82 117 6 3777 82 (No additions) .003 4-hydroxyandrostenedione0.02.001 4.00.4 12 Raloxifene 0.01 .002 3.3 0.1 8 313,5x-tetrahydro 0 0 2 Norethisterone Tamoxifen, 0 0 0 Droloxifene, 0.04.003 5.20.6 14 Idoxifene, 0 0 0 -2g-The present invention has been defined in terms of certain examples, which are not to be construed as limiting. The full scope of the present invention is defined in the following claims.
Claims (41)
1. A use of a sufficient amount of an uncompetitive inhibitor of estrogen-stimulated leucine aminopeptidase (es-LAPase) for reducing HIV infectivity towards T lymphocytes.
2. A use of a sufficient amount of an uncompetitive inhibitor of estrogen-stimulated leucine amino peptidase (es-LAPase) for the production of a medicament for reducing HIV infectivity towards T lymphocytes.
3. The use according to claim 1 or 2 wherein the uncompetitive inhibitor is a thiol containing compound.
4. The use according to claim 3 wherein the thiol containing compound is thioredoxin.
5. A use of a sufficient amount of an antibody specific for es-LAPase for reducing HIV infectivity towards T lymphocytes.
6. A use of a sufficient amount of an antibody specific for es-LAPase for the production of a medicament for reducing HIV infectivity towards T
lymphocytes.
lymphocytes.
7. The use according to claim 5 or 6 wherein the antibody is a monoclonal antibody.
8. The use according to claim 7 wherein the monoclonal antibody is produced by hybridoma cell line 7B6 (International Depositary Authority of Canada #
IDAC 230300-1).
IDAC 230300-1).
9. A use according to claim 5 or 6, wherein the use further comprises the use of at least one LAPase inhibitor.
10. The use according to claim 9 wherein the inhibitor is selected from the group consisting of Bestatin and a thiol containing compound.
11. The use according to claim 10 wherein the thiol containing compound is selected from the group consisting of thioredoxin and glutathione.
12. The use according to claim 11 wherein the antibody is a monoclonal antibody.
13. The use according to claim 12 wherein the monoclonal antibody is produced by hybridoma cell line 7B6 (International Depositary Authority of Canada #
IDAC 230300-1).
IDAC 230300-1).
14. A use according to claim 9, wherein the use further comprises the use of at least one anti-estrogen compound.
15. The use according to claim 14 wherein the anti-estrogen is selected from the group consisting of 4-hydroxyandrostenedione, Raloxifene, 3.beta., 5.alpha.-tetrahydro Norethisterone, Tamoxifen, Droloxifene, Idoxifene.
16. A use of a pharmaceutically effective amount of a composition which comprises an antibody specific to es-LAPase and a pharmaceutically acceptable carrier for reducing the susceptibility of T lymphocytes to HIV
infection in a patient in need thereof.
infection in a patient in need thereof.
17. A use of a pharmaceutically effective amount of a composition which comprises an antibody specific to es-LAPase and a pharmaceutically acceptable carrier for the production of a medicament for reducing the susceptibility of T lymphocytes to HIV infection in a patient in need thereof.
18. The use according to claim 16 or 17 wherein the antibody is a monoclonal antibody.
19. The use according to claim 18 wherein the monoclonal antibody is produced by hybridoma cell line 7B6 (International Depositary Authority of Canada #
IDAC 230300-1).
IDAC 230300-1).
20. A use of a pharmaceutically effective amount of a composition which comprises an uncompetitive inhibitor of LAPase and a pharmaceutically acceptable carrier for reducing the susceptibility of T lymphocytes to HIV
infection in a patient in need thereof.
infection in a patient in need thereof.
21. A use of a pharmaceutically effective amount of a composition which comprises an uncompetitive inhibitor of LAPase and a pharmaceutically acceptable carrier for the production of a medicament for reducing the susceptability of T lymphocytes to HIV infection in a patient in need thereof.
22. The use according to claim 20 or 21 wherein the uncompetitive inhibitor is a thiol containing compound.
23. The use according to claim 22 wherein the thiol containing compound is thioredoxin.
24. The use according to claim 16 wherein the composition further comprises an uncompetitive inhibitor of LAPase.
25. The use according to claim 24 wherein the antibody is a monoclonal antibody.
26. The use according to claim 25 wherein the monoclonal antibody is produced by hybridoma cell line 7B6 (International Depositary Authority of Canada #
IDAC 230300-1).
IDAC 230300-1).
27. The use according to claim 26 wherein the uncompetitive inhibitor is a thiol containing compound.
28. The use according to claim 27 wherein the thiol containing compound is thioredoxin.
29. The use according to claim 24 wherein the composition further comprises an anti-estrogen compound.
30. The use according to claim 29 wherein the anti-estrogen is selected from the group consisting of 4-hydroxyandrostenedione, Raloxifene, 3.beta., 5.alpha.-tetrahydro Norethisterone, Tamoxifen, Droloxifene, Idoxifene.
31. A composition comprising an antibody specific to es-LAPase and an inhibitor of LAPase.
32. The composition according to claim 31 wherein the antibody is a monoclonal antibody.
33. The composition according to claim 32 wherein the monoclonal antibody is produced by hybridoma cell line 7B6 (International Depositary Authority of Canada # IDAC 230300-1).
34. The composition according to claim 31 wherein the inhibitor is selected from the group consisting of Bestatin and a thiol containing compound.
35. The composition according to claim 34 wherein the thiol containing compound is selected from the group consisting of thioredoxin and glutathione.
36. The composition according to claim 31 wherein the composition further comprises an anti-estrogen compound.
37. The composition according to claim 36 wherein the anti-estrogen is selected from the group consisting of 4-hydroxyandrostenedione, Raloxifene, 3.beta., 5.alpha.-tetrahydro Norethisterone, Tamoxifen, Droloxifene, Idoxifene.
38. A use of a sufficient amount of an uncompetitive inhibitor of LAPase, a monoclonal antibody specific for es-LAPase and an anti-estrogen for reducing the susceptibility of T lymphocytes to HIV infection.
39. A use of an uncompetitive inhibitor of LAPase, a competitive inhibitor of LAPase, a monoclonal antibody specific for es-LAPase and an anti-estrogen for reducing the susceptibility of T lymphocytes to HIV infection.
40. A use of a composition comprising a sufficient amount of an uncompetitive inhibitor of LAPase, a monoclonal antibody specific for es-LAPase and an anti-estrogen for making a medicament for reducing the susceptibility of T
lymphocytes to HIV infection.
lymphocytes to HIV infection.
41. A use of a composition comprising a sufficient amount of an uncompetitive inhibitor of LAPase, a competitive inhibitor of LAPase, a monoclonal antibody specific for es-LAPase and an anti-estrogen for malting a medicament for reducing the susceptibility of T lymphocytes to HIV infection.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2303591 CA2303591C (en) | 1999-03-30 | 2000-03-30 | Method and compositions for preventing or reducing hiv infection |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA002267481A CA2267481A1 (en) | 1999-03-30 | 1999-03-30 | Critical interdependency: from the role of estrogen on breast cancer to the susceptibility of women towards hiv infection |
| CA2,267,481 | 2000-03-30 | ||
| CA 2303591 CA2303591C (en) | 1999-03-30 | 2000-03-30 | Method and compositions for preventing or reducing hiv infection |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2303591A1 CA2303591A1 (en) | 2000-09-11 |
| CA2303591C true CA2303591C (en) | 2002-12-17 |
Family
ID=25680865
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2303591 Expired - Fee Related CA2303591C (en) | 1999-03-30 | 2000-03-30 | Method and compositions for preventing or reducing hiv infection |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2303591C (en) |
-
2000
- 2000-03-30 CA CA 2303591 patent/CA2303591C/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CA2303591A1 (en) | 2000-09-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US6406701B1 (en) | Method and compositions for preventing or reducing HIV infection | |
| KR100667121B1 (en) | Compositions and Methods for the Treatment of Viral Infectious Diseases | |
| EP0822941B1 (en) | Monoclonal antibodies against hiv-1 and vaccines made thereof | |
| US6177253B1 (en) | Screening methods for the identification of anti-HIV compounds employing a cyclosporine-specific monoclonal antibody that cross-reacts with HIV-1 p24 | |
| JPH06502539A (en) | Neutralizing human monoclonal antibody specific for the V3 loop and CD-4 binding site of HIV-1 GP120 | |
| Hansen et al. | A mouse aminopeptidase N is a marker for antigen‐presenting cells and appears to be co‐expressed with major histocompatibility complex class II molecules | |
| EP0295803B1 (en) | Monoclonal antibodies | |
| US6140043A (en) | Pharmaceutical compositions for competitively inhibiting the binding of a retrovirus to the IFN-receptor and means for diagnosis of an HIV infection | |
| Edson et al. | Synthesis and processing of the three major envelope glycoproteins of Epstein-Barr virus | |
| JP2888980B2 (en) | HIV immunotherapy | |
| US6241986B1 (en) | Human monoclonal antibodies to the CD4-binding domain of HIV, uses thereof and synergistic neutralization of HIV | |
| US6268484B1 (en) | HIV-vaccines | |
| Stein et al. | Immune-based therapeutics: scientific rationale and the promising approaches to the treatment of the human immunodeficiency virus-infected individual | |
| US5981278A (en) | Chimeric monoclonal antibodies which neutralize HIV-1 infection and their applications in therapy and prevention for AIDS | |
| CN110612120A (en) | Method of treating a pediatric disorder using an antibody specific for α4β7 integrin (vedolizumab) | |
| US6200575B1 (en) | Non-toxic immunogens derived from a retroviral regulatory protein antibodies preparation process and pharmaceutical compositions comprising them | |
| EP2697259B1 (en) | Compositions for preventing and/or treating an infection by an hiv-1 virus | |
| CA2303591C (en) | Method and compositions for preventing or reducing hiv infection | |
| US5567805A (en) | The cellular receptor for the CS3 peptide of human immunodeficiency virus | |
| EP0379295A2 (en) | Anti-tryptase antibody and composition for treatment of AIDS using the same | |
| JP4310498B2 (en) | Human IgM antibody that induces apoptosis in HIV-infected cells and therapeutic agent for HIV infection | |
| JPS63294795A (en) | Method and substance for detection and remedy of human immunodeficiency virus (hiv) | |
| WO1993020104A9 (en) | Monoclonal antibodies against a carbohydrate-dependent epitope related to the v2 region of hiv-1 gp120 | |
| WO1993020104A1 (en) | Monoclonal antibodies against a carbohydrate-dependent epitope related to the v2 region of hiv-1 gp120 | |
| EA010058B1 (en) | Peptides modulating caspase activation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| MKLA | Lapsed |
Effective date: 20130402 |