US20200163924A1 - Compositions and methods for reactivating latent viral infections - Google Patents
Compositions and methods for reactivating latent viral infections Download PDFInfo
- Publication number
- US20200163924A1 US20200163924A1 US16/506,841 US201916506841A US2020163924A1 US 20200163924 A1 US20200163924 A1 US 20200163924A1 US 201916506841 A US201916506841 A US 201916506841A US 2020163924 A1 US2020163924 A1 US 2020163924A1
- Authority
- US
- United States
- Prior art keywords
- hiv
- pep005
- ingenol
- cells
- angelate
- 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.)
- Abandoned
Links
- 239000000203 mixture Substances 0.000 title claims abstract description 84
- 238000000034 method Methods 0.000 title claims abstract description 73
- 230000009385 viral infection Effects 0.000 title description 35
- 208000036142 Viral infection Diseases 0.000 title description 33
- VDJHFHXMUKFKET-UHFFFAOYSA-N Ingenol mebutate Natural products CC1CC2C(C)(C)C2C2C=C(CO)C(O)C3(O)C(OC(=O)C(C)=CC)C(C)=CC31C2=O VDJHFHXMUKFKET-UHFFFAOYSA-N 0.000 claims abstract description 426
- VDJHFHXMUKFKET-WDUFCVPESA-N ingenol mebutate Chemical compound C[C@@H]1C[C@H]2C(C)(C)[C@H]2[C@@H]2C=C(CO)[C@@H](O)[C@]3(O)[C@@H](OC(=O)C(\C)=C/C)C(C)=C[C@]31C2=O VDJHFHXMUKFKET-WDUFCVPESA-N 0.000 claims abstract description 426
- 229960002993 ingenol mebutate Drugs 0.000 claims abstract description 423
- 230000007420 reactivation Effects 0.000 claims abstract description 147
- 241000700605 Viruses Species 0.000 claims abstract description 105
- 230000001939 inductive effect Effects 0.000 claims abstract description 19
- 241000725303 Human immunodeficiency virus Species 0.000 claims description 297
- 210000004027 cell Anatomy 0.000 claims description 167
- 210000001744 T-lymphocyte Anatomy 0.000 claims description 97
- 239000003795 chemical substances by application Substances 0.000 claims description 94
- 239000003112 inhibitor Substances 0.000 claims description 84
- 239000012190 activator Substances 0.000 claims description 81
- 108090000315 Protein Kinase C Proteins 0.000 claims description 79
- 102000003923 Protein Kinase C Human genes 0.000 claims description 79
- 230000003612 virological effect Effects 0.000 claims description 76
- 102000003964 Histone deacetylase Human genes 0.000 claims description 64
- 108090000353 Histone deacetylase Proteins 0.000 claims description 64
- 108010036115 Histone Methyltransferases Proteins 0.000 claims description 60
- 102000011787 Histone Methyltransferases Human genes 0.000 claims description 60
- 108010012271 Positive Transcriptional Elongation Factor B Proteins 0.000 claims description 57
- 102000019014 Positive Transcriptional Elongation Factor B Human genes 0.000 claims description 57
- -1 CG05/CG06 Chemical compound 0.000 claims description 51
- ULNXAWLQFZMIHX-UHFFFAOYSA-N GSK343 Chemical group C1=C(C)NC(=O)C(CNC(=O)C=2C=3C=NN(C=3C=C(C=2)C=2C=C(N=CC=2)N2CCN(C)CC2)C(C)C)=C1CCC ULNXAWLQFZMIHX-UHFFFAOYSA-N 0.000 claims description 48
- 108010057466 NF-kappa B Proteins 0.000 claims description 46
- 102000003945 NF-kappa B Human genes 0.000 claims description 46
- 238000002560 therapeutic procedure Methods 0.000 claims description 41
- 150000002596 lactones Chemical class 0.000 claims description 40
- 150000001982 diacylglycerols Chemical class 0.000 claims description 37
- PHEDXBVPIONUQT-RGYGYFBISA-N phorbol 13-acetate 12-myristate Chemical compound C([C@]1(O)C(=O)C(C)=C[C@H]1[C@@]1(O)[C@H](C)[C@H]2OC(=O)CCCCCCCCCCCCC)C(CO)=C[C@H]1[C@H]1[C@]2(OC(C)=O)C1(C)C PHEDXBVPIONUQT-RGYGYFBISA-N 0.000 claims description 37
- 229960005486 vaccine Drugs 0.000 claims description 36
- 230000035897 transcription Effects 0.000 claims description 30
- 238000013518 transcription Methods 0.000 claims description 30
- WAEXFXRVDQXREF-UHFFFAOYSA-N vorinostat Chemical group ONC(=O)CCCCCCC(=O)NC1=CC=CC=C1 WAEXFXRVDQXREF-UHFFFAOYSA-N 0.000 claims description 30
- 239000002644 phorbol ester Substances 0.000 claims description 28
- 230000004163 JAK-STAT signaling pathway Effects 0.000 claims description 24
- 239000003120 macrolide antibiotic agent Substances 0.000 claims description 24
- 230000001225 therapeutic effect Effects 0.000 claims description 20
- 230000006044 T cell activation Effects 0.000 claims description 18
- 241000701022 Cytomegalovirus Species 0.000 claims description 16
- 239000003968 dna methyltransferase inhibitor Substances 0.000 claims description 16
- 229960000237 vorinostat Drugs 0.000 claims description 16
- 230000008901 benefit Effects 0.000 claims description 15
- BNQSTAOJRULKNX-UHFFFAOYSA-N N-(6-acetamidohexyl)acetamide Chemical compound CC(=O)NCCCCCCNC(C)=O BNQSTAOJRULKNX-UHFFFAOYSA-N 0.000 claims description 14
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 13
- 230000003013 cytotoxicity Effects 0.000 claims description 12
- 231100000135 cytotoxicity Toxicity 0.000 claims description 12
- NIJJYAXOARWZEE-UHFFFAOYSA-N Valproic acid Chemical compound CCCC(C(O)=O)CCC NIJJYAXOARWZEE-UHFFFAOYSA-N 0.000 claims description 10
- 241000701161 unidentified adenovirus Species 0.000 claims description 9
- 229940126190 DNA methyltransferase inhibitor Drugs 0.000 claims description 8
- IDQPVOFTURLJPT-UHFFFAOYSA-N N,N'-dihydroxyoctanediamide Chemical compound ONC(=O)CCCCCCC(=O)NO IDQPVOFTURLJPT-UHFFFAOYSA-N 0.000 claims description 8
- 229960000604 valproic acid Drugs 0.000 claims description 8
- 239000003085 diluting agent Substances 0.000 claims description 6
- OMKHWTRUYNAGFG-IEBDPFPHSA-N 3-deazaneplanocin a Chemical compound C1=NC=2C(N)=NC=CC=2N1[C@@H]1C=C(CO)[C@@H](O)[C@H]1O OMKHWTRUYNAGFG-IEBDPFPHSA-N 0.000 claims description 5
- JWOGUUIOCYMBPV-GMFLJSBRSA-N (3S,6S,9S,12R)-3-[(2S)-Butan-2-yl]-6-[(1-methoxyindol-3-yl)methyl]-9-(6-oxooctyl)-1,4,7,10-tetrazabicyclo[10.4.0]hexadecane-2,5,8,11-tetrone Chemical compound N1C(=O)[C@H](CCCCCC(=O)CC)NC(=O)[C@H]2CCCCN2C(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H]1CC1=CN(OC)C2=CC=CC=C12 JWOGUUIOCYMBPV-GMFLJSBRSA-N 0.000 claims description 4
- QRPSQQUYPMFERG-LFYBBSHMSA-N (e)-5-[3-(benzenesulfonamido)phenyl]-n-hydroxypent-2-en-4-ynamide Chemical compound ONC(=O)\C=C\C#CC1=CC=CC(NS(=O)(=O)C=2C=CC=CC=2)=C1 QRPSQQUYPMFERG-LFYBBSHMSA-N 0.000 claims description 4
- JHSXDAWGLCZYSM-UHFFFAOYSA-N 4-(4-chloro-2-methylphenoxy)-N-hydroxybutanamide Chemical compound CC1=CC(Cl)=CC=C1OCCCC(=O)NO JHSXDAWGLCZYSM-UHFFFAOYSA-N 0.000 claims description 4
- OSXFATOLZGZLSK-UHFFFAOYSA-N 6,7-dimethoxy-2-(4-methyl-1,4-diazepan-1-yl)-N-[1-(phenylmethyl)-4-piperidinyl]-4-quinazolinamine Chemical compound C=12C=C(OC)C(OC)=CC2=NC(N2CCN(C)CCC2)=NC=1NC(CC1)CCN1CC1=CC=CC=C1 OSXFATOLZGZLSK-UHFFFAOYSA-N 0.000 claims description 4
- JTDYUFSDZATMKU-UHFFFAOYSA-N 6-(1,3-dioxo-2-benzo[de]isoquinolinyl)-N-hydroxyhexanamide Chemical compound C1=CC(C(N(CCCCCC(=O)NO)C2=O)=O)=C3C2=CC=CC3=C1 JTDYUFSDZATMKU-UHFFFAOYSA-N 0.000 claims description 4
- JWOGUUIOCYMBPV-UHFFFAOYSA-N OT-Key 11219 Natural products N1C(=O)C(CCCCCC(=O)CC)NC(=O)C2CCCCN2C(=O)C(C(C)CC)NC(=O)C1CC1=CN(OC)C2=CC=CC=C12 JWOGUUIOCYMBPV-UHFFFAOYSA-N 0.000 claims description 4
- 108010082820 apicidin Proteins 0.000 claims description 4
- 229930186608 apicidin Natural products 0.000 claims description 4
- NCNRHFGMJRPRSK-MDZDMXLPSA-N belinostat Chemical compound ONC(=O)\C=C\C1=CC=CC(S(=O)(=O)NC=2C=CC=CC=2)=C1 NCNRHFGMJRPRSK-MDZDMXLPSA-N 0.000 claims description 4
- PZPPOCZWRGNKIR-PNVYSBBASA-N chaetocin Chemical compound N([C@@H]1N2C(=O)[C@]3(CO)SS[C@]2(C(N3C)=O)C2)C3=CC=CC=C3[C@]12[C@@]12C[C@]3(SS4)C(=O)N(C)[C@]4(CO)C(=O)N3[C@H]2NC2=CC=CC=C12 PZPPOCZWRGNKIR-PNVYSBBASA-N 0.000 claims description 4
- PZPPOCZWRGNKIR-UHFFFAOYSA-N chaetocin Natural products C1C2(C(N3C)=O)SSC3(CO)C(=O)N2C2NC3=CC=CC=C3C21C12CC3(SS4)C(=O)N(C)C4(CO)C(=O)N3C2NC2=CC=CC=C12 PZPPOCZWRGNKIR-UHFFFAOYSA-N 0.000 claims description 4
- FKGKZBBDJSKCIS-UHFFFAOYSA-N diethyl-[[6-[[4-(hydroxycarbamoyl)phenyl]carbamoyloxymethyl]naphthalen-2-yl]methyl]azanium;chloride;hydrate Chemical compound O.[Cl-].C1=CC2=CC(C[NH+](CC)CC)=CC=C2C=C1COC(=O)NC1=CC=C(C(=O)NO)C=C1 FKGKZBBDJSKCIS-UHFFFAOYSA-N 0.000 claims description 4
- MFBOGIVSZKQAPD-UHFFFAOYSA-M sodium butyrate Chemical compound [Na+].CCCC([O-])=O MFBOGIVSZKQAPD-UHFFFAOYSA-M 0.000 claims description 4
- RTKIYFITIVXBLE-QEQCGCAPSA-N trichostatin A Chemical compound ONC(=O)/C=C/C(/C)=C/[C@@H](C)C(=O)C1=CC=C(N(C)C)C=C1 RTKIYFITIVXBLE-QEQCGCAPSA-N 0.000 claims description 4
- 150000001875 compounds Chemical class 0.000 abstract description 99
- 230000002195 synergetic effect Effects 0.000 abstract description 14
- 230000014509 gene expression Effects 0.000 description 125
- 101000716102 Homo sapiens T-cell surface glycoprotein CD4 Proteins 0.000 description 98
- 102100036011 T-cell surface glycoprotein CD4 Human genes 0.000 description 98
- DNVXATUJJDPFDM-KRWDZBQOSA-N JQ1 Chemical compound N([C@@H](CC(=O)OC(C)(C)C)C1=NN=C(N1C=1SC(C)=C(C)C=11)C)=C1C1=CC=C(Cl)C=C1 DNVXATUJJDPFDM-KRWDZBQOSA-N 0.000 description 77
- 238000011225 antiretroviral therapy Methods 0.000 description 59
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 51
- 230000000694 effects Effects 0.000 description 45
- 229930004069 diterpene Natural products 0.000 description 36
- VEBVPUXQAPLADL-POYOOMFHSA-N ingenol Chemical class C1=C(CO)[C@@H](O)[C@]2(O)[C@@H](O)C(C)=C[C@]32[C@H](C)C[C@H]2C(C)(C)[C@H]2[C@H]1C3=O VEBVPUXQAPLADL-POYOOMFHSA-N 0.000 description 34
- 230000001965 increasing effect Effects 0.000 description 33
- 239000003814 drug Substances 0.000 description 32
- 238000011282 treatment Methods 0.000 description 32
- 230000037361 pathway Effects 0.000 description 30
- 239000008194 pharmaceutical composition Substances 0.000 description 30
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 25
- 230000037396 body weight Effects 0.000 description 25
- 238000011529 RT qPCR Methods 0.000 description 24
- 230000002708 enhancing effect Effects 0.000 description 23
- 230000027455 binding Effects 0.000 description 22
- 230000004913 activation Effects 0.000 description 21
- 238000000338 in vitro Methods 0.000 description 21
- 108090000623 proteins and genes Proteins 0.000 description 20
- 238000003762 quantitative reverse transcription PCR Methods 0.000 description 20
- 108020004414 DNA Proteins 0.000 description 18
- 108020004999 messenger RNA Proteins 0.000 description 16
- 210000005259 peripheral blood Anatomy 0.000 description 16
- 239000011886 peripheral blood Substances 0.000 description 16
- 230000003213 activating effect Effects 0.000 description 15
- BOJKFRKNLSCGHY-HXGSDTCMSA-N prostratin Chemical compound C1=C(CO)C[C@]2(O)C(=O)C(C)=C[C@H]2[C@@]2(O)[C@H](C)C[C@@]3(OC(C)=O)C(C)(C)[C@H]3[C@@H]21 BOJKFRKNLSCGHY-HXGSDTCMSA-N 0.000 description 15
- 102000004169 proteins and genes Human genes 0.000 description 15
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 14
- 208000031886 HIV Infections Diseases 0.000 description 14
- 229940079593 drug Drugs 0.000 description 14
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 13
- 208000037357 HIV infectious disease Diseases 0.000 description 13
- 238000009472 formulation Methods 0.000 description 13
- 208000033519 human immunodeficiency virus infectious disease Diseases 0.000 description 13
- 230000004044 response Effects 0.000 description 13
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 12
- 230000002255 enzymatic effect Effects 0.000 description 12
- 239000005090 green fluorescent protein Substances 0.000 description 12
- 230000011664 signaling Effects 0.000 description 12
- 241001465754 Metazoa Species 0.000 description 11
- 230000008029 eradication Effects 0.000 description 11
- 150000004633 phorbol derivatives Chemical class 0.000 description 11
- 230000003389 potentiating effect Effects 0.000 description 11
- 235000018102 proteins Nutrition 0.000 description 11
- VERWQPYQDXWOGT-LVJNJWHOSA-N 4-amino-5-fluoro-1-[(2r,5s)-2-(hydroxymethyl)-1,3-oxathiolan-5-yl]pyrimidin-2-one;[[(2r)-1-(6-aminopurin-9-yl)propan-2-yl]oxymethyl-(propan-2-yloxycarbonyloxymethoxy)phosphoryl]oxymethyl propan-2-yl carbonate;(e)-but-2-enedioic acid Chemical compound OC(=O)\C=C\C(O)=O.C1=C(F)C(N)=NC(=O)N1[C@H]1O[C@@H](CO)SC1.N1=CN=C2N(C[C@@H](C)OCP(=O)(OCOC(=O)OC(C)C)OCOC(=O)OC(C)C)C=NC2=C1N VERWQPYQDXWOGT-LVJNJWHOSA-N 0.000 description 10
- 102000004127 Cytokines Human genes 0.000 description 10
- 108090000695 Cytokines Proteins 0.000 description 10
- 102100025137 Early activation antigen CD69 Human genes 0.000 description 10
- 101000934374 Homo sapiens Early activation antigen CD69 Proteins 0.000 description 10
- 238000003556 assay Methods 0.000 description 10
- 230000006698 induction Effects 0.000 description 10
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 10
- 230000004936 stimulating effect Effects 0.000 description 10
- 239000000126 substance Substances 0.000 description 10
- 102100038970 Histone-lysine N-methyltransferase EZH2 Human genes 0.000 description 9
- 101000882127 Homo sapiens Histone-lysine N-methyltransferase EZH2 Proteins 0.000 description 9
- NUQJULCGNZMBEF-UHFFFAOYSA-N Prostratin Natural products COC(=O)C12CC(C)C3(O)C(C=C(CO)CC4(O)C3C=C(C)C4=O)C1C2(C)C NUQJULCGNZMBEF-UHFFFAOYSA-N 0.000 description 9
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 9
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 9
- 239000000556 agonist Substances 0.000 description 9
- 238000004113 cell culture Methods 0.000 description 9
- 238000001990 intravenous administration Methods 0.000 description 9
- KQPYUDDGWXQXHS-UHFFFAOYSA-N juglone Chemical compound O=C1C=CC(=O)C2=C1C=CC=C2O KQPYUDDGWXQXHS-UHFFFAOYSA-N 0.000 description 9
- 230000000284 resting effect Effects 0.000 description 9
- 230000001235 sensitizing effect Effects 0.000 description 9
- 102100035875 C-C chemokine receptor type 5 Human genes 0.000 description 8
- 125000000567 diterpene group Chemical group 0.000 description 8
- 238000000684 flow cytometry Methods 0.000 description 8
- 208000015181 infectious disease Diseases 0.000 description 8
- 230000000670 limiting effect Effects 0.000 description 8
- 239000000523 sample Substances 0.000 description 8
- 230000019491 signal transduction Effects 0.000 description 8
- 101710149870 C-C chemokine receptor type 5 Proteins 0.000 description 7
- VEBVPUXQAPLADL-UHFFFAOYSA-N Ingenol Natural products C1=C(CO)C(O)C2(O)C(O)C(C)=CC32C(C)CC2C(C)(C)C2C1C3=O VEBVPUXQAPLADL-UHFFFAOYSA-N 0.000 description 7
- 102000016397 Methyltransferase Human genes 0.000 description 7
- 108060004795 Methyltransferase Proteins 0.000 description 7
- 241000713311 Simian immunodeficiency virus Species 0.000 description 7
- 102100035100 Transcription factor p65 Human genes 0.000 description 7
- 108010067390 Viral Proteins Proteins 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 7
- MJQUEDHRCUIRLF-TVIXENOKSA-N bryostatin 1 Chemical compound C([C@@H]1CC(/[C@@H]([C@@](C(C)(C)/C=C/2)(O)O1)OC(=O)/C=C/C=C/CCC)=C\C(=O)OC)[C@H]([C@@H](C)O)OC(=O)C[C@H](O)C[C@@H](O1)C[C@H](OC(C)=O)C(C)(C)[C@]1(O)C[C@@H]1C\C(=C\C(=O)OC)C[C@H]\2O1 MJQUEDHRCUIRLF-TVIXENOKSA-N 0.000 description 7
- 230000002301 combined effect Effects 0.000 description 7
- 230000003247 decreasing effect Effects 0.000 description 7
- 239000003276 histone deacetylase inhibitor Substances 0.000 description 7
- 230000002401 inhibitory effect Effects 0.000 description 7
- 230000000977 initiatory effect Effects 0.000 description 7
- 239000002105 nanoparticle Substances 0.000 description 7
- 230000003204 osmotic effect Effects 0.000 description 7
- 230000026731 phosphorylation Effects 0.000 description 7
- 238000006366 phosphorylation reaction Methods 0.000 description 7
- 230000010076 replication Effects 0.000 description 7
- 150000003839 salts Chemical class 0.000 description 7
- GZNWHPFWQMQXII-UHFFFAOYSA-N 1-(2-ethylphenyl)pyrrole-2,5-dione Chemical compound CCC1=CC=CC=C1N1C(=O)C=CC1=O GZNWHPFWQMQXII-UHFFFAOYSA-N 0.000 description 6
- 102100031650 C-X-C chemokine receptor type 4 Human genes 0.000 description 6
- 108010083930 HIV Receptors Proteins 0.000 description 6
- 102000006481 HIV Receptors Human genes 0.000 description 6
- 101000922348 Homo sapiens C-X-C chemokine receptor type 4 Proteins 0.000 description 6
- 102000042846 PKC family Human genes 0.000 description 6
- 108091082203 PKC family Proteins 0.000 description 6
- 230000000996 additive effect Effects 0.000 description 6
- 229960005520 bryostatin Drugs 0.000 description 6
- MUIWQCKLQMOUAT-AKUNNTHJSA-N bryostatin 20 Natural products COC(=O)C=C1C[C@@]2(C)C[C@]3(O)O[C@](C)(C[C@@H](O)CC(=O)O[C@](C)(C[C@@]4(C)O[C@](O)(CC5=CC(=O)O[C@]45C)C(C)(C)C=C[C@@](C)(C1)O2)[C@@H](C)O)C[C@H](OC(=O)C(C)(C)C)C3(C)C MUIWQCKLQMOUAT-AKUNNTHJSA-N 0.000 description 6
- 238000004364 calculation method Methods 0.000 description 6
- 230000003833 cell viability Effects 0.000 description 6
- 230000001413 cellular effect Effects 0.000 description 6
- 230000002354 daily effect Effects 0.000 description 6
- AUZONCFQVSMFAP-UHFFFAOYSA-N disulfiram Chemical compound CCN(CC)C(=S)SSC(=S)N(CC)CC AUZONCFQVSMFAP-UHFFFAOYSA-N 0.000 description 6
- 150000004141 diterpene derivatives Chemical class 0.000 description 6
- 230000002222 downregulating effect Effects 0.000 description 6
- 230000028993 immune response Effects 0.000 description 6
- 230000005764 inhibitory process Effects 0.000 description 6
- 150000007524 organic acids Chemical class 0.000 description 6
- 229940107670 picato Drugs 0.000 description 6
- 239000000843 powder Substances 0.000 description 6
- 102000004196 processed proteins & peptides Human genes 0.000 description 6
- 108090000765 processed proteins & peptides Proteins 0.000 description 6
- 230000000638 stimulation Effects 0.000 description 6
- 230000008685 targeting Effects 0.000 description 6
- MSRILKIQRXUYCT-UHFFFAOYSA-M valproate semisodium Chemical compound [Na+].CCCC(C(O)=O)CCC.CCCC(C([O-])=O)CCC MSRILKIQRXUYCT-UHFFFAOYSA-M 0.000 description 6
- 230000007419 viral reactivation Effects 0.000 description 6
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 5
- 102100031585 ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 1 Human genes 0.000 description 5
- XPOQHMRABVBWPR-UHFFFAOYSA-N Efavirenz Natural products O1C(=O)NC2=CC=C(Cl)C=C2C1(C(F)(F)F)C#CC1CC1 XPOQHMRABVBWPR-UHFFFAOYSA-N 0.000 description 5
- 102000006354 HLA-DR Antigens Human genes 0.000 description 5
- 108010058597 HLA-DR Antigens Proteins 0.000 description 5
- 108010030471 Histamine N-methyltransferase Proteins 0.000 description 5
- 102100039869 Histone H2B type F-S Human genes 0.000 description 5
- 101000777636 Homo sapiens ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 1 Proteins 0.000 description 5
- 101001035372 Homo sapiens Histone H2B type F-S Proteins 0.000 description 5
- 102000038030 PI3Ks Human genes 0.000 description 5
- 108010029485 Protein Isoforms Proteins 0.000 description 5
- 102000001708 Protein Isoforms Human genes 0.000 description 5
- 101710149951 Protein Tat Proteins 0.000 description 5
- 108091008874 T cell receptors Proteins 0.000 description 5
- 239000000427 antigen Substances 0.000 description 5
- 108091007433 antigens Proteins 0.000 description 5
- 102000036639 antigens Human genes 0.000 description 5
- 239000002775 capsule Substances 0.000 description 5
- 239000002299 complementary DNA Substances 0.000 description 5
- 238000012377 drug delivery Methods 0.000 description 5
- XPOQHMRABVBWPR-ZDUSSCGKSA-N efavirenz Chemical compound C([C@]1(C2=CC(Cl)=CC=C2NC(=O)O1)C(F)(F)F)#CC1CC1 XPOQHMRABVBWPR-ZDUSSCGKSA-N 0.000 description 5
- 229960003804 efavirenz Drugs 0.000 description 5
- 150000002148 esters Chemical class 0.000 description 5
- 229940121372 histone deacetylase inhibitor Drugs 0.000 description 5
- 230000002757 inflammatory effect Effects 0.000 description 5
- JVOGDKZMFMSQIS-IQSLPNCHSA-N ingenol-3-hexanoate Chemical compound C[C@@H]1C[C@@]2(C)C(C)(C)[C@@]2(C)[C@]2(C)C=C(CO)[C@@H](O)[C@]3(O)[C@@H](OC(=O)CCCCC)C(C)=C[C@]31C2=O JVOGDKZMFMSQIS-IQSLPNCHSA-N 0.000 description 5
- 238000002347 injection Methods 0.000 description 5
- 239000007924 injection Substances 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 230000007246 mechanism Effects 0.000 description 5
- 238000007069 methylation reaction Methods 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 230000003405 preventing effect Effects 0.000 description 5
- 231100000331 toxic Toxicity 0.000 description 5
- 230000002588 toxic effect Effects 0.000 description 5
- 208000030507 AIDS Diseases 0.000 description 4
- AXRYRYVKAWYZBR-UHFFFAOYSA-N Atazanavir Natural products C=1C=C(C=2N=CC=CC=2)C=CC=1CN(NC(=O)C(NC(=O)OC)C(C)(C)C)CC(O)C(NC(=O)C(NC(=O)OC)C(C)(C)C)CC1=CC=CC=C1 AXRYRYVKAWYZBR-UHFFFAOYSA-N 0.000 description 4
- 108010019625 Atazanavir Sulfate Proteins 0.000 description 4
- JKVOKRQJEYPKQG-UHFFFAOYSA-N Gnidimacrin Natural products CC1CCCCCCC(O)C23OC4C5C6OC6(CO)C(O)C7(O)C(OC(=O)c8ccccc8)C(C)C1C7C5(O2)C(CC4(O3)C(=C)C)OC(=O)c9ccccc9 JKVOKRQJEYPKQG-UHFFFAOYSA-N 0.000 description 4
- 238000000134 MTT assay Methods 0.000 description 4
- 108091007960 PI3Ks Proteins 0.000 description 4
- NCDNCNXCDXHOMX-UHFFFAOYSA-N Ritonavir Natural products C=1C=CC=CC=1CC(NC(=O)OCC=1SC=NC=1)C(O)CC(CC=1C=CC=CC=1)NC(=O)C(C(C)C)NC(=O)N(C)CC1=CSC(C(C)C)=N1 NCDNCNXCDXHOMX-UHFFFAOYSA-N 0.000 description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- 238000000692 Student's t-test Methods 0.000 description 4
- 239000004480 active ingredient Substances 0.000 description 4
- 230000008485 antagonism Effects 0.000 description 4
- 229960003277 atazanavir Drugs 0.000 description 4
- AXRYRYVKAWYZBR-GASGPIRDSA-N atazanavir Chemical compound C([C@H](NC(=O)[C@@H](NC(=O)OC)C(C)(C)C)[C@@H](O)CN(CC=1C=CC(=CC=1)C=1N=CC=CC=1)NC(=O)[C@@H](NC(=O)OC)C(C)(C)C)C1=CC=CC=C1 AXRYRYVKAWYZBR-GASGPIRDSA-N 0.000 description 4
- 230000005754 cellular signaling Effects 0.000 description 4
- 230000007541 cellular toxicity Effects 0.000 description 4
- 238000002487 chromatin immunoprecipitation Methods 0.000 description 4
- 239000003937 drug carrier Substances 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- SSXCVTWCXHGTLK-VFZTWJSWSA-N gnidimacrin Chemical compound O([C@H]1[C@H]([C@H]2[C@@H]3[C@]1(O)[C@@H]([C@@]1(CO)O[C@H]1[C@H]1[C@H]4O[C@]5(O[C@@]31[C@H](COC(=O)C=1C=CC=CC=1)C[C@@]4(O5)C(C)=C)[C@H](O)CCCCCC[C@H]2C)O)C)C(=O)C1=CC=CC=C1 SSXCVTWCXHGTLK-VFZTWJSWSA-N 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 4
- 230000002147 killing effect Effects 0.000 description 4
- 238000012423 maintenance Methods 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 102000006240 membrane receptors Human genes 0.000 description 4
- 230000011987 methylation Effects 0.000 description 4
- 239000013610 patient sample Substances 0.000 description 4
- 230000035755 proliferation Effects 0.000 description 4
- 238000003753 real-time PCR Methods 0.000 description 4
- 230000009467 reduction Effects 0.000 description 4
- 230000001105 regulatory effect Effects 0.000 description 4
- 229960000311 ritonavir Drugs 0.000 description 4
- NCDNCNXCDXHOMX-XGKFQTDJSA-N ritonavir Chemical compound N([C@@H](C(C)C)C(=O)N[C@H](C[C@H](O)[C@H](CC=1C=CC=CC=1)NC(=O)OCC=1SC=NC=1)CC=1C=CC=CC=1)C(=O)N(C)CC1=CSC(C(C)C)=N1 NCDNCNXCDXHOMX-XGKFQTDJSA-N 0.000 description 4
- 229920006395 saturated elastomer Polymers 0.000 description 4
- 230000004797 therapeutic response Effects 0.000 description 4
- 230000000699 topical effect Effects 0.000 description 4
- 230000007733 viral latency Effects 0.000 description 4
- XAUDJQYHKZQPEU-KVQBGUIXSA-N 5-aza-2'-deoxycytidine Chemical compound O=C1N=C(N)N=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 XAUDJQYHKZQPEU-KVQBGUIXSA-N 0.000 description 3
- NMUSYJAQQFHJEW-KVTDHHQDSA-N 5-azacytidine Chemical compound O=C1N=C(N)N=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 NMUSYJAQQFHJEW-KVTDHHQDSA-N 0.000 description 3
- WOVKYSAHUYNSMH-RRKCRQDMSA-N 5-bromodeoxyuridine Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(Br)=C1 WOVKYSAHUYNSMH-RRKCRQDMSA-N 0.000 description 3
- ZYCAGKYWXRKLSN-KLKWOBOISA-N C[C@@H]1C[C@H]2C(C)(C)[C@H]2[C@@H]2C=C(COC(C)=O)[C@@H](O)[C@]3(O)[C@@H](OC(=O)C(\C)=C/C)C(C)=C[C@]31C2=O Chemical compound C[C@@H]1C[C@H]2C(C)(C)[C@H]2[C@@H]2C=C(COC(C)=O)[C@@H](O)[C@]3(O)[C@@H](OC(=O)C(\C)=C/C)C(C)=C[C@]31C2=O ZYCAGKYWXRKLSN-KLKWOBOISA-N 0.000 description 3
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 3
- 108010001857 Cell Surface Receptors Proteins 0.000 description 3
- 229920001661 Chitosan Polymers 0.000 description 3
- 108010077544 Chromatin Proteins 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 229940124602 FDA-approved drug Drugs 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 241000282412 Homo Species 0.000 description 3
- 241000713772 Human immunodeficiency virus 1 Species 0.000 description 3
- 108090001005 Interleukin-6 Proteins 0.000 description 3
- 108010002586 Interleukin-7 Proteins 0.000 description 3
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 3
- 239000004472 Lysine Substances 0.000 description 3
- 231100000002 MTT assay Toxicity 0.000 description 3
- 230000018199 S phase Effects 0.000 description 3
- 229920002472 Starch Polymers 0.000 description 3
- 229930006000 Sucrose Natural products 0.000 description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 3
- 102000000887 Transcription factor STAT Human genes 0.000 description 3
- 108050007918 Transcription factor STAT Proteins 0.000 description 3
- 239000000443 aerosol Substances 0.000 description 3
- 230000000798 anti-retroviral effect Effects 0.000 description 3
- 229960002756 azacitidine Drugs 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- VYTBDSUNRJYVHL-UHFFFAOYSA-N beta-Hydrojuglone Natural products O=C1CCC(=O)C2=C1C=CC=C2O VYTBDSUNRJYVHL-UHFFFAOYSA-N 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 3
- 230000000981 bystander Effects 0.000 description 3
- 230000004663 cell proliferation Effects 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 210000003483 chromatin Anatomy 0.000 description 3
- 238000013270 controlled release Methods 0.000 description 3
- 239000000599 controlled substance Substances 0.000 description 3
- 229960003603 decitabine Drugs 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 229960002563 disulfiram Drugs 0.000 description 3
- 239000000890 drug combination Substances 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 3
- 210000002865 immune cell Anatomy 0.000 description 3
- 210000000987 immune system Anatomy 0.000 description 3
- 238000002513 implantation Methods 0.000 description 3
- 230000000415 inactivating effect Effects 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 238000001802 infusion Methods 0.000 description 3
- CUPXSRUXWRMVAW-NRCSZYEJSA-N ingenol-3-dodecanoate Chemical compound C[C@@H]1C[C@@]2(C)C(C)(C)[C@@]2(C)[C@]2(C)C=C(CO)[C@@H](O)[C@]3(O)[C@@H](OC(=O)CCCCCCCCCCC)C(C)=C[C@]31C2=O CUPXSRUXWRMVAW-NRCSZYEJSA-N 0.000 description 3
- KXVFSPLUBBDMLD-UHFFFAOYSA-N ingenol-3-dodecanoate Natural products CCCCCCCCCCCCOC1C(=CC23C(C)CC4C(C(C=C(CO)C(O)C12O)C3=O)C4(C)C)C KXVFSPLUBBDMLD-UHFFFAOYSA-N 0.000 description 3
- NLZBKAQZGMJUBI-RYIIFTHESA-N ingenol-3-trans-cinnamate Chemical compound O([C@@H]1[C@@]2(O)[C@H](O)C(CO)=C[C@@]3(C)[C@]4(C)C(C)(C)[C@]4(C)C[C@H]([C@@]2(C=C1C)C3=O)C)C(=C)\C=C\C1=CC=CC=C1 NLZBKAQZGMJUBI-RYIIFTHESA-N 0.000 description 3
- 239000004615 ingredient Substances 0.000 description 3
- 238000007918 intramuscular administration Methods 0.000 description 3
- 238000010255 intramuscular injection Methods 0.000 description 3
- 238000007912 intraperitoneal administration Methods 0.000 description 3
- PGHMRUGBZOYCAA-ADZNBVRBSA-N ionomycin Chemical compound O1[C@H](C[C@H](O)[C@H](C)[C@H](O)[C@H](C)/C=C/C[C@@H](C)C[C@@H](C)C(/O)=C/C(=O)[C@@H](C)C[C@@H](C)C[C@@H](CCC(O)=O)C)CC[C@@]1(C)[C@@H]1O[C@](C)([C@@H](C)O)CC1 PGHMRUGBZOYCAA-ADZNBVRBSA-N 0.000 description 3
- PGHMRUGBZOYCAA-UHFFFAOYSA-N ionomycin Natural products O1C(CC(O)C(C)C(O)C(C)C=CCC(C)CC(C)C(O)=CC(=O)C(C)CC(C)CC(CCC(O)=O)C)CCC1(C)C1OC(C)(C(C)O)CC1 PGHMRUGBZOYCAA-UHFFFAOYSA-N 0.000 description 3
- 239000008101 lactose Substances 0.000 description 3
- 230000007774 longterm Effects 0.000 description 3
- 235000018977 lysine Nutrition 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 3
- 238000007911 parenteral administration Methods 0.000 description 3
- 239000001814 pectin Substances 0.000 description 3
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 3
- 230000034190 positive regulation of NF-kappaB transcription factor activity Effects 0.000 description 3
- 239000003755 preservative agent Substances 0.000 description 3
- 230000000770 proinflammatory effect Effects 0.000 description 3
- 230000002685 pulmonary effect Effects 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000003757 reverse transcription PCR Methods 0.000 description 3
- 235000019698 starch Nutrition 0.000 description 3
- 229960004793 sucrose Drugs 0.000 description 3
- 239000000829 suppository Substances 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 238000012353 t test Methods 0.000 description 3
- 230000009258 tissue cross reactivity Effects 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- DOEWDSDBFRHVAP-KRXBUXKQSA-N (E)-3-tosylacrylonitrile Chemical compound CC1=CC=C(S(=O)(=O)\C=C\C#N)C=C1 DOEWDSDBFRHVAP-KRXBUXKQSA-N 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 2
- PYMYPHUHKUWMLA-UHFFFAOYSA-N 2,3,4,5-tetrahydroxypentanal Chemical compound OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 2
- FAWLNURBQMTKEB-URDPEVQOSA-N 213546-53-3 Chemical compound N([C@@H](C)C(=O)N[C@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@H](C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N1[C@@H](CCC1)C(O)=O)C(C)C)C(C)C)C(=O)[C@@H]1CCCN1C(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)N)C(C)C FAWLNURBQMTKEB-URDPEVQOSA-N 0.000 description 2
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 238000010599 BrdU assay Methods 0.000 description 2
- 208000024172 Cardiovascular disease Diseases 0.000 description 2
- 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 2
- 230000007067 DNA methylation Effects 0.000 description 2
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 2
- 208000030453 Drug-Related Side Effects and Adverse reaction Diseases 0.000 description 2
- 208000032928 Dyslipidaemia Diseases 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 241000709661 Enterovirus Species 0.000 description 2
- 241000991587 Enterovirus C Species 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 239000001856 Ethyl cellulose Substances 0.000 description 2
- ZZSNKZQZMQGXPY-UHFFFAOYSA-N Ethyl cellulose Chemical compound CCOCC1OC(OC)C(OCC)C(OCC)C1OC1C(O)C(O)C(OC)C(CO)O1 ZZSNKZQZMQGXPY-UHFFFAOYSA-N 0.000 description 2
- 108010010803 Gelatin Proteins 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 241000711549 Hepacivirus C Species 0.000 description 2
- 241000700721 Hepatitis B virus Species 0.000 description 2
- 241000709721 Hepatovirus A Species 0.000 description 2
- 108010033040 Histones Proteins 0.000 description 2
- 101001057504 Homo sapiens Interferon-stimulated gene 20 kDa protein Proteins 0.000 description 2
- 101001055144 Homo sapiens Interleukin-2 receptor subunit alpha Proteins 0.000 description 2
- 101000617830 Homo sapiens Sterol O-acyltransferase 1 Proteins 0.000 description 2
- 241000598436 Human T-cell lymphotropic virus Species 0.000 description 2
- 241000701085 Human alphaherpesvirus 3 Species 0.000 description 2
- 241000701044 Human gammaherpesvirus 4 Species 0.000 description 2
- 206010022489 Insulin Resistance Diseases 0.000 description 2
- 102100037850 Interferon gamma Human genes 0.000 description 2
- 108010074328 Interferon-gamma Proteins 0.000 description 2
- 102100027268 Interferon-stimulated gene 20 kDa protein Human genes 0.000 description 2
- 108010002350 Interleukin-2 Proteins 0.000 description 2
- 208000017170 Lipid metabolism disease Diseases 0.000 description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 2
- 241000282560 Macaca mulatta Species 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 229930195725 Mannitol Natural products 0.000 description 2
- 241000712079 Measles morbillivirus Species 0.000 description 2
- 241000711386 Mumps virus Species 0.000 description 2
- 108010052419 NF-KappaB Inhibitor alpha Proteins 0.000 description 2
- 102100039337 NF-kappa-B inhibitor alpha Human genes 0.000 description 2
- 102100033104 NF-kappa-B inhibitor epsilon Human genes 0.000 description 2
- 101710093997 NF-kappa-B inhibitor epsilon Proteins 0.000 description 2
- 108091000080 Phosphotransferase Proteins 0.000 description 2
- 241000711798 Rabies lyssavirus Species 0.000 description 2
- 241000710799 Rubella virus Species 0.000 description 2
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 2
- 102100021993 Sterol O-acyltransferase 1 Human genes 0.000 description 2
- 101000697584 Streptomyces lavendulae Streptothricin acetyltransferase Proteins 0.000 description 2
- 230000006052 T cell proliferation Effects 0.000 description 2
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 description 2
- GWEVSGVZZGPLCZ-UHFFFAOYSA-N Titan oxide Chemical compound O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 description 2
- 241000700618 Vaccinia virus Species 0.000 description 2
- 208000009621 actinic keratosis Diseases 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 235000010443 alginic acid Nutrition 0.000 description 2
- 229920000615 alginic acid Polymers 0.000 description 2
- UIERETOOQGIECD-ARJAWSKDSA-N angelic acid Chemical compound C\C=C(\C)C(O)=O UIERETOOQGIECD-ARJAWSKDSA-N 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 229940125763 bromodomain inhibitor Drugs 0.000 description 2
- CJZGTCYPCWQAJB-UHFFFAOYSA-L calcium stearate Chemical compound [Ca+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O CJZGTCYPCWQAJB-UHFFFAOYSA-L 0.000 description 2
- OSGAYBCDTDRGGQ-UHFFFAOYSA-L calcium sulfate Chemical compound [Ca+2].[O-]S([O-])(=O)=O OSGAYBCDTDRGGQ-UHFFFAOYSA-L 0.000 description 2
- 229910002092 carbon dioxide Inorganic materials 0.000 description 2
- 230000030833 cell death Effects 0.000 description 2
- 210000003855 cell nucleus Anatomy 0.000 description 2
- 235000010980 cellulose Nutrition 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 210000001072 colon Anatomy 0.000 description 2
- 239000002577 cryoprotective agent Substances 0.000 description 2
- 231100000433 cytotoxic Toxicity 0.000 description 2
- 230000001472 cytotoxic effect Effects 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000002552 dosage form Substances 0.000 description 2
- 231100000673 dose–response relationship Toxicity 0.000 description 2
- 230000007750 drug combination effect Effects 0.000 description 2
- 230000000857 drug effect Effects 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 230000008030 elimination Effects 0.000 description 2
- 238000003379 elimination reaction Methods 0.000 description 2
- 239000003995 emulsifying agent Substances 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 235000019325 ethyl cellulose Nutrition 0.000 description 2
- 229920001249 ethyl cellulose Polymers 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 235000003599 food sweetener Nutrition 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 239000008273 gelatin Substances 0.000 description 2
- 229920000159 gelatin Polymers 0.000 description 2
- 239000007903 gelatin capsule Substances 0.000 description 2
- 235000019322 gelatine Nutrition 0.000 description 2
- 235000011852 gelatine desserts Nutrition 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 235000010979 hydroxypropyl methyl cellulose Nutrition 0.000 description 2
- 239000001866 hydroxypropyl methyl cellulose Substances 0.000 description 2
- 229920003088 hydroxypropyl methyl cellulose Polymers 0.000 description 2
- UFVKGYZPFZQRLF-UHFFFAOYSA-N hydroxypropyl methyl cellulose Chemical compound OC1C(O)C(OC)OC(CO)C1OC1C(O)C(O)C(OC2C(C(O)C(OC3C(C(O)C(O)C(CO)O3)O)C(CO)O2)O)C(CO)O1 UFVKGYZPFZQRLF-UHFFFAOYSA-N 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 239000007927 intramuscular injection Substances 0.000 description 2
- 238000010253 intravenous injection Methods 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 239000002502 liposome Substances 0.000 description 2
- 239000000314 lubricant Substances 0.000 description 2
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 235000010355 mannitol Nutrition 0.000 description 2
- 239000000594 mannitol Substances 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 210000004379 membrane Anatomy 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 231100000324 minimal toxicity Toxicity 0.000 description 2
- 230000000869 mutational effect Effects 0.000 description 2
- 230000017128 negative regulation of NF-kappaB transcription factor activity Effects 0.000 description 2
- 150000002894 organic compounds Chemical class 0.000 description 2
- 235000010987 pectin Nutrition 0.000 description 2
- 229920001277 pectin Polymers 0.000 description 2
- 230000002688 persistence Effects 0.000 description 2
- 102000020233 phosphotransferase Human genes 0.000 description 2
- 229920001606 poly(lactic acid-co-glycolic acid) Polymers 0.000 description 2
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 2
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 2
- 238000002203 pretreatment Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 230000035939 shock Effects 0.000 description 2
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 235000010356 sorbitol Nutrition 0.000 description 2
- 239000000600 sorbitol Substances 0.000 description 2
- 239000007921 spray Substances 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 238000010561 standard procedure Methods 0.000 description 2
- 229940032147 starch Drugs 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000001629 suppression Effects 0.000 description 2
- 239000003765 sweetening agent Substances 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 238000007910 systemic administration Methods 0.000 description 2
- 230000009885 systemic effect Effects 0.000 description 2
- 108700004027 tat Genes Proteins 0.000 description 2
- 101150098170 tat gene Proteins 0.000 description 2
- 238000011285 therapeutic regimen Methods 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000002103 transcriptional effect Effects 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 2
- 241001529453 unidentified herpesvirus Species 0.000 description 2
- 241000712461 unidentified influenza virus Species 0.000 description 2
- 230000003827 upregulation Effects 0.000 description 2
- 239000003981 vehicle Substances 0.000 description 2
- 230000029812 viral genome replication Effects 0.000 description 2
- 230000017613 viral reproduction Effects 0.000 description 2
- 239000000080 wetting agent Substances 0.000 description 2
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
- QGVLYPPODPLXMB-UBTYZVCOSA-N (1aR,1bS,4aR,7aS,7bS,8R,9R,9aS)-4a,7b,9,9a-tetrahydroxy-3-(hydroxymethyl)-1,1,6,8-tetramethyl-1,1a,1b,4,4a,7a,7b,8,9,9a-decahydro-5H-cyclopropa[3,4]benzo[1,2-e]azulen-5-one Chemical compound C1=C(CO)C[C@]2(O)C(=O)C(C)=C[C@H]2[C@@]2(O)[C@H](C)[C@@H](O)[C@@]3(O)C(C)(C)[C@H]3[C@@H]21 QGVLYPPODPLXMB-UBTYZVCOSA-N 0.000 description 1
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 1
- VBMOHECZZWVLFJ-GXTUVTBFSA-N (2s)-2-[[(2s)-6-amino-2-[[(2s)-6-amino-2-[[(2s,3r)-2-[[(2s,3r)-2-[[(2s)-6-amino-2-[[(2s)-2-[[(2s)-6-amino-2-[[(2s)-2-[[(2s)-2-[[(2s)-2,6-diaminohexanoyl]amino]-5-(diaminomethylideneamino)pentanoyl]amino]propanoyl]amino]hexanoyl]amino]propanoyl]amino]hexan Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H]([C@H](O)C)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](N)CCCCN VBMOHECZZWVLFJ-GXTUVTBFSA-N 0.000 description 1
- DDMOUSALMHHKOS-UHFFFAOYSA-N 1,2-dichloro-1,1,2,2-tetrafluoroethane Chemical compound FC(F)(Cl)C(F)(F)Cl DDMOUSALMHHKOS-UHFFFAOYSA-N 0.000 description 1
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 1
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- 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 1
- SQDAZGGFXASXDW-UHFFFAOYSA-N 5-bromo-2-(trifluoromethoxy)pyridine Chemical compound FC(F)(F)OC1=CC=C(Br)C=N1 SQDAZGGFXASXDW-UHFFFAOYSA-N 0.000 description 1
- 206010001258 Adenoviral infections Diseases 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 235000019489 Almond oil Nutrition 0.000 description 1
- UIERETOOQGIECD-UHFFFAOYSA-N Angelic acid Natural products CC=C(C)C(O)=O UIERETOOQGIECD-UHFFFAOYSA-N 0.000 description 1
- 101100366892 Anopheles gambiae Stat gene Proteins 0.000 description 1
- 241000416162 Astragalus gummifer Species 0.000 description 1
- 241000972773 Aulopiformes Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 108091052242 Bromo- and Extra-Terminal domain (BET) family Proteins 0.000 description 1
- 108050009021 Bromodomains Proteins 0.000 description 1
- 102000001805 Bromodomains Human genes 0.000 description 1
- NEOVTYQDNTUFDO-SFHVURJKSA-N CC1=CC=C(C2=N[C@@H](CC(=O)OC(C)(C)C)C3=NN=C(C)N3C3=C2C(C)=C(C)S3)C=C1 Chemical compound CC1=CC=C(C2=N[C@@H](CC(=O)OC(C)(C)C)C3=NN=C(C)N3C3=C2C(C)=C(C)S3)C=C1 NEOVTYQDNTUFDO-SFHVURJKSA-N 0.000 description 1
- 108010017088 CCR5 Receptors Proteins 0.000 description 1
- HCIDMOZXKOHYFH-UHFFFAOYSA-N CNC(=O)CCCCCCC(=O)CC1=CC=CC=C1 Chemical compound CNC(=O)CCCCCCC(=O)CC1=CC=CC=C1 HCIDMOZXKOHYFH-UHFFFAOYSA-N 0.000 description 1
- 108010061299 CXCR4 Receptors Proteins 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 241000700199 Cavia porcellus Species 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 229920002567 Chondroitin Polymers 0.000 description 1
- 229920001287 Chondroitin sulfate Polymers 0.000 description 1
- 229920002261 Corn starch Polymers 0.000 description 1
- 102100024457 Cyclin-dependent kinase 9 Human genes 0.000 description 1
- 229920000858 Cyclodextrin Polymers 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 230000004568 DNA-binding Effects 0.000 description 1
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 1
- 102100030012 Deoxyribonuclease-1 Human genes 0.000 description 1
- 239000004338 Dichlorodifluoromethane Substances 0.000 description 1
- 101100366894 Drosophila melanogaster Stat92E gene Proteins 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- XQSPYNMVSIKCOC-NTSWFWBYSA-N Emtricitabine Chemical compound C1=C(F)C(N)=NC(=O)N1[C@H]1O[C@@H](CO)SC1 XQSPYNMVSIKCOC-NTSWFWBYSA-N 0.000 description 1
- 241000792859 Enema Species 0.000 description 1
- 229920003148 Eudragit® E polymer Polymers 0.000 description 1
- 229920003136 Eudragit® L polymer Polymers 0.000 description 1
- 229920003141 Eudragit® S 100 Polymers 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 240000001837 Euphorbia peplus Species 0.000 description 1
- 244000256297 Euphorbia tirucalli Species 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 1
- 208000031448 Genomic Instability Diseases 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- AEMRFAOFKBGASW-UHFFFAOYSA-M Glycolate Chemical compound OCC([O-])=O AEMRFAOFKBGASW-UHFFFAOYSA-M 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 101000980930 Homo sapiens Cyclin-dependent kinase 9 Proteins 0.000 description 1
- 101000979342 Homo sapiens Nuclear factor NF-kappa-B p105 subunit Proteins 0.000 description 1
- 101000605639 Homo sapiens Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform Proteins 0.000 description 1
- 101001051777 Homo sapiens Protein kinase C alpha type Proteins 0.000 description 1
- 108010070875 Human Immunodeficiency Virus tat Gene Products Proteins 0.000 description 1
- XQFRJNBWHJMXHO-RRKCRQDMSA-N IDUR Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(I)=C1 XQFRJNBWHJMXHO-RRKCRQDMSA-N 0.000 description 1
- 102000000588 Interleukin-2 Human genes 0.000 description 1
- 102000004889 Interleukin-6 Human genes 0.000 description 1
- RRHGJUQNOFWUDK-UHFFFAOYSA-N Isoprene Chemical group CC(=C)C=C RRHGJUQNOFWUDK-UHFFFAOYSA-N 0.000 description 1
- 102000015617 Janus Kinases Human genes 0.000 description 1
- 108010024121 Janus Kinases Proteins 0.000 description 1
- OFFWOVJBSQMVPI-RMLGOCCBSA-N Kaletra Chemical compound N1([C@@H](C(C)C)C(=O)N[C@H](C[C@H](O)[C@H](CC=2C=CC=CC=2)NC(=O)COC=2C(=CC=CC=2C)C)CC=2C=CC=CC=2)CCCNC1=O.N([C@@H](C(C)C)C(=O)N[C@H](C[C@H](O)[C@H](CC=1C=CC=CC=1)NC(=O)OCC=1SC=NC=1)CC=1C=CC=CC=1)C(=O)N(C)CC1=CSC(C(C)C)=N1 OFFWOVJBSQMVPI-RMLGOCCBSA-N 0.000 description 1
- 208000032420 Latent Infection Diseases 0.000 description 1
- 235000010643 Leucaena leucocephala Nutrition 0.000 description 1
- 240000007472 Leucaena leucocephala Species 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 108700018351 Major Histocompatibility Complex Proteins 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 229920000168 Microcrystalline cellulose Polymers 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- WHNWPMSKXPGLAX-UHFFFAOYSA-N N-Vinyl-2-pyrrolidone Chemical compound C=CN1CCCC1=O WHNWPMSKXPGLAX-UHFFFAOYSA-N 0.000 description 1
- 102100033457 NF-kappa-B inhibitor beta Human genes 0.000 description 1
- 101710204094 NF-kappa-B inhibitor beta Proteins 0.000 description 1
- 102100023050 Nuclear factor NF-kappa-B p105 subunit Human genes 0.000 description 1
- 229940122313 Nucleoside reverse transcriptase inhibitor Drugs 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- 229920000375 Poly(ethylene glycol)-block-poly(ε−caprolactone) methyl ether Polymers 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 102000001253 Protein Kinase Human genes 0.000 description 1
- 102100024924 Protein kinase C alpha type Human genes 0.000 description 1
- 102100033810 RAC-alpha serine/threonine-protein kinase Human genes 0.000 description 1
- 101710113459 RAC-alpha serine/threonine-protein kinase Proteins 0.000 description 1
- 239000012083 RIPA buffer Substances 0.000 description 1
- 102000009572 RNA Polymerase II Human genes 0.000 description 1
- 108010009460 RNA Polymerase II Proteins 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 238000013381 RNA quantification Methods 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 206010038997 Retroviral infections Diseases 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 102000039471 Small Nuclear RNA Human genes 0.000 description 1
- 108020004688 Small Nuclear RNA Proteins 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 235000021355 Stearic acid Nutrition 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 210000000662 T-lymphocyte subset Anatomy 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 229920001615 Tragacanth Polymers 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 1
- RTKIYFITIVXBLE-UHFFFAOYSA-N Trichostatin A Natural products ONC(=O)C=CC(C)=CC(C)C(=O)C1=CC=C(N(C)C)C=C1 RTKIYFITIVXBLE-UHFFFAOYSA-N 0.000 description 1
- 102100040247 Tumor necrosis factor Human genes 0.000 description 1
- 206010058874 Viraemia Diseases 0.000 description 1
- 108020005202 Viral DNA Proteins 0.000 description 1
- 108700005077 Viral Genes Proteins 0.000 description 1
- 108020000999 Viral RNA Proteins 0.000 description 1
- 101710201961 Virion infectivity factor Proteins 0.000 description 1
- LMRFCUFNKZOVOW-IEMNKQQPSA-N [H][C@@]12C=C(CC)[C@@H](O)[C@]3(O)[C@@H](OC(=O)/C(C)=C\C)C(C)=C[C@@]3(C1=O)[C@H](C)C[C@@]1([H])C(C)(C)[C@@]21[H] Chemical compound [H][C@@]12C=C(CC)[C@@H](O)[C@]3(O)[C@@H](OC(=O)/C(C)=C\C)C(C)=C[C@@]3(C1=O)[C@H](C)C[C@@]1([H])C(C)(C)[C@@]21[H] LMRFCUFNKZOVOW-IEMNKQQPSA-N 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 description 1
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000033289 adaptive immune response Effects 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 239000000783 alginic acid Substances 0.000 description 1
- 229960001126 alginic acid Drugs 0.000 description 1
- 150000004781 alginic acids Chemical class 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 239000008168 almond oil Substances 0.000 description 1
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 1
- SNAAJJQQZSMGQD-UHFFFAOYSA-N aluminum magnesium Chemical compound [Mg].[Al] SNAAJJQQZSMGQD-UHFFFAOYSA-N 0.000 description 1
- 235000019824 amidated pectin Nutrition 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 239000003708 ampul Substances 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 229940019748 antifibrinolytic proteinase inhibitors Drugs 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000009118 appropriate response Effects 0.000 description 1
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 238000012742 biochemical analysis Methods 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 229920001400 block copolymer Polymers 0.000 description 1
- 230000036760 body temperature Effects 0.000 description 1
- 125000001246 bromo group Chemical group Br* 0.000 description 1
- 229960005539 bryostatin 1 Drugs 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000000337 buffer salt Substances 0.000 description 1
- NEDGUIRITORSKL-UHFFFAOYSA-N butyl 2-methylprop-2-enoate;2-(dimethylamino)ethyl 2-methylprop-2-enoate;methyl 2-methylprop-2-enoate Chemical compound COC(=O)C(C)=C.CCCCOC(=O)C(C)=C.CN(C)CCOC(=O)C(C)=C NEDGUIRITORSKL-UHFFFAOYSA-N 0.000 description 1
- FUFJGUQYACFECW-UHFFFAOYSA-L calcium hydrogenphosphate Chemical compound [Ca+2].OP([O-])([O-])=O FUFJGUQYACFECW-UHFFFAOYSA-L 0.000 description 1
- 159000000007 calcium salts Chemical class 0.000 description 1
- 235000013539 calcium stearate Nutrition 0.000 description 1
- 239000008116 calcium stearate Substances 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229960004424 carbon dioxide Drugs 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000020411 cell activation Effects 0.000 description 1
- 230000006369 cell cycle progression Effects 0.000 description 1
- 239000002771 cell marker Substances 0.000 description 1
- 108091092356 cellular DNA Proteins 0.000 description 1
- 230000033077 cellular process Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- DLGJWSVWTWEWBJ-HGGSSLSASA-N chondroitin Chemical compound CC(O)=N[C@@H]1[C@H](O)O[C@H](CO)[C@H](O)[C@@H]1OC1[C@H](O)[C@H](O)C=C(C(O)=O)O1 DLGJWSVWTWEWBJ-HGGSSLSASA-N 0.000 description 1
- 229940059329 chondroitin sulfate Drugs 0.000 description 1
- 239000013611 chromosomal DNA Substances 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 238000011260 co-administration Methods 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- ZCIGNRJZKPOIKD-CQXVEOKZSA-N cobicistat Chemical compound S1C(C(C)C)=NC(CN(C)C(=O)N[C@@H](CCN2CCOCC2)C(=O)N[C@H](CC[C@H](CC=2C=CC=CC=2)NC(=O)OCC=2SC=NC=2)CC=2C=CC=CC=2)=C1 ZCIGNRJZKPOIKD-CQXVEOKZSA-N 0.000 description 1
- 229960002402 cobicistat Drugs 0.000 description 1
- 229940110456 cocoa butter Drugs 0.000 description 1
- 235000019868 cocoa butter Nutrition 0.000 description 1
- 239000008119 colloidal silica Substances 0.000 description 1
- 229940075614 colloidal silicon dioxide Drugs 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 238000011284 combination treatment Methods 0.000 description 1
- 238000013329 compounding Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 239000008120 corn starch Substances 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 230000001351 cycling effect Effects 0.000 description 1
- 230000000120 cytopathologic effect Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 239000003405 delayed action preparation Substances 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 235000019700 dicalcium phosphate Nutrition 0.000 description 1
- PXBRQCKWGAHEHS-UHFFFAOYSA-N dichlorodifluoromethane Chemical compound FC(F)(Cl)Cl PXBRQCKWGAHEHS-UHFFFAOYSA-N 0.000 description 1
- 235000019404 dichlorodifluoromethane Nutrition 0.000 description 1
- 229940042935 dichlorodifluoromethane Drugs 0.000 description 1
- 229940087091 dichlorotetrafluoroethane Drugs 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 235000013681 dietary sucrose Nutrition 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 description 1
- 235000011180 diphosphates Nutrition 0.000 description 1
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 239000007884 disintegrant Substances 0.000 description 1
- PXEDJBXQKAGXNJ-QTNFYWBSSA-L disodium L-glutamate Chemical compound [Na+].[Na+].[O-]C(=O)[C@@H](N)CCC([O-])=O PXEDJBXQKAGXNJ-QTNFYWBSSA-L 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 229960004679 doxorubicin Drugs 0.000 description 1
- 229940000406 drug candidate Drugs 0.000 description 1
- 229940112141 dry powder inhaler Drugs 0.000 description 1
- 229960003586 elvitegravir Drugs 0.000 description 1
- JUZYLCPPVHEVSV-LJQANCHMSA-N elvitegravir Chemical compound COC1=CC=2N([C@H](CO)C(C)C)C=C(C(O)=O)C(=O)C=2C=C1CC1=CC=CC(Cl)=C1F JUZYLCPPVHEVSV-LJQANCHMSA-N 0.000 description 1
- 229960000366 emtricitabine Drugs 0.000 description 1
- 239000007920 enema Substances 0.000 description 1
- 229940079360 enema for constipation Drugs 0.000 description 1
- 230000004049 epigenetic modification Effects 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- BEFDCLMNVWHSGT-UHFFFAOYSA-N ethenylcyclopentane Chemical compound C=CC1CCCC1 BEFDCLMNVWHSGT-UHFFFAOYSA-N 0.000 description 1
- VJJPUSNTGOMMGY-MRVIYFEKSA-N etoposide Chemical compound COC1=C(O)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3[C@@H](O[C@H]3[C@@H]([C@@H](O)[C@@H]4O[C@H](C)OC[C@H]4O3)O)[C@@H]3[C@@H]2C(OC3)=O)=C1 VJJPUSNTGOMMGY-MRVIYFEKSA-N 0.000 description 1
- 229960005420 etoposide Drugs 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 238000013401 experimental design Methods 0.000 description 1
- 239000003777 experimental drug Substances 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000003925 fat Substances 0.000 description 1
- 235000019197 fats Nutrition 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229960001031 glucose Drugs 0.000 description 1
- 125000005456 glyceride group Chemical group 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000005745 host immune response Effects 0.000 description 1
- 239000008172 hydrogenated vegetable oil Substances 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000005934 immune activation Effects 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 238000012606 in vitro cell culture Methods 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 239000007972 injectable composition Substances 0.000 description 1
- 229940124524 integrase inhibitor Drugs 0.000 description 1
- 239000002850 integrase inhibitor Substances 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000001361 intraarterial administration Methods 0.000 description 1
- 238000007917 intracranial administration Methods 0.000 description 1
- 238000007913 intrathecal administration Methods 0.000 description 1
- 230000002601 intratumoral effect Effects 0.000 description 1
- 238000007914 intraventricular administration Methods 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 239000000644 isotonic solution Substances 0.000 description 1
- 238000009533 lab test Methods 0.000 description 1
- 125000000686 lactone group Chemical group 0.000 description 1
- 229960001375 lactose Drugs 0.000 description 1
- JCQLYHFGKNRPGE-FCVZTGTOSA-N lactulose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 JCQLYHFGKNRPGE-FCVZTGTOSA-N 0.000 description 1
- 229960000511 lactulose Drugs 0.000 description 1
- PFCRQPBOOFTZGQ-UHFFFAOYSA-N lactulose keto form Natural products OCC(=O)C(O)C(C(O)CO)OC1OC(CO)C(O)C(O)C1O PFCRQPBOOFTZGQ-UHFFFAOYSA-N 0.000 description 1
- 235000010445 lecithin Nutrition 0.000 description 1
- 239000000787 lecithin Substances 0.000 description 1
- 229940067606 lecithin Drugs 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 229940113983 lopinavir / ritonavir Drugs 0.000 description 1
- 239000006210 lotion Substances 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 150000002669 lysines Chemical class 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 108010068904 lysyl-arginyl-alanyl-lysyl-alanyl-lysyl-threonyl-threonyl-lysyl-lysyl-arginine Proteins 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 108020004084 membrane receptors Proteins 0.000 description 1
- 230000006996 mental state Effects 0.000 description 1
- 229920000609 methyl cellulose Polymers 0.000 description 1
- 235000010270 methyl p-hydroxybenzoate Nutrition 0.000 description 1
- 235000010981 methylcellulose Nutrition 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- 235000019813 microcrystalline cellulose Nutrition 0.000 description 1
- 239000008108 microcrystalline cellulose Substances 0.000 description 1
- 229940016286 microcrystalline cellulose Drugs 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 244000309715 mini pig Species 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 230000003990 molecular pathway Effects 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 235000013923 monosodium glutamate Nutrition 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 239000006199 nebulizer Substances 0.000 description 1
- 239000002687 nonaqueous vehicle Substances 0.000 description 1
- 231100001160 nonlethal Toxicity 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 230000005937 nuclear translocation Effects 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 1
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- 239000002674 ointment Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000006072 paste Substances 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- 229940021222 peritoneal dialysis isotonic solution Drugs 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 239000008180 pharmaceutical surfactant Substances 0.000 description 1
- QGVLYPPODPLXMB-QXYKVGAMSA-N phorbol Natural products C[C@@H]1[C@@H](O)[C@]2(O)[C@H]([C@H]3C=C(CO)C[C@@]4(O)[C@H](C=C(C)C4=O)[C@@]13O)C2(C)C QGVLYPPODPLXMB-QXYKVGAMSA-N 0.000 description 1
- 230000000865 phosphorylative effect Effects 0.000 description 1
- XNGIFLGASWRNHJ-UHFFFAOYSA-L phthalate(2-) Chemical compound [O-]C(=O)C1=CC=CC=C1C([O-])=O XNGIFLGASWRNHJ-UHFFFAOYSA-L 0.000 description 1
- 230000006461 physiological response Effects 0.000 description 1
- 229920000058 polyacrylate Polymers 0.000 description 1
- 230000008488 polyadenylation Effects 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229940093429 polyethylene glycol 6000 Drugs 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 150000004804 polysaccharides Chemical class 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 229920001592 potato starch Polymers 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 210000004206 promonocyte Anatomy 0.000 description 1
- 230000001902 propagating effect Effects 0.000 description 1
- 239000003380 propellant Substances 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 235000010232 propyl p-hydroxybenzoate Nutrition 0.000 description 1
- QELSKZZBTMNZEB-UHFFFAOYSA-N propylparaben Chemical class CCCOC(=O)C1=CC=C(O)C=C1 QELSKZZBTMNZEB-UHFFFAOYSA-N 0.000 description 1
- 108060006633 protein kinase Proteins 0.000 description 1
- 108010061269 protein kinase D Proteins 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 229960004742 raltegravir Drugs 0.000 description 1
- CZFFBEXEKNGXKS-UHFFFAOYSA-N raltegravir Chemical compound O1C(C)=NN=C1C(=O)NC(C)(C)C1=NC(C(=O)NCC=2C=CC(F)=CC=2)=C(O)C(=O)N1C CZFFBEXEKNGXKS-UHFFFAOYSA-N 0.000 description 1
- 238000011552 rat model Methods 0.000 description 1
- 230000037425 regulation of transcription Effects 0.000 description 1
- 230000008844 regulatory mechanism Effects 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 239000003419 rna directed dna polymerase inhibitor Substances 0.000 description 1
- 235000019515 salmon Nutrition 0.000 description 1
- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- WXMKPNITSTVMEF-UHFFFAOYSA-M sodium benzoate Chemical compound [Na+].[O-]C(=O)C1=CC=CC=C1 WXMKPNITSTVMEF-UHFFFAOYSA-M 0.000 description 1
- 239000004299 sodium benzoate Substances 0.000 description 1
- 235000010234 sodium benzoate Nutrition 0.000 description 1
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 description 1
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 description 1
- 229940073490 sodium glutamate Drugs 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 229940080313 sodium starch Drugs 0.000 description 1
- 239000002195 soluble material Substances 0.000 description 1
- 239000012453 solvate Substances 0.000 description 1
- 239000004334 sorbic acid Substances 0.000 description 1
- 235000010199 sorbic acid Nutrition 0.000 description 1
- 229940075582 sorbic acid Drugs 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 239000008117 stearic acid Substances 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 description 1
- 239000002511 suppository base Substances 0.000 description 1
- 230000020382 suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I Effects 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 230000009044 synergistic interaction Effects 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 235000012222 talc Nutrition 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 229960004556 tenofovir Drugs 0.000 description 1
- VCMJCVGFSROFHV-WZGZYPNHSA-N tenofovir disoproxil fumarate Chemical compound OC(=O)\C=C\C(O)=O.N1=CN=C2N(C[C@@H](C)OCP(=O)(OCOC(=O)OC(C)C)OCOC(=O)OC(C)C)C=NC2=C1N VCMJCVGFSROFHV-WZGZYPNHSA-N 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 231100001274 therapeutic index Toxicity 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 239000004408 titanium dioxide Substances 0.000 description 1
- 238000011200 topical administration Methods 0.000 description 1
- 235000010487 tragacanth Nutrition 0.000 description 1
- 239000000196 tragacanth Substances 0.000 description 1
- 229940116362 tragacanth Drugs 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- ODLHGICHYURWBS-LKONHMLTSA-N trappsol cyclo Chemical compound CC(O)COC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](COCC(C)O)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](COCC(C)O)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](COCC(C)O)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](COCC(C)O)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)COCC(O)C)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1COCC(C)O ODLHGICHYURWBS-LKONHMLTSA-N 0.000 description 1
- 229940074410 trehalose Drugs 0.000 description 1
- CYRMSUTZVYGINF-UHFFFAOYSA-N trichlorofluoromethane Chemical compound FC(Cl)(Cl)Cl CYRMSUTZVYGINF-UHFFFAOYSA-N 0.000 description 1
- 229940029284 trichlorofluoromethane Drugs 0.000 description 1
- 231100000402 unacceptable toxicity Toxicity 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 230000007501 viral attachment Effects 0.000 description 1
- 230000007502 viral entry Effects 0.000 description 1
- 230000005727 virus proliferation Effects 0.000 description 1
- 230000003442 weekly effect Effects 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
- 229940061261 zolinza Drugs 0.000 description 1
Images
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
- A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
- A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
- A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
-
- 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
- A61K31/16—Amides, e.g. hydroxamic acids
- A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
- A61K31/167—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the nitrogen of a carboxamide group directly attached to the aromatic ring, e.g. lidocaine, paracetamol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
- A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
Definitions
- Anti-retroviral therapy is effective in suppressing HIV replication but it fails to eliminate latent viral reservoirs in HIV infected resting CD4+ T cells which, in blood, consist mainly of central and transitional memory CD4+ T cells [1-4].
- Current ART options do not eradicate HIV from infected cells. In addition, these cells are invisible to the virus-specific immune responses in the setting of viral latency [5,6]. The viral reservoir is rapidly seeded and HIV latency might be established immediately after virus infection [7,8]. Despite initiation of ART in infants within hours of birth to HIV infected mothers, stable viral reservoirs were established and viral rebound occurred when therapy was interrupted [9].
- the present invention provides a composition comprising ingenol-3-angelate (PEP005) and one or more additional latency reactivation agents.
- the one or more additional latency reactivation agents is selected from the group consisting of a positive transcription elongation factor b activator, a histone methyltransferase (HMT) inhibitor, a histone deacetylase (HDAC) inhibitor, a DNA methyltransferase inhibitor, an NF- ⁇ B activator, an Akt/HEXIM-1 modulator, a Jak/Stat pathway modulator, a diterpene compound (e.g., an ingenol derivative, a phorbol ester), a macrolide lactone, a diacylglycerol (DAG) lactone, a protein kinase C (PKC) activator, and combinations thereof.
- HMT histone methyltransferase
- HDAC histone deacetylase
- the composition comprises a combination of ingenol-3-angelate (PEP005) and JQ1. In other embodiments, the composition comprises a combination of ingenol-3-angelate (PEP005) and GSK343. In yet other embodiments, the composition comprises a combination of ingenol-3-angelate (PEP005) and vorinostat (suberanilohydroxamic acid; SAHA). In certain embodiments, the composition further comprises a viral therapy vaccine. In certain embodiments, the composition further comprises a pharmaceutically acceptable carrier, excipient, or diluent.
- the present invention provides a method for reactivating a latent virus in a subject infected with the virus, the method comprising administering to the subject an effective amount of ingenol-3-angelate (PEP005) alone or in combination with one or more additional latency reactivation agents.
- PEP005 ingenol-3-angelate
- the one or more additional latency reactivation agents is selected from the group consisting of a positive transcription elongation factor b activator, a histone methyltransferase (HMT) inhibitor, a histone deacetylase (HDAC) inhibitor, a DNA methyltransferase inhibitor, an NF- ⁇ B activator, an Akt/HEXIM-1 modulator, a Jak/Stat pathway modulator, a diterpene compound (e.g., an ingenol derivative, a phorbol ester), a macrolide lactone, a diacylglycerol (DAG) lactone, a protein kinase C (PKC) activator, and combinations thereof.
- the method further comprises co-administering a viral therapy vaccine.
- the method comprises co-administering a combination of ingenol-3-angelate (PEP005) and JQ1. In other embodiments, the method comprises co-administering a combination of ingenol-3-angelate (PEP005) and GSK343. In yet other embodiments, the method comprises co-administering a combination of ingenol-3-angelate (PEP005) and vorinostat (suberanilohydroxamic acid; SAHA).
- a combination of ingenol-3-angelate (PEP005) and one or more additional latency reactivation agents is capable of synergistically inducing reactivation of the latent virus.
- the present invention provides a kit comprising: (a) ingenol-3-angelate (PEP005); and (b) one or more additional latency reactivation agents.
- the one or more additional latency reactivation agents is selected from the group consisting of a positive transcription elongation factor b activator, a histone methyltransferase (HMT) inhibitor, a histone deacetylase (HDAC) inhibitor, a DNA methyltransferase inhibitor, an NF- ⁇ B activator, an Akt/HEXIM-1 modulator, a Jak/Stat pathway modulator, a diterpene compound (e.g., an ingenol derivative, a phorbol ester), a macrolide lactone, a diacylglycerol (DAG) lactone, a protein kinase C (PKC) activator, and combinations thereof.
- the kit further comprises a viral therapy vaccine.
- the kit comprises a combination of ingenol-3-angelate (PEP005) and JQ1. In other embodiments, the kit comprises a combination of ingenol-3-angelate (PEP005) and GSK343. In yet other embodiments, the kit comprises a combination of ingenol-3-angelate (PEP005) and vorinostat (suberanilohydroxamic acid; SAHA).
- FIG. 1 PEP005 induces reactivation of HIV latency in vitro. J-Lat A1 cells were exposed to different concentrations of PEP005 and virus reactivation was measured by GFP expression using flow cytometry. Cell viability was determined by Live/Dead dye staining.
- A Chemical Structure of PEP005.
- B, C PEP005 reactivates HIV in a dose-dependent manner and displays minimal cytotoxicity.
- D PEP005 potently reactivates latent HIV in cell lines compared with other LRAs.
- J-Lat A1 cells were treated with 10 nM PEP005, 500 nM SAHA, 2 M JQ1, 2 ⁇ M GSK343, 10 ng/ml TNF, 2 ⁇ M Prostratin, or 5 ng/ml PMA for 24 hours and the percentage of GFP expression was evaluated using flow cytometry. *, p ⁇ 0.05; **, p ⁇ 0.01.
- FIG. 2 PEP005 synergizes with other latency reversing agents in reactivating latent HIV.
- J-Lat A1 cells were treated with 5 ng/ml PMA, 6 nM PEP005, 500 nM SAHA, 2 ⁇ M JQ1, 2 ⁇ M GSK343, or 10 ⁇ M Prostratin alone or in combination with 6 nM PEP005 for 24 hours and the percentage of GFP expressing cells was determined using flow cytometry. **, p ⁇ 0.01; *, p ⁇ 0.05 compared with control treatment; **, p ⁇ 0.01; *, p ⁇ 0.05 compared with PEP005 treatment alone.
- FIG. 3 PEP005 activates PKC ⁇ / ⁇ -I ⁇ B ⁇ / ⁇ -NF- ⁇ B signaling.
- A J-Lat A1 cells or PBMCs isolated from peripheral blood of healthy HIV-negative individuals were treated with 12 nM of PEP005 for up to 6 hours. Western blot analysis was performed to detect the expression of PKC isoforms, I ⁇ B isoforms, as well as expression of NF- ⁇ B/p65.
- B Quantitation of phosphorylation of I ⁇ B ⁇ or I ⁇ B ⁇ in J-Lat A1 cells after 2 hr treatment with PEP005 in panel A.
- Relative band intensities from three independent experiments in J-Lat A1 cells as determined using ImageJ (NIH) are shown in the bar graph. * p ⁇ 0.05.
- C Quantitation of PKC ⁇ / ⁇ S643/S676 phosphorylation after 2 hr treatment of PEP005 in panel A. Relative band intensities from three independent experiments in J-Lat A1 cells (Left panel) or PBMCs (Right panel) as determined using ImageJ (NIH) are shown in the bar graph. * p ⁇ 0.05, ** p ⁇ 0.01.
- FIG. 4 PEP005-induced HIV reactivation is mediated through PKC ⁇ / ⁇ -I ⁇ B ⁇ / ⁇ -NF- ⁇ B signaling.
- A PEP005-induced HIV reactivation is suppressed by inhibition of the PKC ⁇ / ⁇ . J-Lat A1 cells were treated with 6 nM of PEP005 in the presence of 1, 2 or 5 ⁇ M of PKC inhibitor (PKC ⁇ / ⁇ inhibitor; Milipore/Calbiochem (539649)) and evaluated for GFP expression by RT-qPCR.
- PKC ⁇ / ⁇ inhibitor Milipore/Calbiochem (539649)
- NF- ⁇ B inhibition partially suppresses PEP005-induced HIV reactivation in J-Lat A1 cells.
- J-Lat A1 cells were treated with 6 nM of PEP005 in the presence of 2.5 ⁇ M Bay 11-7082, an NF- ⁇ B inhibitor, and were evaluated for GFP expression by RT-qPCR.
- C J-Lat A1 cells were treated with PEP005 or alternatively with PKC inhibitor alone or in the presence of PEP005 and the relative binding of NF- ⁇ B to the HIV LTR was determined using ChIP-qPCR.
- FIG. 5 Expression of T cell activation markers in PEP005-treated primary CD4+ T cells.
- CD4+ T cells were isolated from uninfected control subjects and treated with 12 nM PEP005 for 24 hrs. Total RNA was extracted, and gene expression of CD38, CD69, CD25, or HLA-DR was analyzed by RT-qPCR.
- B-F PBMCs isolated from peripheral blood of healthy HIV-negative donors were treated for 24 or 72 hours with 12 nM of PEP005, and the expression of CD38 (B), CD69 (C, D), and HLA-DR (E, F) was evaluated using flow cytometry after co-staining with the CD3 T cell marker.
- FIG. 6 PEP005 does not induce expression of pro-inflammatory cytokines in primary CD4+ T cells from peripheral blood of HIV-negative donors.
- CD4+ T cells were isolated from healthy donors and treated with 6 or 12 nM of PEP005 for 24 or 72 hours, and the relative expression of TNF- ⁇ (A), IFN- ⁇ (B), IL-2 (C), and IL-6 (D) was quantified using RT-qPCR and normalized to GAPDH internal control.
- FIG. 7 PEP005 causes minimal cytotoxicity and T cell proliferation.
- U1 cells (A), J-Lat A1 cells (B) and CD4+ T cells (C) were treated with 12 nM of PEP005 for 24 hours, the cell viability was examined with the MTT assay and the S-phase cell cycle progression was evaluated after BrdU incorporation using BrdU ELISA. 25-50 ⁇ M Etoposide or 100-150 ⁇ M Doxorubicin (Doxo) served as positive controls for MTT and BrdU assays respectively [48-50].
- Statistical analysis was performed in comparison with controls. **, p ⁇ 0.01.
- FIG. 8 PEP005 induces full-length HIV transcripts in primary CD4+ T cells from HIV infected individuals on suppressive ART.
- Primary CD4+ T cells were isolated from peripheral blood of HIV infected individuals on suppressive ART and treated with 6 or 12 nM PEP005, 200 ng/ml PMA plus 2 ⁇ M Ionomycin, or DMSO for 6 hours.
- Induction of HIV transcription was measured using RT-qPCR for the 5′ LTR region or Poly A region of the virus. **, p ⁇ 0.01.
- FIG. 9 PEP005 and JQ1 synergistically induce HIV transcription 6 hrs after treatment in primary CD4+ T cells from HIV infected individuals on suppressive ART.
- Primary CD4+ T cells were isolated from the peripheral blood of HIV positive individuals on suppressive ART and treated with 12 nM PEP005 alone or in combination with JQ1 for 6 hours, and HIV transcription was quantified using RT-qPCR for the 5′ LTR region or Poly A region of the virus. The amount of cDNA was only enough to amplify either Poly A region or LTR region during PCR in donor 1 and donor 9.
- FIG. 10 PEP005 and JQ1 synergistically induce HIV transcription 48 hrs after treatment in primary CD4+ T cells from HIV infected individuals on suppressive ART.
- Primary CD4+ T cells were isolated from the peripheral blood of HIV positive individuals on suppressive ART and treated with 12 nM PEP005 alone or in combination with JQ1 for 48 hours, and HIV transcription was quantified using RT-qPCR for the 5′ LTR region or Poly A region of the virus. The amount of cDNA was only enough to amplify LTR region during PCR in donor 9.
- FIG. 11 PEP005 and JQ1 synergistically induce HIV transcription in primary CD4+ T cells from HIV infected individuals on suppressive ART.
- Primary CD4+ T cells were isolated from the peripheral blood of HIV positive individuals on suppressive ART and treated with 12 nM PEP005 alone or in combination with JQ1 for 6 or 48 hours, and HIV transcription was quantified using RT-qPCR for the 5′ LTR region or Poly A region of the virus.
- FIG. 12 PEP005 down-modulates the expression of CD4, CCR5 and CXCR4 and inhibits HIV infection of primary CD4+ T cells ex vivo.
- A PEP005 inhibits expression of HIV receptors/co-receptors. The CD4+ T cells were isolated from the peripheral blood of HIV-negative uninfected controls and treated with 12 nM PEP005 for 72 hours. The expression of CD4, CCR5 and CXCR4 genes was evaluated using RT-qPCR after normalizing for GAPDH.
- the CD4+ T cells were incubated for 5 days without PEP005.
- the cells were collected for RT-qPCR targeting the HIV LTR region (B), or supernatants were collected for p24 ELISA (C).
- Stable latent viral reservoirs in HIV infected individuals are rapidly reactivated following the interruption of anti-retroviral therapy (ART).
- ART anti-retroviral therapy
- viral reservoirs are established and persist as demonstrated in the case of the Mississippi baby and from recent studies of the SIV model of AIDS. Therefore, new strategies are needed for the eradication of the latent HIV reservoirs.
- the present inventors have discovered that ingenol-3-angelate (PEP005), a member of a new class of anti-cancer ingenol compounds, effectively reactivated HIV from latency in primary CD4+ T cells from HIV infected individuals receiving ART.
- the terms “subject”, “patient” or “individual” are used herein interchangeably to include a human or animal.
- the animal subject may be a mammal, a primate (e.g., a monkey), a livestock animal (e.g., a horse, a cow, a sheep, a pig, or a goat), a companion animal (e.g., a dog, a cat), a laboratory test animal (e.g., a mouse, a rat, a guinea pig, a bird), an animal of veterinary significance, or an animal of economic significance.
- a primate e.g., a monkey
- livestock animal e.g., a horse, a cow, a sheep, a pig, or a goat
- a companion animal e.g., a dog, a cat
- a laboratory test animal e.g., a mouse, a rat, a guinea pig, a bird
- a “therapeutically effective amount” includes an amount or quantity effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
- administering includes oral administration, topical contact, administration as a suppository, intravenous, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal, or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to a subject.
- Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal).
- Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial.
- co-administer it is meant that a first compound described herein is administered at the same time, just prior to, or just after the administration of a second compound described herein, e.g., the sequential or simultaneous administration of ingenol-3-angelate (PEP005) and one or more latency reactivation agents.
- two or more compounds can be co-administered by administering a pharmaceutical composition adapted for oral administration that contains the two or more compounds.
- two or more compounds can be co-administered by administering one compound and then administering the other compound.
- the co-administered compounds are administered by the same route.
- the co-administered compounds are administered via different routes.
- one compound can be administered orally, and the other compound can be administered, e.g., sequentially or simultaneously, via intravenous or intramuscular injection.
- anti-retroviral therapy or “ART” include administering one or more compounds simultaneously or sequentially to reduce or suppress the replication of a virus within an infected individual. This can be achieved, for example, by administering various classes of anti-retroviral compounds to an infected individual that inhibit various steps of the replication cycle. These classes of compounds include, but are not limited to, fusion inhibitors, nucleoside reverse transcriptase inhibitors, integrase inhibitors, protease inhibitors, and combinations thereof.
- anti-retroviral therapy can be used to treat viral infections caused by human immunodeficiency virus (HIV), cytomegalovirus (CMV), or adenovirus.
- HIV human immunodeficiency virus
- CMV cytomegalovirus
- adenovirus adenovirus
- latent virus includes a state of the viral life cycle where the viral genome has integrated into the chromosomal DNA of the infected cell, but replication of the genome and viral proliferation in the infected cell is dormant. In some instances, the dormant viral replication impedes the treatment of viral infection with anti-retroviral therapy because anti-retroviral therapy targets various stages of the viral replication cycle. For example, latent HIV virus cannot be treated using anti-retroviral therapy, and is known problem inhibiting the functionality of anti-retroviral treatment techniques.
- latent reservoir includes a cell or group of cells that are infected with a latent virus such as, for example, latent HIV.
- reactivating a latent virus includes administering a compound or combination of compounds to induce a latent virus infected cell into an active replication and proliferation state.
- the term includes, inter alia, administering one or more latency reactivation agents described herein.
- latency reactivation agent or “latency reversing agent” include a compound or combination of compounds capable of reactivating a latent virus.
- the terms include a single compound that is capable of inducing reactivation of a latent virus.
- the terms also include a combination of two or more compounds that are capable of synergistically or additively inducing reactivation of a latent virus.
- Latency reactivation agents are useful for treating a variety of latent viral infections including, but not limited to, latent HIV, CMV, and adenoviral infections.
- Latency reactivation agents include, but are not limited to, ingenol-3-angelate (PEP005), as well as any compound belonging to a class selected from a positive transcription elongation factor b activator, a histone methyltransferase (HMT) inhibitor, a histone deacetylase (HDAC) inhibitor, a DNA methyltransferase inhibitor, an NF- ⁇ B activator, an Akt/HEXIM-1 modulator, a Jak/Stat pathway modulator, a diterpene compound (e.g., an ingenol derivative, a phorbol ester), a macrolide lactone, a diacylglycerol (DAG) lactone, a protein kinase C (PKC) activator, and combinations thereof.
- PEP005 ingenol-3-angelate
- HMT histone methyltransferase
- HDAC histone deacetylase
- DNA methyltransferase inhibitor e
- an effective amount includes a dosage sufficient to produce a desired result with respect to the indicated disorder, condition, or mental state.
- the desired result may comprise a subjective or objective improvement in the recipient of the dosage.
- an effective amount of PEP005 and JQ1 includes an amount sufficient to reactivate latent HIV expression.
- an effective amount includes an amount sufficient to suppress HIV infection of primary CD4+ T cells.
- an effective amount can be an amount that down-modulates cell surface expression of HIV co-receptors comprising CD4, CCR5, and/or CXCR4. The effective amount will vary with the type of subject being treated, the compound or combination of compounds applied, and/or the amount of anti-retroviral treatments administered to the subject.
- enhancing a therapeutic benefit includes any of a number of subjective or objective factors indicating a beneficial response or improvement of the condition being treated as discussed herein.
- enhancing the therapeutic benefit comprises administering two or more compounds to produce a desired clinical result greater than any one of the compounds administered separately.
- enhancing the therapeutic benefit comprises administering two or more latency reactivating agents to produce the desired clinical result greater than any one of the latency reactivating agents administered separately.
- enhancing the therapeutic benefit comprises administerting two or more latency reactivation agents in combination with a viral therapy vaccine to more effectively combat and kill reactivated virally infected cells.
- pharmaceutically acceptable includes a substance which does not interfere with the effectiveness or the biological activity of the active ingredients and which is not toxic to the hosts in the amounts used, and which hosts may be either humans or animals to which it is to be administered.
- low to minimal cytotoxicity includes measuring the toxic effect of an administered compound or compounds in host cells based on the amount of the compound(s) administered. Thus, low to minimal cytotoxicity can be an amount that is not toxic to the cells or at a level of toxicity that the majority of cells are unaffected.
- global T cell activation includes inducing a large scale immune response in a host organism. In some instances, global T cell activation could trigger a fatal systemic immune response.
- synergistic or “synergistically” include an enhanced therapeutic benefit or effect where a combination of two or more compounds produces a result that exceeds their expected additive effect.
- synergistic latency reactivation agents enhance latent viral reactivation to an extent that exceeds their expected additive effect.
- viral therapy vaccine includes compositions that are administered to a subject that increase the subject's response to a viral infection.
- a viral therapy vaccine can be administered to a subject to increase the subject's immune response to a viral infection.
- a viral therapy vaccine can be administered to a subject to increase the subject's ability to combat and kill virally infected cells that have been reactivated from their latent state.
- cytotoxic T lymphocytes (CTL) booster includes compositions comprising proteins, DNA, RNA, and/or other biologic or non-biologic matter that can be used to improve CTL response and proliferation to combat and kill virally infected cells that have been reactivated from their latent state.
- the present invention provides compositions comprising ingenol-3-angelate (PEP005) and one or more additional latency reactivation agents.
- the present invention also provides methods for reactivating a latent virus in a subject infected with the virus, the method comprising administering to the subject an effective amount of ingenol-3-angelate (PEP005) optionally in combination with one or more additional latency reactivation agents.
- a combination of ingenol-3-angelate (PEP005) with one or more additional latency reactivation agents advantageously provides a synergistic effect at inducing reactivation of a latent virus such as HIV.
- the present invention further provides kits comprising ingenol-3-angelate (PEP005) and one or more additional latency reactivation agents.
- the present invention provides a composition comprising ingenol-3-angelate (PEP005) and one or more additional latency reactivation agents.
- the ingenol-3-angelate (PEP005) is present in an amount that is effective to enhance a therapeutic benefit of the one or more additional latency reactivation agents.
- the therapeutic benefit comprises reactivation of a latent virus in a subject infected with the virus.
- the latent virus is present as viral reservoirs in virus-infected cells (e.g., HIV-infected resting CD4+ T cells).
- the virus is selected from the group consisting of a human immunodeficiency virus (HIV), a cytomegalovirus (CMV), adenoviruses, papovaviruses, herpesviruses, varicella-zoster virus, Epstein-Barr virus, pox viruses, vaccinia virus, hepatitis B virus, rhinoviruses, hepatitis A virus, poliovirus, rubellavirus, hepatitis C virus, arboviruses, rabiesvirus, influenza viruses A and B, measles virus, mumps virus, and HTLV I and II.
- the virus is selected from the group consisting of a human immunodeficiency virus (HIV), a cytomegalovirus (CMV), and an adenovirus.
- the one or more additional latency reactivation agents is selected from the group consisting of a positive transcription elongation factor b activator, a histone methyltransferase (HMT) inhibitor, a histone deacetylase (HDAC) inhibitor, a DNA methyltransferase inhibitor, an NF- ⁇ B activator, an Akt/HEXIM-1 modulator, a Jak/Stat pathway modulator, a diterpene compound (e.g., an ingenol derivative, a phorbol ester), a macrolide lactone, a diacylglycerol (DAG) lactone, a protein kinase C (PKC) activator, and combinations thereof.
- HMT histone methyltransferase
- HDAC histone deacetylase
- Non-limiting examples of other latency reactivation agents include juglone (5HN, 5-hydroxynaphthalene-1,4-dione), disulfiram, AV6 (4-3′,4′-dichloroanilino-6-methoxyquinoline), and combinations thereof.
- Non-limiting examples of positive transcription elongation factor b activators include JQ1, hexamethylene bisacetamide (HMBA), and combinations thereof.
- HMT histone methyltransferase
- HDAC histone deacetylase
- SAHA suberoyl bis-hydroxamic acid
- SBHA suberoyl bis-hydroxamic acid
- TsA trichostatin A
- ITF2357 oxamflatin
- PXD101 belinostat
- VPA valproic acid
- sodium butyrate sodium butyrate
- apicidin and combinations thereof.
- Non-limiting examples of DNA methyltransferase inhibitors include decitabine, azacitidine, and combinations thereof.
- Non-limiting examples of NF- ⁇ B activators include 12-deoxyphorbol-13-acetate (prostratin), phorbol myristate acetate (PMA), TNF ⁇ , and combinations ther
- Non-limiting examples of the Akt/HEXIM-1 modulator is hexamethylene bisacetamide (HMBA).
- a non-limiting example of a Jak/Stat pathway modulator includes IL-7.
- Non-limiting examples of diterpene compounds include an ingenol derivative, a phorbol ester, SJ23B, gnidimacrin, and combinations thereof.
- the ingenol derivative is an ester of the diterpene ingenol and an organic acid.
- the organic acid is a saturated or unsaturated optionally substituted C 1 -C 22 organic acid.
- the ingenol derivative is selected from the group consisting of ingenol-3-trans-cinnamate (ING A), ingenol-3-hexanoate (ING B), ingenol-3-dodecanoate (ING C), 3-(2,4,6-dodecatrienoyl)-ingenol, 3-(2,4,6,8-tetradecatetranoyl)-ingenol, 20-hydroxy-ingenol-3-angelate, 20-O-acetyl-ingenol-3-angelate, and combinations thereof.
- the ingenol derivative is any one of the compounds described in US 2015/0030638 and US 2003/0166613, the disclosures of which are hereby incorporated by reference in their entirety for all purposes.
- phorbol esters include phorbol myristate acetate (PMA), 12-deoxyphorbol-13-acetate (prostratin), 12-deoxyphorbol 13-phenylacetate (DPP), and combinations thereof.
- a non-limiting example of a macrolide lactone includes bryostatin.
- the one or more additional latency reactivation agents is a positive transcription elongation factor b activator.
- the positive transcription elongation factor b activator is JQ1, i.e., the composition comprises a combination of ingenol-3-angelate (PEP005) and JQ1.
- the one or more additional latency reactivation agents is a histone methyltransferase (HMT) inhibitor.
- HMT histone methyltransferase
- the histone methyltransferase (HMT) inhibitor is GSK343, i.e., the composition comprises a combination of ingenol-3-angelate (PEP005) and GSK343.
- the one or more additional latency reactivation agents is a histone deacetylase (HDAC) inhibitor.
- HDAC histone deacetylase
- the histone deacetylase (HDAC) inhibitor is vorinostat (suberanilohydroxamic acid; SAHA), i.e., the composition comprises a combination of ingenol-3-angelate (PEP005) and SAHA.
- the composition further comprises a viral therapy vaccine.
- the viral therapy vaccine is present in an amount effective to enhance a therapeutic benefit of the latency reactivation agents.
- the therapeutic benefit comprises increasing a subject's immune respone (e.g., cytotoxic T lymphocyte (CTL) response) by activating the proliferation of CTLs in the subject to more effectively combat and kill reactivated virally infected cells.
- CTL cytotoxic T lymphocyte
- the activated CTLs target viral proteins or fragments thereof that are presented on the surface of reactivated virally infected cells.
- Non-limiting examples of viral therapy vaccines include CTL boosters.
- Non-limiting examples of CTL boosters include viral proteins that are presented on the surface of infected cells, such as B Gag, Nef, Rev, Tat, and/or Env peptides.
- the composition further comprises a pharmaceutically acceptable excipient or diluent.
- the present invention provides a method for reactivating a latent virus in a subject infected with the virus, the method comprising administering to the subject an effective amount of ingenol-3-angelate (PEP005).
- PEP005 ingenol-3-angelate
- the effective amount of the ingenol-3-angelate (PEP005) is an amount that is capable of inducing RNA transcription from the latent virus in an infected cell (e.g., CD4+ T cell) from the subject.
- the latent virus is present as viral reservoirs in virus-infected cells (e.g., HIV-infected resting CD4+ T cells).
- the effective amount of the ingenol-3-angelate is an amount that displays low to minimal cytotoxicity without inducing global T cell activation (e.g., in the absence of major global T cell activation).
- the effective amount of the ingenol-3-angelate (PEP005) is an amount that suppresses or prevents the reactivated latent virus from infecting uninfected CD4+ T cells in the subject.
- the effective amount of the ingenol-3-angelate (PEP005) down-modulates the expression of cell surface receptors that are known to mediate viral (e.g., HIV) attachment and entry, thereby preventing the spread of viral infection to uninfected bystander CD4+ T cells.
- the ingenol-3-angelate (PEP005) is administered orally, topically, intravenously, intraperitoneally, intramuscularly, intralesionally, intrathecally, intranasally, subcutaneously, parenterally, or transmucosally.
- the method of the present invention further comprises administering one or more additional latency reactivation agents selected from the group consisting of a positive transcription elongation factor b activator, a histone methyltransferase (HMT) inhibitor, a histone deacetylase (HDAC) inhibitor, a DNA methyltransferase inhibitor, an NF- ⁇ B activator, an Akt/HEXIM-1 modulator, a Jak/Stat pathway modulator, a diterpene compound (e.g., an ingenol derivative, a phorbol ester), a macrolide lactone, a diacylglycerol (DAG) lactone, a protein kinase C (PKC) activator, and combinations thereof.
- HMT histone methyltransferase
- HDAC histone deacetylase
- Non-limiting examples of other latency reactivation agents include juglone (5HN, 5-hydroxynaphthalene-1,4-dione), disulfiram, AV6 (4-3′,4′-dichloroanilino-6-methoxyquinoline), and combinations thereof.
- Non-limiting examples of positive transcription elongation factor b activators include JQ1, hexamethylene bisacetamide (HMBA), and combinations thereof.
- HMT histone methyltransferase
- HDAC histone deacetylase
- SAHA suberoyl bis-hydroxamic acid
- SBHA suberoyl bis-hydroxamic acid
- TsA trichostatin A
- ITF2357 oxamflatin
- PXD101 belinostat
- VPA valproic acid
- sodium butyrate sodium butyrate
- apicidin and combinations thereof.
- Non-limiting examples of DNA methyltransferase inhibitors include decitabine, azacitidine, and combinations thereof.
- Non-limiting examples of NF- ⁇ B activators include 12-deoxyphorbol-13-acetate (prostratin), phorbol myristate acetate (PMA), TNF ⁇ , and combinations ther
- Non-limiting examples of the Akt/HEXIM-1 modulator is hexamethylene bisacetamide (HMBA).
- a non-limiting example of a Jak/Stat pathway modulator includes IL-7.
- Non-limiting examples of diterpene compounds include an ingenol derivative, a phorbol ester, SJ23B, gnidimacrin, and combinations thereof.
- the ingenol derivative is an ester of the diterpene ingenol and an organic acid.
- the organic acid is a saturated or unsaturated optionally substituted C 1 -C 22 organic acid.
- the ingenol derivative is selected from the group consisting of ingenol-3-trans-cinnamate (ING A), ingenol-3-hexanoate (ING B), ingenol-3-dodecanoate (ING C), 3-(2,4,6-dodecatrienoyl)-ingenol, 3-(2,4,6,8-tetradecatetranoyl)-ingenol, 20-hydroxy-ingenol-3-angelate, 20-O-acetyl-ingenol-3-angelate, and combinations thereof.
- the ingenol derivative is any one of the compounds described in US 2015/0030638 and US 2003/0166613, the disclosures of which are hereby incorporated by reference in their entirety for all purposes.
- phorbol esters include phorbol myristate acetate (PMA), 12-deoxyphorbol-13-acetate (prostratin), 12-deoxyphorbol 13-phenylacetate (DPP), and combinations thereof.
- a non-limiting example of a macrolide lactone includes bryostatin.
- the method of the present invention further comprises administering one or more additional latency reactivation agents comprising a positive transcription elongation factor b activator.
- the positive transcription elongation factor b activator is JQ1, i.e., the method comprises co-administering a combination of ingenol-3-angelate (PEP005) and JQ1.
- the method of the present invention further comprises administering one or more additional latency reactivation agents comprising a histone methyltransferase (HMT) inhibitor.
- HMT histone methyltransferase
- the positive transcription elongation factor b activator is GSK343, i.e., the method comprises co-administering a combination of ingenol-3-angelate (PEP005) and GSK343.
- the method of the present invention further comprises administering one or more additional latency reactivation agents comprising a histone deacetylase (HDAC) inhibitor.
- HDAC histone deacetylase
- the positive transcription elongation factor b activator is vorinostat (suberanilohydroxamic acid; SAHA), i.e., the method comprises co-administering a combination of ingenol-3-angelate (PEP005) and SAHA.
- a combination of the ingenol-3-angelate (PEP005) and the one or more additional latency reactivation agents is capable of synergistically inducing reactivation of the latent virus.
- the one or more additional latency reactivation agents is each independently administered orally, topically, intravenously, intraperitoneally, intramuscularly, intralesionally, intrathecally, intranasally, subcutaneously, parenterally, or transmucosally.
- the method comprises administering an effective amount of ingenol-3-angelate (PEP005) alone or with one or more additional latency reactivation agents for reactivating a latent virus in a virally infected cell in a subject infected with the virus in combination with administering an effective amount of a viral therapy vaccine for increasing a subject's immune respone (e.g., CTL response) to more effectively combat and kill the reactivated virally infected cell.
- PEP005 ingenol-3-angelate
- additional latency reactivation agents for reactivating a latent virus in a virally infected cell in a subject infected with the virus in combination with administering an effective amount of a viral therapy vaccine for increasing a subject's immune respone (e.g., CTL response) to more effectively combat and kill the reactivated virally infected cell.
- the effective amount of the viral therapy vaccine is an amount that is capable of increasing the subject's immune respone (e.g., cytotoxic T lymphocyte (CTL) response) by activating the proliferation of CTLs in the subject to more effectively combat and kill reactivated virally infected cells.
- the activated CTLs target viral proteins or fragments thereof that are presented on the surface of reactivated virally infected cells.
- the effective amount of the viral therapy vaccine is an amount that displays killing of virally infected cells that have been reactivated from their latent state.
- Non-limiting examples of viral therapy vaccines include cytotoxic T lymphocytes (CTL) boosters.
- CTL boosters include viral proteins that are presented on the surface of infected cells, such as B Gag, Nef, Rev, Tat, and/or Env peptides.
- the method comprises co-administering a combination of ingenol-3-angelate (PEP005), JQ1, and a cytotoxic T lymphocyte (CTL) booster.
- the method comprises co-administering a combination of ingenol-3-angelate (PEP005), GSK343, and a CTL booster.
- the method comprises co-administering a combination of ingenol-3-angelate (PEP005), vorinostat (suberanilohydroxamic acid; SAHA), and a CTL booster.
- a combination of ingenol-3-angelate (PEP005) with one or more additional latency reactivation agents (e.g., JQ1, GSK343, and/or SAHA) and a viral therapy vaccine is capable of synergistically inducing reactivation of the latent virus and increasing an immune response to reactivated virally infected cells.
- PEP005 ingenol-3-angelate
- additional latency reactivation agents e.g., JQ1, GSK343, and/or SAHA
- a viral therapy vaccine is capable of synergistically inducing reactivation of the latent virus and increasing an immune response to reactivated virally infected cells.
- the viral therapy vaccine is administered before ingenol-3-angelate (PEP005) and/or the one or more additional latency reactivation agents is administered.
- the viral therapy vaccine is administered orally, topically, intravenously, intraperitoneally, intramuscularly, intralesionally, intrathecally, intranasally, subcutaneously, parenterally, or transmucosally.
- the virus is selected from the group consisting of a human immunodeficiency virus (HIV), a cytomegalovirus (CMV), adenoviruses, papovaviruses, herpesviruses, varicella-zoster virus, Epstein-Barr virus, pox viruses, vaccinia virus, hepatitis B virus, rhinoviruses, hepatitis A virus, poliovirus, rubellavirus, hepatitis C virus, arboviruses, rabiesvirus, influenza viruses A and B, measles virus, mumps virus, and HTLV I and II.
- the virus is selected from the group consisting of a human immunodeficiency virus (HIV), a cytomegalovirus (CMV), and an adenovirus.
- the subject is a human.
- the subject is receiving anti-retroviral therapy (ART), e.g., highly active anti-retroviral therapy (HAART), suppressive anti-retroviral therapy, and the like.
- ART anti-retroviral therapy
- HAART highly active anti-retroviral therapy
- suppressive anti-retroviral therapy and the like.
- the present invention provides a kit comprising: (a) ingenol-3-angelate (PEP005); and (b) one or more additional latency reactivation agents.
- the kit further comprises a label with instructions for administering the ingenol-3-angelate (PEP005) and/or the one or more additional latency reactivation agents.
- the one or more additional latency reactivation agents is selected from the group consisting of a positive transcription elongation factor b activator, a histone methyltransferase (HMT) inhibitor, a histone deacetylase (HDAC) inhibitor, a DNA methyltransferase inhibitor, an NF- ⁇ B activator, an Akt/HEXIM-1 modulator, a Jak/Stat pathway modulator, a diterpene compound (e.g., an ingenol derivative, a phorbol ester), a macrolide lactone, a diacylglycerol (DAG) lactone, a protein kinase C (PKC) activator, and combinations thereof.
- HMT histone methyltransferase
- HDAC histone deacetylase
- the one or more additional latency reactivation agents is a positive transcription elongation factor b activator.
- the positive transcription elongation factor b activator is JQ1, i.e., the kit comprises a combination of ingenol-3-angelate (PEP005) and JQ1.
- the one or more additional latency reactivation agents is a histone methyltransferase (HMT) inhibitor.
- HMT histone methyltransferase
- the positive transcription elongation factor b activator is GSK343, i.e., the kit comprises a combination of ingenol-3-angelate (PEP005) and GSK343.
- the one or more additional latency reactivation agents is a histone deacetylase (HDAC) inhibitor.
- HDAC histone deacetylase
- the positive transcription elongation factor b activator is vorinostat (suberanilohydroxamic acid; SAHA), i.e., the kit comprises a combination of ingenol-3-angelate (PEP005) and SAHA.
- the kit further comprises a viral therapy vaccine.
- viral therapy vaccines include cytotoxic T lymphocytes (CTL) boosters.
- CTL boosters include viral proteins that are presented on the surface of infected cells, such as B Gag, Nef, Rev, Tat, and/or Env peptides.
- the kit comprises a combination of ingenol-3-angelate (PEP005), JQ1, and a cytotoxic T lymphocytes (CTL) booster.
- the kit comprises a combination of ingenol-3-angelate (PEP005), GSK343, and a CTL booster.
- the kit comprises a combination of ingenol-3-angelate (PEP005), vorinostat (suberanilohydroxamic acid; SAHA), and a CTL booster.
- the present invention provides compositions and kits comprising ingenol-3-angelate (PEP005) as a latency reactivation agent and one or more additional latency reactivation agents.
- the present invention also provides methods for reactivating a latent virus by administering an effective amount of ingenol-3-angelate (PEP005) as a latency reactivation agent alone or in combination with one or more additional latency reactivation agents.
- Non-limiting examples of additional latency reactivation agents include positive transcription elongation factor b activators, histone methyltransferase (HMT) inhibitors, histone deacetylase (HDAC) inhibitors, DNA methyltransferase inhibitors, NF- ⁇ B activators, Akt/HEXIM-1 modulators, Jak/Stat pathway modulators, diterpene compounds (e.g., ingenol derivatives, phorbol esters), macrolide lactones, diacylglycerol (DAG) lactones, protein kinase C (PKC) activators, juglone (5HN, 5-hydroxynaphthalene-1,4-dione), disulfiram, AV6 (4-3′,4′-dichloroanilino-6-methoxyquinoline), pharmaceutically acceptable salts thereof, derivatives thereof, and combinations thereof.
- HMT histone methyltransferase
- HDAC histone deacetylase
- Ingenol-3-angelate also known as ingenol mebutate, PEP005, or PICATO®, is a natural product found in the plant Euphorbia peplus .
- Ingenol-3-angelate is an ester of the diterpene ingenol and angelic acid.
- ingenol-3-angelate is (1aR,2S,5R,5aS,6S,8aS,9R,1 OaR)-5,5a-Dihydroxy-4-(hydroxymethyl)-1,1,7,9-tetramethyl-11-oxo-1a,2,5,5a,6,9,10,10a-octahydro-1H-2,8a-methanocyclopenta[a]cyclpropa[e][10]annulen-6-yl (2Z)-2-methylbut-2-enoate.
- the structure of ingenol-3-angelate is shown below:
- Positive transcription elongation factor b also known as P-TEFb
- P-TEFb plays an essential role in the regulation of transcription by RNA polymerase II (Pol II) in eukaryotes.
- RNA polymerase II RNA polymerase II
- P-TEFb is targeted by the HIV Tat protein which bypasses normal cellular P-TEFb control and directly brings P-TEFb to the promoter proximal paused polymerase in the HIV genome.
- activators of P-TEFb are capable of binding to, stimulating, increasing, activating, facilitating, enhancing activation or enzymatic activity, sensitizing or upregulating the activity or expression of P-TEFb.
- activators of P-TEFb are capable of increasing, enhancing, or upregulating the expression of an mRNA that encodes P-TEFb.
- positive transcription elongation factor b activators include JQ1, hexamethylene bisacetamide (HMBA), and combinations thereof.
- the positive transcription elongation factor b activator is JQ1.
- the full IUPAC chemical name of JQ1 is (S)-tert-butyl 2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate.
- JQ1 is an inhibitor of the bromo and extra terminal (BET) family of bromodomain proteins. BET proteins are key mediators for the assembly of the P-TEFb complex. The structure of JQ1 is shown below:
- HMT Histone Methyltransferase
- Histone methyltransferases are histone-modifying enzymes that catalyze the transfer of methyl groups to lysine and arginine residues of histone proteins.
- HMTs play an important biological role in the epigenetic modification of chromatin that directly impacts cellular activities including gene expression, DNA methylation, and genomic stability.
- Methylation of histone protein H3 at lysines 9 and 27 has been implicated in HIV latency (see, Shirakawa et al., Trends Microbiol, 21(6):277-285 (2013)).
- inhibitors of HMTs are capable of binding to, inhibiting, partially or totally blocking stimulation or enzymatic activity, decreasing, preventing, delaying activation, inactivating, desensitizing, or down regulating the activity or expression of HMTs. In certain other embodiments, inhibitors of HMTs are capable of decreasing, down regulating, or inhibiting the expression of an mRNA that encodes HMT.
- HMT inhibitors include GSK343, BIX01294, chaetocin, 3-deazaneplanocin A (DZNep), G9a, and combinations thereof.
- the HMT inhibitor is GSK343.
- GSK343 is an inhibitor of EZH2, which is a histone H3-lysine 27 methyltransferase. Inhibition of EZH2 has been implicated in reactivating latent HIV-infected cells.
- the chemical structure of GSK343 is shown below:
- HDAC Histone Deacetylase
- Histone deacetylases also known as HDACs, are enzymes that remove acetyl groups from N-acetylated lysine amino acids on histone proteins. This process allows the histone proteins to more tightly wrap around DNA molecules. HDAC inhibitors have been shown to induce reactivation of virally infected latent cells, including herpes simples virus and HIV (see, Danaher et al., J Neurovirol, 11(3):306-17 (2005) and Shirakawa et al., Trends Microbiol, 21(6):277-85 (2013)).
- inhibitors of HDACs are capable of binding to, inhibiting, partially or totally blocking stimulation or enzymatic activity, decreasing, preventing, delaying activation, inactivating, desensitizing, or down regulating the activity or expression of HDACs. In certain other embodiments, inhibitors of HDACs are capable of decreasing, down regulating, or inhibiting the expression of an mRNA that encodes HDAC.
- HDAC inhibitors include, but are not limited to, vorinostat, suberoyl bis-hydroxamic acid (SBHA), trichrostatin A (TsA), scriptaid, oxamflatin, givinostat (ITF2357), belinostat (PXD101), droxinostat, CG05/CGO6, valproic acid (VPA), sodium butyrate, apicidin, and combinations thereof.
- the HDAC inhibitor is vorinostat.
- Vorinostat is also known as suberanilohydroxamic acid, SAHA, or ZOLINZA®.
- SAHA suberanilohydroxamic acid
- ZOLINZA® ZOLINZA®
- the full IUPAC chemical name for SAHA is N-Hydroxy-N′-phenyloctanediamide.
- the chemical structure of vorinostat is shown below:
- DNA methyltransferases are enzymes that transfer a methyl group to DNA.
- DNA methylation typically acts to suppress gene transcription, and methylation patterns play an important role in epigenetics.
- inhibitors of DNA methyltransferas can directly alter cellular gene expression. Indeed, DNA methyltransfase inhibitors, in altering the gene expression pattern, can induce reactivation of HIV infected cells (see, Blazkova et al., PLoS Pathog, 5(8):e1000554 (2009)).
- inhibitors of DNA methyltransferases are capable of binding to, inhibiting, partially or totally blocking stimulation or enzymatic activity, decreasing, preventing, delaying activation, inactivating, desensitizing, or down regulating the activity or expression of DNA methyltransferases.
- inhibitors of DNA methyltransferases are capable of decreasing, down regulating, or inhibiting the expression of an mRNA that encodes DNA methyltransferase.
- Non-limiting examples of DNA methyltransferases include decitabine, azacitidine, and combinations thereof.
- NF- ⁇ B is a protein complex that controls, among other things, transcription of cellular DNA; thus, it is a transcription factor. In some instances, activated NF- ⁇ B binds to DNA-binding sites where expression of specific genes is turned on. Several viruses, including HIV, have binding sites of NF- ⁇ B that control the expression of viral genes. NF- ⁇ B activation has been implicated in the reactivation of infected cells from their latent state (see, Hiscott et al., J. Clin. Invest., 107(2):143-151 (2001)).
- activators of NF- ⁇ B are capable of binding to, stimulating, increasing, activating, facilitating, enhancing activation or enzymatic activity, sensitizing or upregulating the activity or expression of NF- ⁇ B or members of the NF- ⁇ B signaling pathway.
- activators of NF- ⁇ B are capable of increasing, enhancing, or upregulating the expression of an mRNA that encodes NF- ⁇ B or members of the NF- ⁇ B signaling pathway.
- Non-limiting examples of NF- ⁇ B activators include 12-deoxyphorbol-13-acetate (prostratin), phorbol myristate acetate (PMA), TNF ⁇ , and combinations thereof.
- Non-phosphorylated HEXIM-1 in combination with other cellular factors including 7SK snRNA, forms a complex with positive transcription elongation factor b (P-TEFb) that sequesters P-TEFb and represses PTEFb activity.
- P-TEFb positive transcription elongation factor b
- PI3K/Akt phosphoinositide 3-kinase/RAC-alpha serine/threonine-protein kinase
- modulators of Akt/HEXIM-1 are capable of binding to, stimulating, increasing, activating, facilitating, enhancing activation or enzymatic activity, sensitizing or upregulating the activity or expression of members of the PI3K/Akt pathway.
- modulators of Akt/HEXIM-1 are capable of increasing, enhancing, or upregulating the expression of an mRNA that encodes members of the PI3K/Akt pathway.
- Non-limiting examples of Akt/HEXIM-1 modulators include hexamethylene bisacetamide (HMBA).
- the Jak/Stat pathway is a transcription regulation pathway that transmits information from chemical signals outside the cell to gene promoters on the DNA in the cell nucleus.
- the pathway is made up of three components, a cellular membrane receptor, a Janus kinase (Jak), and a signal transducer and activator of transcription (Stat).
- the receptor is activated by a signal from a chemical messenger, thereby activating the kinase function of Jak, which then autophosphorylates itself.
- the STAT protein then binds to the phosphorylated receptor, where STAT is phosphorylated by JAK.
- the phosphorylated STAT protein binds to another phosphorylated STAT protein (dimerizes) and translocates into the cell nucleus.
- Jak/Stat pathway modulators are capable of binding to, stimulating, increasing, activating, facilitating, enhancing activation or enzymatic activity, sensitizing or upregulating the activity or expression of members of the Jak/Stat pathway.
- modulators of the Jak/Stat pathway are capable of increasing, enhancing, or upregulating the expression of an mRNA that encodes members of the Jak/Stat pathway.
- Jak/Stat pathway modulators include IL-7.
- Diterpenes are a class of organic compounds based off of four isoprene units which are derived from genanylgeraniol pyrophosphate, and most diterpenes are of fungal or plant origin. In some instances, diterpenes have been shown to activate the protein kinase C (PKC) pathway. In certain embodiments, diterpenes are capable modulating the PKC pathway by binding to, stimulating, increasing, activating, facilitating, enhancing activation or enzymatic activity, sensitizing or upregulating the activity or expression of the members of the PKC family. In certain other embodiments, diterpenes are capable modulating the PKC pathway by increasing, enhancing, or upregulating the expression of an mRNA that encodes members of the PKC family. Non-limiting examples of diterpenes include ingenol derivatives, phorbol esters, SJ23B, gnidimacrin, and combinations thereof.
- the ingenol derivative is an ester of the diterpene ingenol and an organic acid.
- the organic acid is a saturated or unsaturated optionally substituted C 1 -C 22 organic acid.
- the ingenol derivative is selected from the group consisting of ingenol-3-trans-cinnamate (ING A), ingenol-3-hexanoate (ING B), ingenol-3-dodecanoate (ING C), 3-(2,4,6-dodecatrienoyl)-ingenol, 3-(2,4,6,8-tetradecatetranoyl)-ingenol, 20-hydroxy-ingenol-3-angelate, 20-O-acetyl-ingenol-3-angelate, and combinations thereof.
- the ingenol derivative is any one of the compounds described in US 2015/0030638 and US 2003/0166613, the disclosures of which are hereby incorporated by reference in their entirety for all purposes.
- Phorbol esters are tetracyclic members of the diterpene family. Phorbol is the parent diterpene of phorbol esters. Biologically, phorbol esters mimic the action of diacylglycerol, an activator of protein kinase C (PKC). In certain embodiments, phorbol esters are capable modulating the PKC pathway by binding to, stimulating, increasing, activating, facilitating, enhancing activation or enzymatic activity, sensitizing or upregulating the activity or expression of the members of the PKC family. In certain other embodiments, phorbol esters are capable modulating the PKC pathway by increasing, enhancing, or upregulating the expression of an mRNA that encodes members of the PKC family. Non-limiting examples of phorbol esters include phorbol myristate acetate (PMA), 12-deoxyphorbol-13-acetate (prostratin), 12-deoxyphorbol 13-phenylacetate (DPP), and combinations thereof.
- PMA phorbol myristate acetate
- Macrolide lactones are organic compounds that contain a large macrocyclic lactone ring.
- the lactone rings of macrolide lactones are usually 14, 15, or 16-membered.
- macrolide lactones such as bryostatin and analogs thereof have been shown to activate the protein kinase C (PKC) pathway and can induce HIV reactivation in latently infected cells as described in DeChristopher et al., Nature Chemistry, 4, 705-710 (2012).
- macrolide lactones are capable of modulating the PKC pathway by binding to, stimulating, increasing, activating, facilitating, enhancing activation or enzymatic activity, sensitizing or upregulating the activity or expression of members of the PKC family.
- macrolide lactones are capable modulating the PKC pathway by increasing, enhancing, or upregulating the expression of an mRNA that encodes members of the PKC pathway.
- macrolide lactones include bryostatin.
- Diacylglycerol (DAG) lactones are effective modulators of critical cellular signaling pathways.
- Diacylglycerol (DAG) is a known activator of protein kinase C (PKC) and the PKC pathway, and DAG lactones are structural mimetics of DAG.
- PKC protein kinase C
- DAG lactones are also capable of activating the PKC pathway and can induce HIV reactivation in latently infected cells.
- DAG lactones are capable of modulating the PKC pathway by binding to, stimulating, increasing, activating, facilitating, enhancing activation or enzymatic activity, sensitizing or upregulating the activity or expression of members of the PKC family.
- DAG lactones are capable modulating the PKC pathway by increasing, enhancing, or upregulating the expression of an mRNA that encodes members of the PKC pathway.
- DAG lactones are described in Hamer et al. ( J. Virology, 77(19):10227-10236 (2003)).
- PKC Protein Kinase C
- Protein kinase C also known as PKC, is a family of protein kinase enzymes that are involved in controlling the function of many other proteins and cellular processes by phosphorylating the hydroxyl groups of serine and threonine amino acid residues of these proteins. Activation of PKC has many downstream effects, one of them being NF- ⁇ B activation, which can lead to HIV reactivation in latently infected cells. PKC activity is activated by, among other things, diacylglycerol (DAG) or DAG mimetics. In certain embodiments, activators of PKC are capable of binding to, stimulating, increasing, activating, facilitating, enhancing activation or enzymatic activity, sensitizing or upregulating the activity or expression of PKC.
- DAG diacylglycerol
- activators of PKC are capable of binding to, stimulating, increasing, activating, facilitating, enhancing activation or enzymatic activity, sensitizing or upregulating the activity or expression of PKC.
- activators of PKC are capable of increasing, enhancing, or upregulating the expression of an mRNA that encodes PKC.
- PKC activators include diterpenes, phorbol esters, macrolide lactones, and DAG lactones such as those compounds described herein.
- the present invention provides compositions and kits comprising ingenol-3-angelate (PEP005) alone or with one or more additional latency reactivation agents to reactivate latent virally infected cells in combination with a viral therapy vaccine to increase a subject's immune respone (e.g., cytotoxic T lymphocyte (CTL) response) to more effectively combat and kill the reactivated virally infected cells.
- PEP005 ingenol-3-angelate
- additional latency reactivation agents to reactivate latent virally infected cells in combination with a viral therapy vaccine to increase a subject's immune respone (e.g., cytotoxic T lymphocyte (CTL) response) to more effectively combat and kill the reactivated virally infected cells.
- CTL cytotoxic T lymphocyte
- the present invention provides methods for reactivating a latent virus in a virally infected cell and increasing a subject's immune respone (e.g., CTL response) to more effectively combat and kill the reactivated virally infected cells comprising administering ingenol-3-angelate (PEP005) as a latency reactivation agent alone or with one or more additional latency reactivation agents in combination a viral therapy vaccine.
- a subject's immune respone e.g., CTL response
- Non-limiting examples of viral therapy vaccines include cytotoxic T lymphocytes (CTL) boosters.
- CTL cytotoxic T lymphocytes
- Non-limiting examples of cytotoxic T lymphocytes (CTL) boosters include viral proteins that are presented on the surface of infected cells. In HIV, those proteins include B Gag, Nef, Rev, Tat, Env, Pol, Vpr, Vpu, and Vif peptides.
- Other non-limiting examples of CTL boosters include viral DNA, RNA, and other biologic or non-biologic material.
- Cytotoxic T lymphocytes are part of the adaptive immune response, and play a critical role in fighting viral infections. CTLs recognize and bind infected cells using T-cell receptors (TCR). TCRs contain a highly variable binding region that allow them to recognize a large range of antigens. TCRs bind to the major histocompatibility complex I (MHC I) of infected cells presenting an appropriate antigen. TCRs binding is highly specific, so only a small number of CTLs will be able to recognize a particular antigen. Once an antigen is recognized by CTLs binding to the MHC I complex of the infected cell, they activate to induce cellular death. Activated CTLs proliferate to fight the detected infection.
- MHC I major histocompatibility complex I
- CTL response is limited by viral mutational rate.
- the high mutational rate in the proteins expressed on the cellular MHC complex creates ‘escape mutations’ that can avoid detection of activated CTLs, making the viral infection difficult to irradicate.
- the use of a viral therapy vaccine to induce and boost CTL response to better combat the infection, particularly in latently reactivated virally infected cells, is provided herein.
- viral therapy vaccines stimulate CTL response to more effectively combat and kill the reactivated virally infected cells.
- CTLs are stimulated with a mixture of consensus HIV peptides known to be exhibited on the MHC I complex of infected cells.
- a pharmaceutical composition or medicament can be administered to a subject for the treatment of a viral infection such as an HIV infection that leads to the reduction or elimination of the virus from latent reservoirs in infected resting cells such as central and/or transitional memory CD4+ T cells.
- a pharmaceutical composition or medicament can be administered to a subject for the treatment of a viral infection such as an HIV infection that leads to the reactivation of virally infected cells from latent reservoirs and activation of the subject's immune system to combat and kill the recently reactivated virally infected cells.
- compositions or medicaments for use in the present invention can be formulated by standard techniques or methods well-known in the art of pharmacy using one or more physiologically acceptable carriers or excipients. Suitable pharmaceutical carriers are described herein and in, e.g., “Remington's Pharmaceutical Sciences” by E. W. Martin.
- Compounds and agents of the present invention and their physiologically acceptable salts and solvates can be formulated for administration by any suitable route, including, but not limited to, orally, topically, nasally, rectally, pulmonary, parenterally (e.g., intravenously, subcutaneously, intramuscularly, etc.), and combinations thereof.
- the therapeutic agent is dissolved in a liquid, for example, water.
- the most suitable route of administration in any given case will depend in part on the nature, severity, and optionally, the stage of the viral infection.
- Co-administration of a plurality or combination of compounds may be by the same or different route of administration or together in the same pharmaceutical formulation.
- compositions of the present invention can include ingenol-3-angelate (PEP005) in combination with positive transcription elongation factor b activators, histone methyltransferase (HMT) inhibitors, histone deacetylase (HDAC) inhibitors, DNA methyltransferase inhibitors, NF- ⁇ B activators, Akt/HEXIM-1 modulators, Jak/Stat pathway modulators, diterpene compounds (e.g., ingenol derivatives, phorbol esters), macrolide lactones, diacylglycerol (DAG) lactones, protein kinase C (PKC) activators, and combinations thereof, or any pharmaceutically acceptable salts thereof, as an active ingredient and a pharmaceutically acceptable carrier and/or excipient or diluent.
- PEP005 ingenol-3-angelate
- HMT histone methyltransferase
- HDAC histone deacetylase
- DNA methyltransferase inhibitors NF
- the pharmaceutical composition can include ingenol-3-angelate (PEP005) in combination with JQ1, GSK343, SAHA, and combinations thereof.
- the pharmaceutical composition can include ingenol-3-angelate (PEP005) in combination with one or more latency reactivation agents (e.g., JQ1, GSK343, and/or SAHA) and one or more viral therapy vaccines (e.g., a CTL booster).
- a pharmaceutical composition of the invention may optionally contain additional therapeutic ingredients, e.g., one or more compounds for use in anti-retroviral therapy.
- the compounds of the present invention can be combined as the active ingredient in intimate admixture with a suitable pharmaceutical carrier and/or excipient according to conventional pharmaceutical compounding techniques. Any carrier and/or excipient suitable for the form of preparation desired for administration is contemplated for use with the compounds disclosed herein.
- the pharmaceutical compositions are suitable for systemic administration.
- Systemic administration includes enteral administration (e.g., absorption of the compound through the gastrointestinal tract) or parenteral administration (e.g., injection, infusion, or implantation).
- parenteral administration e.g., injection, infusion, or implantation.
- the compositions may be administered via a syringe or intravenously.
- the present invention provides a pharmaceutical composition including ingenol-3-angelate (PEP005) in combination with a positive transcription elongation factor b activator, such as JQ1, and a pharmaceutically acceptable excipient.
- the present invention provides a pharmaceutical composition including ingenol-3-angelate (PEP005) in combination with a histone methyltransferase (HMT) inhibitor, such as GSK343, and a pharmaceutically acceptable excipient.
- HMT histone methyltransferase
- the present invention provides a pharmaceutical composition including ingenol-3-angelate (PEP005) and a histone deacetylase (HDAC) inhibitor, such as SAHA, and a pharmaceutically acceptable excipient.
- HDAC histone deacetylase
- the present invention provides a pharmaceutical composition including ingenol-3-angelate (PEP005) in combination with one or more latency reactivation agents (e.g., JQ1, GSK343, and/or SAHA), one or more viral therapy vaccines (e.g., a CTL booster), and a pharmaceutically acceptable excipient.
- the pharmaceutically acceptable excipient includes a salt or a diluent.
- a pharmaceutical composition or a medicament can take the form of, e.g., a tablet or a capsule prepared by conventional means with a pharmaceutically acceptable excipient.
- Liquid preparations for oral administration can take the form of, for example, solutions, syrups, or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use.
- Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives, for example, suspending agents, for example, sorbitol syrup, cellulose derivatives, or hydrogenated edible fats; emulsifying agents, for example, lecithin or acacia; non-aqueous vehicles, for example, almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils; and preservatives, for example, methyl or propyl-p-hydroxybenzoates or sorbic acid.
- the preparations can also contain buffer salts, flavoring, coloring, and/or sweetening agents as appropriate. If desired, preparations for oral administration can be suitably formulated to give controlled release of the active compound.
- Typical formulations for topical administration include creams, ointments, sprays, lotions, and patches.
- the pharmaceutical composition can, however, be formulated for any type of administration, e.g., intradermal, subdermal, intravenous, intramuscular, intranasal, intracerebral, intratracheal, intraarterial, intraperitoneal, intravesical, intrapleural, intracoronary or intratumoral injection, with a syringe or other devices.
- Formulation for administration by inhalation e.g., aerosol
- oral, rectal, or vaginal administration is also contemplated.
- compositions for pulmonary administration include, but are not limited to, dry powder compositions consisting of the powder of a compound described herein, or a salt thereof, and the powder of a suitable carrier and/or lubricant.
- the compositions for pulmonary administration can be inhaled from any suitable dry powder inhaler device known to a person skilled in the art.
- the compositions may be conveniently delivered in the form of an aerosol spray from pressurized packs or a nebulizer, with the use of a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas.
- the dosage unit can be determined by providing a valve to deliver a metered amount.
- Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound(s) and a suitable powder base, for example, lactose or starch.
- the compounds can also be formulated in rectal compositions, for example, suppositories or retention enemas, for example, containing conventional suppository bases, for example, cocoa butter or other glycerides.
- compositions set forth herein can be formulated for parenteral administration by injection, for example by bolus injection or continuous infusion.
- Formulations for injection can be presented in unit dosage form, for example, in ampoules or in multi-dose containers, with an added preservative.
- Injectable compositions are preferably aqueous isotonic solutions or suspensions, and suppositories are preferably prepared from fatty emulsions or suspensions.
- the compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers.
- the compound(s) can be in powder form for reconstitution with a suitable vehicle, for example, sterile pyrogen-free water, before use.
- a suitable vehicle for example, sterile pyrogen-free water
- they may also contain other therapeutically valuable substances.
- the compositions are prepared according to conventional mixing, granulating or coating methods, respectively, and contain about 0.1 to 75%, preferably about 1 to 50%, of the compound(s).
- the compound(s) can be formulated as a depot preparation.
- Such long-acting formulations can be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection.
- one or more of the compounds described herein can be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
- the compounds are prepared with a polysaccharide such as chitosan or derivatives thereof (e.g., chitosan succinate, chitosan phthalate, etc.), pectin and derivatives thereof (e.g., amidated pectin, calcium pectinate, etc.), chondroitin and derivatives thereof (e.g., chondroitin sulfate), and alginates.
- a polysaccharide such as chitosan or derivatives thereof (e.g., chitosan succinate, chitosan phthalate, etc.), pectin and derivatives thereof (e.g., amidated pectin, calcium pectinate, etc.), chondroitin and derivatives thereof (e.g., chondroitin sulfate), and alginates.
- chitosan or derivatives thereof e.g., chitosan succinate, chitosan phthalate, etc.
- the compounds are loaded onto polymeric nanoparticles that can target the site of infection.
- nanoparticles include biodegradable nanoparticles, pH-sensitive nanoparticles (e.g., comprising Eudragit® S100), trimethylchitosan nanoparticles, polymeric nanoparticles (e.g., comprising PLGA, PEG-PLGA and/or PEG-PCL), and mannose-grafted polymeric nanoparticles. See, e.g., Coco et al., Int. J. Pharm. 440:3-12 (2013).
- the compounds can be encapsulated in a controlled drug-delivery system such as a pressure controlled delivery capsule (see, e.g., Takaya et al., J. Control Rel., 50:111-122 (1998)), a colon targeted delivery system, a osmotic controlled drug delivery system, and the like.
- the pressure controlled delivery capsule can contain an ethylcellulose membrane.
- the colon target delivery system can contain a tablet core containing lactulose which is over coated with an acid soluble material, e.g., Eudragit E®, and then overcoated with an enteric material, e.g., Eudragit L®.
- the osmotic controlled drug delivery system can be a single or more osmotic unit encapsulated with a hard gelatin capsule (e.g., capsule osmotic pump; commercially available from, e.g., Alzet, Cupertino, Calif.).
- a hard gelatin capsule e.g., capsule osmotic pump; commercially available from, e.g., Alzet, Cupertino, Calif.
- the osmotic unit contains an osmotic push layer and a drug layer, both surrounded by a semipermeable membrane.
- THERAPEUTIC PEPTIDES AND PROTEINS FORMULATION, PROCESSING, AND DELIVERY SYSTEMS, Technomic Publishing Company, Inc., Lancaster, Pa. (1995), incorporated herein by reference.
- Polymers can be used for ion-controlled release of compositions of the present invention.
- Various degradable and non-degradable polymeric matrices for use in controlled drug delivery are known in the art (see, Langer R., Accounts Chem. Res., 26:537-542 (1993)).
- the block copolymer, polaxamer 407 exists as a viscous yet mobile liquid at low temperatures but forms a semisolid gel at body temperature.
- liposomes are used for controlled release as well as drug targeting of the lipid-capsulated drug (Betageri et al., LIPOSOME DRUG DELIVERY SYSTEMS, Technomic Publishing Co., Inc., Lancaster, Pa. (1993)).
- compositions further include a pharmaceutical surfactant.
- compositions further include a cryoprotectant.
- cryoprotectants include glucose, sucrose, trehalose, lactose, sodium glutamate, PVP, cyclodextrin, 2-hydroxypropyl- ⁇ -cyclodextrin (HPI3CD) glycerol, maltose, mannitol, saccharose, and mixtures thereof.
- compositions or medicaments can be administered to a subject at a therapeutically effective dose to reactivate latent infections by viruses, as described herein.
- the pharmaceutical composition or medicament is administered to a subject in an amount sufficient to elicit an effective therapeutic response in the subject.
- the pharmaceutical composition or medicament can be administered to a subject at a therapeutically effective dose to reactivate virally infected cells from latent reservoirs and increase the subject's immune system to combat and kill reactivated virally infected cells.
- the methods of administration comprise administering ingenol-3-angelate (PEP005) alone or in combination with one or more additional latency reactivation agents including positive transcription elongation factor b activators, histone methyltransferase (HMT) inhibitors, histone deacetylase (HDAC) inhibitors, DNA methyltransferase inhibitors, NF- ⁇ B activators, Akt/HEXIM-1 modulators, Jak/Stat pathway modulators, diterpene compounds (e.g., ingenol derivatives, phorbol esters), macrolide lactones, diacylglycerol (DAG) lactones, protein kinase C (PKC) activators, and combinations thereof to a patient in need thereof.
- the methods of administration can include PEP005 in combination with JQ1, GSK343, SAHA, and combinations thereof.
- ingenol-3-angelate (PEP005) is administered to a patient infected with a virus, such as HIV, to reactivate a latent viral infection.
- a virus such as HIV
- ingenol-3-angelate (PEP005) is administered in combination with a positive transcription elongation factor b activator, such as JQ1, to a patient infected with a virus, such as HIV, to reactivate a latent viral infection.
- a positive transcription elongation factor b activator such as JQ1
- ingenol-3-angelate (PEP005) is administered in combination with a histone methyltransferase (HMT) inhibitor, such as GSK343, to a patient infected with a virus, such as HIV, to reactivate a latent viral infection.
- HMT histone methyltransferase
- ingenol-3-angelate (PEP005) is administered in combination with a histone deacetylase (HDAC) inhibitor, such as SAHA, to a patient infected with a virus, such as HIV, to reactivate a latent viral infection.
- HDAC histone deacetylase
- ingenol-3-angelate (PEP005) is administered in combination with a DNA methyltransfase inhibitor to a patient infected with a virus, such as HIV, to reactivate a latent viral infection.
- a virus such as HIV
- ingenol-3-angelate (PEP005) is administered in combination with an NF- ⁇ B activator to a patient infected with a virus, such as HIV, to reactivate a latent viral infection.
- a virus such as HIV
- ingenol-3-angelate (PEP005) is administered in combination with a Akt/HEXIM-1 modulator to a patient infected with a virus, such as HIV, to reactivate a latent viral infection.
- a virus such as HIV
- ingenol-3-angelate (PEP005) is administered in combination with a Jak/Stat pathway modulator to a patient infected with a virus, such as HIV, to reactivate a latent viral infection.
- a virus such as HIV
- ingenol-3-angelate (PEP005) is administered in combination with a diterpene compound to a patient infected with a virus, such as HIV, to reactivate a latent viral infection.
- a virus such as HIV
- ingenol-3-angelate (PEP005) is administered in combination with an ingenol derivative to a patient infected with a virus, such as HIV, to reactivate a latent viral infection.
- a virus such as HIV
- ingenol-3-angelate (PEP005) is administered in combination with a phorbol ester to a patient infected with a virus, such as HIV, to reactivate a latent viral infection.
- a virus such as HIV
- ingenol-3-angelate (PEP005) is administered in combination with a macrolide lactone to a patient infected with a virus, such as HIV, to reactivate a latent viral infection.
- a virus such as HIV
- ingenol-3-angelate (PEP005) is administered in combination with a diacylglycerol lactone to a patient infected with a virus, such as HIV, to reactivate a latent viral infection.
- a virus such as HIV
- ingenol-3-angelate is administered in combination with a protein kinase C activator, such as a diterpene compound, to a patient infected with a virus, such as HIV, to reactivate a latent viral infection.
- a protein kinase C activator such as a diterpene compound
- ingenol-3-angelate is administered with a viral therapy vaccine and optionally one or more latency reactivation agents to a patient infected with a virus, such as HIV, to reactivate a latent viral infection (i.e., latent virally infected cells) and to increase the patient's immune system to combat and kill the reactivated virally infected cells.
- a virus such as HIV
- compositions of the present invention are useful in the manufacture of a pharmaceutical composition or a medicament.
- a pharmaceutical composition or medicament can be administered to a subject in need thereof, e.g. a patient infected with a viral infection, such as, but not limited to HIV, CMV, and adenovirus.
- the methods comprise first administering ingenol-3-angelate (PEP005), to a patient infected with a latent virus, and then administering a positive transcription elongation factor b activator, such as JQ1, to the patient.
- the methods comprise first administering a positive transcription elongation factor b activator, such as JQ1, to a patient infected with a latent virus, and then administering ingenol-3-angelate (PEP005) to the patient.
- ingenol-3-angelate PEP005
- a positive transcription elongation factor b activator such as JQ1
- the methods comprise first administering ingenol-3-angelate (PEP005), to a patient infected with a latent virus, and then administering a histone methyltransferase (HMT) inhibitor, such as GSK343, to the patient.
- HMT histone methyltransferase
- the methods comprise first administering a histone methyltransferase (HMT) inhibitor, such as GSK343, to a patient infected with a latent virus, and then administering ingenol-3-angelate (PEP005) to the patient.
- HMT histone methyltransferase
- ingenol-3-angelate PEP005
- HMT histone methyltransferase
- the methods comprise first administering ingenol-3-angelate (PEP005), to a patient infected with a latent virus, and then administering a histone deacetylase (HDAC) inhibitor, such as SAHA, to the patient.
- HDAC histone deacetylase
- the methods comprise first administering a histone deacetylase (HDAC) inhibitor, such as SAHA, to a patient infected with a latent virus, and then administering ingenol-3-angelate (PEP005) to the patient.
- HDAC histone deacetylase
- ingenol-3-angelate PEP005
- HDAC histone deacetylase
- the methods comprise first administering a viral therapy vaccine to a patient infected with a latent virus, and then administering ingenol-3-angelate (PEP005) to the patient.
- the methods comprise first administering a viral therapy vaccine to a patient infected with a latent virus, and then administering ingenol-3-angelate (PEP005) as well as one or more latency reactivating agents to the patient.
- the methods comprise simultaneously administering a viral therapy vaccine, the ingenol-3-angelate (PEP005), and optionally one or more latency reactivating agents to a patient infected with a latent virus.
- a viral therapy vaccine the ingenol-3-angelate (PEP005)
- PEP005 the ingenol-3-angelate
- the present invention provides a method of delivering an effective amount of ingenol-3-angelate (PEP005) alone or in combination with one or more additional latency reactivation agents including positive transcription elongation factor b activators, histone methyltransferase (HMT) inhibitors, histone deacetylase (HDAC) inhibitors, DNA methyltransferase inhibitors, NF- ⁇ B activators, Akt/HEXIM-1 modulators, Jak/Stat pathway modulators, diterpene compounds (e.g., ingenol derivatives, phorbol esters), macrolide lactones, diacylglycerol (DAG) lactones, protein kinase C (PKC) activators, and combinations thereof to a patient in need thereof.
- PEP005 ingenol-3-angelate
- additional latency reactivation agents including positive transcription elongation factor b activators, histone methyltransferase (HMT) inhibitors, histone deacet
- the present invention provides a method of delivering an effective amount of a viral therapy vaccine and ingenol-3-angelate (PEP005) alone or in combination with one or more additional latency reactivation agents described herein.
- PEP005 ingenol-3-angelate
- compositions or medicaments can be administered to a subject at a therapeutically effective dose to reactivate latent viral infections as described herein.
- the pharmaceutical composition or medicament is administered to a subject in an amount sufficient to elicit an effective therapeutic response in the subject.
- the dosage of compounds administered is dependent on the subject's body weight, age, individual condition, surface area or volume of the area to be treated, and/or on the form of administration.
- the size of the dose will also be determined by the existence, nature, and extent of any adverse effects that accompany the administration of a particular compound in a particular subject.
- a dosage of the active compounds is a dosage that is sufficient to achieve the desired effect.
- Optimal dosing schedules can be calculated from measurements of compound accumulation in the body of a subject. In general, dosage may be given once or more daily, weekly, or monthly. Persons of ordinary skill in the art can easily determine optimum dosages, dosing methodologies, and repetition rates.
- a unit dosage for oral administration to a subject (e.g., human) of about 50 to about 70 kg may contain between about 1 and about 500 mg, about 5 and about 500 mg, about 5 and about 250 mg, about 25 to about 250 mg, about 100 and about 1000 mg, about 200 and about 2000 mg, about 500 and about 5000 mg, or about 1000 and about 2000 mg of the compound(s).
- a unit dosage for oral administration to a subject (e.g., human) of about 50 to about 70 kg may contain about 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 75 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1250 mg, 1500 mg, 2000 mg, 2500 mg, 3000 mg, or more of the compound(s).
- a unit dosage for intravenous administration to a subject (e.g., human) of about 50 to about 70 kg may contain between about 0.1 and about 100 mg, about 0.5 and about 100 mg, about 0.5 and about 50 mg, about 0.5 and about 25 mg, about 0.5 and about 10 mg, about 0.25 to about 50 mg, about 0.25 to about 25 mg, about 0.1 to about 50 mg, about 0.1 to about 25 mg, or about 0.1 to about 10 mg of the compound(s).
- a unit dosage for intravenous administration to a subject (e.g., human) of about 50 to about 70 kg may contain about 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 75 mg, 100 mg, or more of the compound(s).
- Exemplary doses of the compositions described herein include milligram or microgram amounts of the composition per kilogram of subject weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 10 micrograms per kilogram to about 5 milligrams per kilogram, about 10 microgram per kilogram to about 500 micrograms per kilogram, or about 1 microgram per kilogram to about 100 micrograms per kilogram. It is furthermore understood that appropriate doses of a composition depend upon the potency of the composition with respect to the desired effect to be achieved. When one or more of these compositions is to be administered to a mammal, a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained.
- the specific dose level for any particular subject will depend upon a variety of factors including the activity of the specific composition employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.
- a pharmaceutical composition or medicament of the present invention is administered orally, e.g., in a dose in the range of from about 1 to about 1000 micrograms ( ⁇ g) of compound per kg of subject body weight, from about 1 to about 500 ⁇ g/kg body weight, from about 10 to about 1000 ⁇ g/kg body weight, from about 10 to about 500 ⁇ g/kg body weight, from about 50 to about 1000 ⁇ g/kg body weight, from about 50 to about 500 ⁇ g/kg body weight, from about 100 to about 1000 ⁇ g/kg body weight, or from about 100 to about 500 ⁇ g/kg body weight.
- ⁇ g micrograms
- the dose is about 10, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 ⁇ g/kg body weight.
- the dose can be administered once per day or divided into sub-doses and administered in multiple doses, e.g., twice, three times, or four times per day.
- oral compositions described herein may be administered in different amounts and at different times.
- a pharmaceutical composition or medicament of the present invention is administered intravenously, e.g., in a dose in the range of from about 1 to about 500 micrograms ( ⁇ g) of compound per kg of subject body weight, from about 1 to about 200 ⁇ g/kg body weight, from about 1 to about 100 ⁇ g/kg body weight, from about 10 to about 500 ⁇ g/kg body weight, from about 10 to about 200 ⁇ g/kg body weight, from about 10 to about 100 ⁇ g/kg body weight, or from about 10 to about 80 ⁇ g/kg body weight.
- ⁇ g micrograms
- the dose is about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, or 500 ⁇ g/kg body weight.
- the dose can be administered once per day or divided into sub-doses and administered in multiple doses, e.g., twice, three times, or four times per day.
- intravenous compositions described herein may be administered in different amounts and at different times.
- ingenol-3-angelate is administered orally at a dose between about 100 to about 500 ⁇ g/kg body weight. In other embodiments, ingenol-3-angelate (PEP005) is administered intravenously at a dose between about 10 to about 80 ⁇ g/kg body weight. In particular embodiments, ingenol-3-angelate (PEP005) is adminstered in combination with one or more additional latency reactivation agents described herein. In some embodiments, each compound may be administered at a dose of the individual compounds that is effective for reactivating a latent virus. In other embodiments, each compound may be administered at a lower dose relative to the dose of the individual compounds that is effective for reactivating a latent virus.
- the lower dose of ingenol-3-angelate (PEP005) when used in combination with one or more additional latency reactivation agents is about 100 ⁇ g/kg body weight or less for oral administration (e.g., about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 ⁇ g/kg body weight) or about 10 ⁇ g/kg body weight of less for intravenous administration (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ⁇ g/kg body weight).
- the lower dose of the one or more additional latency reactivation agents is reduced by about 25% to about 35% (e.g., by about 25%, 30%, or 35%) compared to the dose of the additional latency reactivation agent alone.
- the compounds are administered one or more times a day, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times a day.
- the compounds are administered for about 1 to about 31 days, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. In some embodiments, the compounds are administered for at least 1 day. In other embodiments, the compounds are administered for one or more weeks, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more weeks. In yet other embodiments, the compounds are administered for one or more months, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months.
- compounds may be administered for multiple days at the therapeutically effective daily dose.
- therapeutically effective administration of compounds to treat a pertinent condition or disease described herein in a subject requires periodic (e.g., daily or twice daily) administration that continues for a period ranging from three days to two weeks or longer. While consecutive daily doses are a preferred route to achieve a therapeutically effective dose, a therapeutically beneficial effect can be achieved even if the agents are not administered daily, so long as the administration is repeated frequently enough to maintain a therapeutically effective concentration of the agents in the subject. For example, one can administer the agents every day, every other day, or, if higher dose ranges are employed and tolerated by the subject, twice a week.
- Optimum dosages, toxicity, and therapeutic efficacy of such compounds may vary depending on the relative potency of individual compounds and can be determined by standard pharmaceutical procedures in experimental animals, for example, by determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population).
- the dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio, LD 50 /ED 50 .
- Agents that exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side-effects can be used, care should be taken to design a delivery system that targets such compounds to the affected site to minimize potential damage to normal cells and, thereby, reduce side-effects.
- the data obtained from, for example, animal studies can be used to formulate a dosage range for use in humans.
- the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
- the dosage can vary within this range depending upon the dosage form employed and the route of administration.
- a dose can be formulated in animal models to achieve a concentration range that includes the IC 50 (the concentration of the agent that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in stool or an enteric tissue sample can be measured, for example, by high performance liquid chromatography (HPLC). In general, the dose equivalent of compounds is from about 1 ng/kg to about 100 mg/kg for a typical subject.
- HPLC high performance liquid chromatography
- the dosage of a pharmaceutical composition of the present invention can be monitored and adjusted throughout treatment, depending on severity of symptoms, frequency of recurrence, and/or the physiological response to the therapeutic regimen. Those of skill in the art commonly engage in such adjustments in therapeutic regimens.
- a pharmaceutical composition or medicament is administered to a patient at a therapeutically effective dose to induce reactivation of latent virally infected cells.
- the pharmaceutical composition or medicament is administered to a patient in an amount sufficient to elicit an effective therapeutic response in the patient.
- An effective therapeutic response is a response that at least partially reactivates latent virally infected cells. An amount adequate to accomplish this is defined as a “therapeutically effective dose.”
- compositions are administered depending on the dosage and frequency as required and tolerated by the patient.
- the composition should provide a sufficient quantity of the compounds of the invention to effectively treat the patient.
- the dose is sufficient to induce viral reactivation from latent virally infected cells without producing unacceptable toxicity or side-effects to the patient.
- kits and systems can be prepared according to the present invention, depending upon the intended user of the kit and system and the particular needs of the user.
- the present invention provides a kit that includes ingenol-3-angelate (PEP005) and one or more additional latency reactivation agents selected from positive transcription elongation factor b activators, histone methyltransferase (HMT) inhibitors, histone deacetylase (HDAC) inhibitors, DNA methyltransferase inhibitors, NF- ⁇ B activators, Akt/HEXIM-1 modulators, Jak/Stat pathway modulators, diterpene compounds (e.g., ingenol derivatives, phorbol esters), macrolide lactones, diacylglycerol (DAG) lactones, protein kinase C (PKC) activators, and combinations thereof.
- PEP005 ingenol-3-angelate
- additional latency reactivation agents selected from positive transcription elongation factor b activators, histone
- the kit includes ingenol-3-angelate (PEP005) and a compound selected from JQ1, GSK343, SAHA, and combinations thereof.
- the kit includes ingenol-3-angelate (PEP005) and JQ1.
- the kit includes ingenol-3-angelate (PEP005) and GSK343.
- the kit includes ingenol-3-angelate (PEP005) and SAHA.
- the kit includes ingenol-3-angelate (PEP005), one or more additional latency reactivation agents (e.g., JQ1, GSK343, and/or SAHA), and one or more viral therapy vaccines (e.g., CTL booster).
- kits described herein include a label describing a method of administering ingenol-3-angelate (PEP005) and/or one or more additional latency reactivation agents described herein.
- kits described herein include a label describing a method of reactivating a latent virus, e.g., HIV, CMV, or adenovirus.
- compositions of the present invention including but not limited to compositions including ingenol-3-angelate (PEP005), one or more additional latency reactivation agents, and optionally a viral therapy vaccine described herein may, if desired, be presented in a bottle, jar, vial, ampoule, tube, or other container-closure system approved by the Food and Drug Administration (FDA) or other regulatory body, which may provide one or more dosages containing the compounds.
- FDA Food and Drug Administration
- the package or dispenser may also be accompanied by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, the notice indicating approval by the agency.
- the kit may include a formulation or composition as described herein, a container closure system including the formulation or a dosage unit form including the formulation, and a notice or instructions describing a method of use as described herein.
- the kit includes a container which is compartmentalized for holding the various elements of a formulation (e.g., the dry ingredients and the liquid ingredients) or composition, instructions for making the formulation or composition, and instructions for administering the formulation or composition for reactivating a latent virus such as, e.g., HIV, CMV, adenovirus, or combinations thereof.
- a formulation e.g., the dry ingredients and the liquid ingredients
- instructions for making the formulation or composition e.g., instructions for making the formulation or composition
- instructions for administering the formulation or composition for reactivating a latent virus such as, e.g., HIV, CMV, adenovirus, or combinations thereof.
- the kit may include the pharmaceutical preparation in dehydrated or dry form, with instructions for its rehydration (or reconstitution) and administration.
- Kits with unit doses of the compounds described herein, e.g. in oral, rectal, transdermal, or injectable doses (e.g., for intramuscular, intravenous, or subcutaneous injection), are provided.
- an informational package insert describing the use and attendant benefits of the composition for reactivating a latent virus may be included in addition to the containers containing the unit doses.
- Some embodiments of the present invention include packages that include ingenol-3-angelate (PEP005) and one or more additional latency reactivation agents.
- PEP005 ingenol-3-angelate
- PEP005 additional latency reactivation agents
- Some other embodiments of the present invention include packages that include ingenol-3-angelate (PEP005), one or more additional latency reactivation agents (e.g., JQ1, GSK343, and/or SAHA), and one or more viral therapy vaccines (e.g., CTL booster).
- PEP005 ingenol-3-angelate
- additional latency reactivation agents e.g., JQ1, GSK343, and/or SAHA
- one or more viral therapy vaccines e.g., CTL booster.
- Example 1 Synergistic Reactivation of Latent HIV Expression by Ingenol-3-Angelate, PEP005, Targeted NF-kB Signaling in Combination with JQ1 Induced p-TEFb Activation
- PEP005 reactivated latent HIV through the induction of the pS643/S676-PKC ⁇ / ⁇ -I ⁇ B ⁇ / ⁇ -NF- ⁇ B signaling pathway.
- PEP005 alone was sufficient to induce expression of fully elongated and processed HIV RNAs in primary CD4+ T cells from HIV infected individuals receiving suppressive ART.
- PEP005 and the P-TEFb agonist, JQ1 exhibited synergism in reactivation of latent HIV with a combined effect that is 7.5-fold higher than the effect of PEP005 alone.
- PEP005 suppressed HIV infection of primary CD4+ T cells through down-modulation of cell surface expression of HIV co-receptors.
- this example demonstrates that PEP005 is a safe and effective compound for HIV eradication strategies.
- Anti-retroviral therapy is effective in suppressing HIV replication but it fails to eliminate latent viral reservoirs in HIV infected resting CD4+ T cells which, in blood, consist mainly of central and transitional memory CD4+ T cells [1-4].
- Current ART options do not eradicate HIV from infected cells. In addition, these cells are invisible to the virus-specific immune responses in the setting of viral latency [5,6]. The viral reservoir is rapidly seeded and HIV latency might be established immediately after virus infection [7,8]. Despite initiation of ART in infants within hours of birth to HIV infected mothers, stable viral reservoirs were established and viral rebound occurred when therapy was interrupted [9].
- LRAs functioning through the PKC-NF- ⁇ B signaling, are able to reactivate latent HIV across a broad range of HIV latency models [20].
- LRAs stimulating PKC-NF- ⁇ B signaling may be most effective in inducing complete transcription of HIV from resting CD4+ T cells of HIV infected individuals on suppressive ART [26].
- these compounds cause down-modulation of the expression of cell surface receptors, CD4, CXCR4 or CCR5, and protect cells against HIV infection [22]. Therefore, LRAs that activate PKC-NF- ⁇ B signaling are potential candidates for HIV cure studies.
- ingenol ester ingenol-3-hexanoate or IngB
- IngB ingenol-3-hexanoate
- the modified ingenol-3-hexanoate was originally isolated from an Amazonian plant, Euphorbia tirucalli . It exerts low toxicity in CD4+ T cells and does not induce global T-cell activation. It caused reactivation of latent HIV at nanomolar levels [24].
- IngB induces expression and activation of both NF- ⁇ B and CyclinT1/CDK9, and stimulates IFN ⁇ expression in primary CD4+ T cells, further search for new ingenol compounds with better HIV reactivation potential and lower cellular toxicity is needed [24,27,28].
- ingenol-3-angelate (PEP005) is currently approved for clinical use.
- a recently FDA approved drug, PICATO, for the topical treatment of precancerous actinic keratosis contains ingenol-3-angelate as an active component [29].
- PICATO for the topical treatment of precancerous actinic keratosis contains ingenol-3-angelate as an active component [29].
- ingenol-3-angelate could induce HIV expression from the U1 monocyte cell line harboring HIV genome [30].
- PEP005 can effectively reactivate latent HIV through the activation of the pS643/S676-PKC ⁇ / ⁇ -I ⁇ B ⁇ / ⁇ -NF- ⁇ B pathway in an HIV latency model in vitro but does not induce or increase NF- ⁇ B protein production by itself.
- J-Lat A1 cells (harboring a single copy of latent HIV LTR and one copy of green fluorescent protein gene under the HIV LTR control) or U1 cells (harboring two latent HIV genomes with defective Tat gene) were cultured in RPMI1640 medium with 10% fetal bovine serum (FBS) and 1% Pen/strep in a 37° C. incubator containing 5% CO 2 [24,31]. Both of the cell lines were obtained from NIH AIDS Reagent Program.
- FBS fetal bovine serum
- HIV LTR For reactivation of HIV LTR, cells were treated with PMA (Sigma), JQ1 (Biovision), Prostatin (Sigma), SAHA (Santa Cruz), TNF- ⁇ (BD), GSK343 (Sigma), or PEP005 (Tocris Bioscience) for 24 h. HIV reactivation was quantified by GFP expression using flow cytometry and the data were analyzed using FlowJo Software for J-Lat A1 cells, or by quantitative RT-PCR (RT-qPCR) for J-Lat A1 and U1 cells. Cell viability was evaluated using Live/Dead dye (Life Technologies) by flow cytometry. So far, there is not a single in vitro cell culture model available that captures all the features of HIV latency.
- J-Lat A1 clone has been widely used by researchers for HIV latency studies.
- the T-cell-derived J-Lat A1 cells harbor one copy of a construct containing a Tat and a green fluorescent protein (GFP) gene framed by the 5′ and 3′ HIV LTR [32].
- the J-Lat A1 cells also contain the TAR loop located within the R region of HIV LTR (nt+1 to +60), which provides an opportunity for Tat-TAR interactions for the modulation of transcription and transcriptional elongation. Consequently, the J-Lat A1 cell model is a suitable cell culture model to investigate latency reversal by PKC agonists.
- Peripheral blood samples were collected from 13 HIV-infected individuals receiving suppressive ART for >3 years except for one patient. All subjects had suppression of plasma viremia for more than 6 months (Average 5.84 years).
- CD4+ T cell counts in peripheral blood samples ranged from 264 to 1100 cells/mm 3 (Average 639 cell/mm 3 ) and plasma viral loads were ⁇ 20 copies per ml as measured by qPCR (Table 1). Isolation of the peripheral blood mononuclear cells (PBMC) and purification of CD4+ T cells using the EasySep kit (STEMCELL Technologies Inc. Vancouver, BC, Canada) were performed as previously described [24].
- the purified CD4+ T cells were plated at a density of 1 ⁇ 10 6 cells/ml and treated with 200 ng/ml PMA plus 2 ⁇ M Ionomycin, 6-12 nM PEP005, 2 ⁇ M of JQ1, or 12 nM PEP005 plus 2 ⁇ M JQ1 for 6 hrs or 48 hrs and the cells were collected for RNA purification.
- PBMCs were isolated from uninfected controls and 2 ⁇ 10 6 cells were incubated with DMSO, 200 ng/ml PMA plus 2 M Ionomycin, 6-12 nM PEP005 for 24 hrs or 72 hrs, and immunostained with anti-CD3, anti-CD38, anti-CD69, or anti-HLA-DR antibodies (Biolegend) for 20 min at 4° C. Cells were fixed in 1% PFA and analyzed by flow cytometry (FlowJo software from TreeStar). In addition, PBMCs from HIV-negative uninfected controls were similarly treated for 24 or 72 hrs and cells were collected for cytokine analysis using RT-qPCR.
- Cells were placed in 96-well plates and incubated for 24 or 72 h hrs with compounds. Cell viability was measured using MTT assay (Roche Laboratories), and cell proliferation/S-phase progression was determined by using BrdU ELISA kit (Cell signaling, #1863).
- the level of phosphorylation of PKC was determined using anti-Phospho-Ser664-PKCS, anti-Phospho-Ser643/676 PKCS or anti-Phospho-T538-PKC ⁇ (Millipore), and the level of phosphorylation of I ⁇ B was determined using p-I ⁇ B ⁇ (Ser32), p-I ⁇ B ⁇ (Thr19/Ser23) or p-I ⁇ B ⁇ (Ser18/22) antibodies (Millipore).
- fa x (HIV RNA copies with drug x background copies with DMSO)/(HIV RNA copies with PMA ⁇ background copies with DMSO).
- fa x (HIV RNA copies with drug x background copies with DMSO)/(HIV RNA copies with PMA ⁇ background copies with DMSO).
- Chromatin Immunoprecipitation (ChIP):
- ChIP assay was performed as previously described [24,37]. Briefly, 1 ⁇ 10 6 J-Lat A1 cells were incubated with PEP005, or PKC ⁇ /inhibitor (PKC ⁇ i, Millipore/Calbiochem), fixed in 1% formaldehyde then resuspended in lysis buffer containing 1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.1 (ChIP Assay Kit, Millipore) and protease inhibitor cocktail (Sigma-Aldrich). Lysates were sonicated to obtain DNA fragments of 200-1500 bp.
- PEP005 PKC ⁇ /inhibitor
- PKC ⁇ i PKC ⁇ /inhibitor
- the immune complex was retrieved by incubating for 45 min with 50 ⁇ l of protein A/G-agarose beads saturated with BSA/salmon sperm DNA. Following the washes, the chromatin was eluted and reverse cross-linked overnight. DNA was extracted (Qiagen PCR purification kit) and quantitative real-time PCR was performed using Agilent Brilliant Ultra-Fast SYBR Green QPCR reagent using the 7500 real-time PCR System.
- the upstream primer sequence was 5′-AGCTTGCTACAAGGGACTTTCC-3′ (SEQ ID NO:3)
- the downstream primer sequence was 5′-ACCCAGTACAGGCAAAAAGCAG-3′ (SEQ ID NO:4).
- the GAPDH primer/probe set was purchased from Applied Biosystems.
- CD4+ T cells were isolated from peripheral blood samples of healthy HIV-negative donors and were pre-treated with PEP005 overnight.
- the CD4+ T cells were infected with HIV-1 (HIV-1 IIIB expanded in Jurkat T cells, 100 ng p24-gag) through spinoculation as described previously [38]. Following an exposure of 24 hrs to HIV, the cells were washed. Cell supernatants were collected and HIV p24 levels were measured by ELISA. The cells were also collected for RNA extraction and measurement of viral transcripts by RT-qPCR.
- PEP005 Induces HIV Expression in an HIV Latency Cell Culture Model In Vitro.
- J-Lat A1 cells an established HIV latency lymphocyte cell culture model in vitro [24,32,39], were treated with increasing concentrations (2-40 nM) of PEP005 ( FIG. 1A ).
- PEP005 induced HIV expression in a dose-dependent manner and in the absence of any apparent cellular toxicity.
- a 7-fold increase in the reactivation of HIV latency was detected in J-Lat A1 cells at 20 nM of PEP005 as compared to untreated controls ( FIGS. 1B and 1C ).
- the effect of PEP005 on HIV expression was evident even at the 2 nM level.
- PEP005 appeared to be more potent than SAHA (a histone deacetylase inhibitor), JQ1 (a BET bromodomain inhibitor) and GSK343 (an inhibitor of EZH2) ( FIG. 1D ).
- SAHA histone deacetylase inhibitor
- JQ1 a BET bromodomain inhibitor
- GSK343 an inhibitor of EZH2
- PEP005 induced reactivation of latent HIV and was highly effective in combination with JQ1 in J-Lat A1 cells ( FIG. 2A ).
- GSK343 alone could not effectively induce HIV expression but was able to enhance the magnitude of latent HIV reactivation by PEP005.
- Prostratin also showed a trend for enhancing effect of PEP005 on latent HIV reactivation. However, this was not statistically significant.
- PEP005 demonstrates significant synergism with JQ1 or EZH2 inhibitors to induce GFP expression in vitro ( FIG. 2C ).
- HDAC inhibitor SAHA and another PKC agonist Prostratin did not exhibit synergy with PEP005.
- PEP005 synergized significantly with JQ1 or SAHA to induce HIV mRNA expression in vitro ( FIG. 2D ), but GSK343 did not exhibit synergy with PEP005.
- PEP005 exerted a synergistic effect with JQ1 on reactivation of HIV from latency in both J-Lat A1 and U1 cell lines.
- PEP005 Disrupts HIV Latency Through I ⁇ B/-pSer664/Ser676-PKC ⁇ / ⁇ -NF- ⁇ B Signaling.
- PEP005 can activate the PKC-NF- ⁇ B pathway, it is not known at which step of the PKC-NF- ⁇ B pathway is modulated during the reactivation of latent HIV. Therefore, we examined protein expression of several components of the PKC pathway in J-Lat A1 cells by Western blot analysis using antibodies specific for four PKC super families, including PKC ⁇ /D, PKC ⁇ , PKC ⁇ , and PKC ⁇ . There was no significant induction of expression of these PKC proteins except for a modest up-regulation of PKC ⁇ that was detected at 1 hr following 12 nM PEP005 treatment.
- PEP005 Displays Minimal Toxicity in Primary CD4+ T Cells.
- FIGS. 5B, 5E and 5F There was no significant change in the expression of CD38 (24 hr) and HLA-DR in CD4+ T cells ( FIGS. 5B, 5E and 5F ). However, there was an increase in expression of CD69, an inducible glycoprotein that is expressed early during T lymphocytes activation ( FIGS. 5C and 5D ). These findings provide additional evidence that PEP005 reverses HIV latency through PKC-NF- ⁇ B signaling in vivo. Since the expression of CD69 is dependent on NF- ⁇ B binding to its promoter region, it is understandable that PEP005 induced PKC-NF- ⁇ B signaling would up-regulate CD69 expression in CD4+ T cells [44].
- TNF- ⁇ , IFN- ⁇ , IL-2, and IL-6 cytokines were determined by RT-qPCR ( FIGS. 6A-D ). There was no significant increase in the expression of these cytokines, except for TNF- ⁇ that showed a tendency towards an up-regulation. However, the increase was not statistically significant.
- PEP005 In order to further evaluate the effects of PEP005 on T cell activation, we determined the ability of PEP005 to induce T cell proliferation and cytotoxicity using BrdU incorporation and MTT assays. PEP005 induced minimal levels of cellular toxicity in both J-Lat A1 and U1 cell lines, as well as in primary CD4+ T cells from peripheral blood samples of healthy HIV-negative donors ( FIG. 7 ). Importantly, PEP005 treatment did not induce any significant increase in the proportion of cells in S-phase with J-Lat A1 cells, U1 cells or primary CD4+ T cells. In summary, despite the increased CD69 expression, minimal to no induction was found for the expression of pro-inflammatory cytokines and no significant impact was seen on cell cycling, indicating that PEP005 is a potential LRA candidate for evaluation in vivo.
- PEP005 Induces Latent HIV Expression in Primary CD4+ T Cells Ex Vivo from Individuals with Suppressive ART.
- HIV Induction in Primary CD4+ T Cells from HIV Infected Individuals on ART is Enhanced by the Combination of PEP005 with JQ1.
- PEP005 Down-Regulates Cell Surface Expression of HIV Receptors on Primary CD4+ T Cells.
- Diterpenes can down-modulate the expression of cell surface receptors that are known to mediate HIV attachment and entry and can potentially prevent the spread of viral infection to bystander CD4+ T cells [27,52-54]. Therefore, it is useful to identify diterpene compounds suitable for HIV cure studies that could reactivate HIV from the latent reservoirs with low to minimal cytotoxicity while preventing new viral infections of CD4+ T cells.
- NF- ⁇ B is a master regulator for multiple signaling pathways
- P-TEFb is a general activator of transcriptional elongation, that also contributes to multiple signaling pathways [58,59].
- Further studies are warranted to investigate whether IngB or PEP005 alone can induce protein expression of NF- ⁇ B or P-TEFb in resting CD4+ T cells, and not just in proliferating CD4+ T cells.
- IngB induced IFN ⁇ protein expression [24,27] while PEP005 barely caused any increase in this or other inflammatory cytokine expression.
- PEP005 shares a similar core structure with IngB, its C3 side chain structure is different and quite distinct from IngB ( FIG. 1 ). Substitution of the C3-ester by different aliphatic and aromatic side chains confer markedly different biological properties [60,61], so it is likely that differences in molecular mechanisms of action and different T cell activation properties between PEP005 and Ingenol B are conferred by the structural differences of the C3-R. Consequently, PEP005 may offer several advantages over IngB. High levels of ingenols, up to 20 ⁇ M, induced NF- ⁇ B-independent cell death in Jurkat cells [62].
- PEP005 is the only active component with PKC agonist activity.
- Pharmacokinetic studies of PEP005 have been performed in small animals in vivo. Similar to the safety of the topical application of PICATO, the systemic (intravenous) use of PEP005 in small animals (mini pig and rat model) was reported to be relatively safe, with the maximum nonlethal dose >73 ⁇ g/kg (See, Assessment report of PICATO to European Medicines Agency, Sep. 20, 2012). These data support the use of PEP005 in HIV cure studies.
- SAHA an HDAC inhibitor
- the efficacy of SAHA varies among HIV infected individuals indicating that SAHA alone may not be sufficient for effective reactivation of latent HIV for all HIV infected individuals [16,17,63]. Therefore, a combination of compounds should be explored to induce viral expression from latently infected cells [43].
- Our data show that PEP005 alone potently reactivates latent HIV expression in vitro.
- PEP005 and JQ1 were substantially more effective in inducing latent HIV in J-Lat A1 cells or U1 cells and in ex vivo primary CD4+ T cells from infected patients receiving suppressive ART. This could be attributed to potentiating effects of the PKC-NF-kB pathway on p-TEFb mechanism for reactivating.
- the range of drug concentrations that provides the best drug synergy using PEP005 can be determined [64].
- Synergistic reactivation of latent HIV is also achieved by combining JQ1 with other ingenol compounds, as it has been observed that JQ1 boosted the reversal of HIV latency by IngB in J-Lat A1 cells [24].
- JQ1 boosted the reversal of HIV latency by IngB in J-Lat A1 cells [24].
- PKC agonists such as Prostratin or Bryostatin 1
- GSK343, an EZH2 inhibitor had no capacity to reactivate latent HIV by itself, but displayed increased potency in combination with PEP005 in J-Lat A1 cells. These observations are complemented by a study showing that GSK343 boosts SAHA or JQ1 reactivation of latent HIV in a primary resting T-cell model of HIV latency [65]. These findings indicate that pre-disruption of methylation of histone tails may facilitate efficient reactivation of latent HIV in combination with a second LRA.
- PEP005 displayed potent activity to reactivate HIV from latency, producing polyadenylated viral transcripts while exerting minimal to low cytotoxicity and in the absence of major global T cell activation.
- Bryostatin and PMA have been shown to stimulate comparable transcription of polyadenylated HIV RNA in primary CD4+ T cells isolated from HIV infected individuals on suppressive ART [26].
- PEP005 had a similar capacity as PMA to reactivate latent HIV both in vitro and ex vivo ( FIG. 1D and FIG. 8 ), and that PEP005 could induce GFP protein expression in J-Lat A1 cells ( FIG. 1 ), PEP005 is useful for inducing HIV virus expression in vivo.
- PEP005 to reduce cell surface expression of CD4, CCR5, and CXCR4 on T cells directly impacts susceptibility of primary CD4+ T cells to in vitro HIV infection. This supports the concept that reactivation of latent HIV by PEP005 may block viral spread to uninfected bystander CD4+ T cells, making it an attractive compound for advancing to clinical HIV cure studies. This study demonstrates that ingenols, including PEP005, represent a new group of compounds for combating HIV latency for viral eradication.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Medicinal Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Emergency Medicine (AREA)
- Pain & Pain Management (AREA)
- Virology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
Description
- This application is a continuation of U.S. Nonprovisional application Ser. No. 15/426,437 filed Feb. 7, 2017, which is a continuation of International Patent Application No. PCT/US2015/042716 filed Jul. 29, 2015, which claims priority to U.S. Provisional Application No. 62/035,333, filed Aug. 8, 2014, which applications are incorporated herein by reference in their entirety for all purposes.
- This invention was made with Government support under Grant Nos. AI043274 and DK061297, awarded by the National Institutes of Health. The Government has certain rights in this invention.
- The Sequence Listing written in file SequenceListing_070772-219030US-1147192.txt created on Jul. 5, 2019, 1,882 bytes, machine format IBM-PC, MS-Windows operating system, is hereby incorporated by reference in its entirety for all purposes.
- Anti-retroviral therapy (ART) is effective in suppressing HIV replication but it fails to eliminate latent viral reservoirs in HIV infected resting CD4+ T cells which, in blood, consist mainly of central and transitional memory CD4+ T cells [1-4]. Current ART options do not eradicate HIV from infected cells. In addition, these cells are invisible to the virus-specific immune responses in the setting of viral latency [5,6]. The viral reservoir is rapidly seeded and HIV latency might be established immediately after virus infection [7,8]. Despite initiation of ART in infants within hours of birth to HIV infected mothers, stable viral reservoirs were established and viral rebound occurred when therapy was interrupted [9]. In the simian immunodeficiency virus (SIV) model of AIDS, stable viral reservoirs are established within 2.5 days of infection [10]. The viral reactivation was detected in rhesus macaques following therapy interruption despite the initiation of ART at 3 days post SIV infection [10,11]. Collectively, these studies demonstrate that a very early initiation of ART may not be sufficient to prevent nor eliminate latent virus reservoirs [9,11,12]. It has been observed that the morbidity of HIV persistence in HIV-positive individuals on long-term ART includes drug toxicities and a higher risk of developing complications including dyslipidemia, cardiovascular disease and insulin resistance [13-15]. Therefore, a therapeutic cure of HIV is urgently needed that leads to viral eradication and experimental strategies for directly targeting HIV latent reservoirs need to be developed. The present invention satisfies these needs and provides related advantages as well.
- In certain aspects, the present invention provides a composition comprising ingenol-3-angelate (PEP005) and one or more additional latency reactivation agents.
- In some embodiments, the one or more additional latency reactivation agents is selected from the group consisting of a positive transcription elongation factor b activator, a histone methyltransferase (HMT) inhibitor, a histone deacetylase (HDAC) inhibitor, a DNA methyltransferase inhibitor, an NF-κB activator, an Akt/HEXIM-1 modulator, a Jak/Stat pathway modulator, a diterpene compound (e.g., an ingenol derivative, a phorbol ester), a macrolide lactone, a diacylglycerol (DAG) lactone, a protein kinase C (PKC) activator, and combinations thereof.
- In some embodiments, the composition comprises a combination of ingenol-3-angelate (PEP005) and JQ1. In other embodiments, the composition comprises a combination of ingenol-3-angelate (PEP005) and GSK343. In yet other embodiments, the composition comprises a combination of ingenol-3-angelate (PEP005) and vorinostat (suberanilohydroxamic acid; SAHA). In certain embodiments, the composition further comprises a viral therapy vaccine. In certain embodiments, the composition further comprises a pharmaceutically acceptable carrier, excipient, or diluent.
- In certain other aspects, the present invention provides a method for reactivating a latent virus in a subject infected with the virus, the method comprising administering to the subject an effective amount of ingenol-3-angelate (PEP005) alone or in combination with one or more additional latency reactivation agents.
- In some embodiments, the one or more additional latency reactivation agents is selected from the group consisting of a positive transcription elongation factor b activator, a histone methyltransferase (HMT) inhibitor, a histone deacetylase (HDAC) inhibitor, a DNA methyltransferase inhibitor, an NF-κB activator, an Akt/HEXIM-1 modulator, a Jak/Stat pathway modulator, a diterpene compound (e.g., an ingenol derivative, a phorbol ester), a macrolide lactone, a diacylglycerol (DAG) lactone, a protein kinase C (PKC) activator, and combinations thereof. In certain embodiments, the method further comprises co-administering a viral therapy vaccine.
- In some embodiments, the method comprises co-administering a combination of ingenol-3-angelate (PEP005) and JQ1. In other embodiments, the method comprises co-administering a combination of ingenol-3-angelate (PEP005) and GSK343. In yet other embodiments, the method comprises co-administering a combination of ingenol-3-angelate (PEP005) and vorinostat (suberanilohydroxamic acid; SAHA). In particular embodiments, a combination of ingenol-3-angelate (PEP005) and one or more additional latency reactivation agents (e.g., JQ1, GSK343, and/or SAHA) is capable of synergistically inducing reactivation of the latent virus.
- In yet other aspects, the present invention provides a kit comprising: (a) ingenol-3-angelate (PEP005); and (b) one or more additional latency reactivation agents.
- In some embodiments, the one or more additional latency reactivation agents is selected from the group consisting of a positive transcription elongation factor b activator, a histone methyltransferase (HMT) inhibitor, a histone deacetylase (HDAC) inhibitor, a DNA methyltransferase inhibitor, an NF-κB activator, an Akt/HEXIM-1 modulator, a Jak/Stat pathway modulator, a diterpene compound (e.g., an ingenol derivative, a phorbol ester), a macrolide lactone, a diacylglycerol (DAG) lactone, a protein kinase C (PKC) activator, and combinations thereof. In certain embodiments, the kit further comprises a viral therapy vaccine.
- In some embodiments, the kit comprises a combination of ingenol-3-angelate (PEP005) and JQ1. In other embodiments, the kit comprises a combination of ingenol-3-angelate (PEP005) and GSK343. In yet other embodiments, the kit comprises a combination of ingenol-3-angelate (PEP005) and vorinostat (suberanilohydroxamic acid; SAHA).
- Other objects, features, and advantages of the present invention will be apparent to one of skill in the art from the following detailed description and figures.
-
FIG. 1 : PEP005 induces reactivation of HIV latency in vitro. J-Lat A1 cells were exposed to different concentrations of PEP005 and virus reactivation was measured by GFP expression using flow cytometry. Cell viability was determined by Live/Dead dye staining. (A) Chemical Structure of PEP005. (B, C) PEP005 reactivates HIV in a dose-dependent manner and displays minimal cytotoxicity. (D) PEP005 potently reactivates latent HIV in cell lines compared with other LRAs. J-Lat A1 cells were treated with 10 nM PEP005, 500 nM SAHA, 2 M JQ1, 2 μM GSK343, 10 ng/ml TNF, 2 μM Prostratin, or 5 ng/ml PMA for 24 hours and the percentage of GFP expression was evaluated using flow cytometry. *, p<0.05; **, p<0.01. -
FIG. 2 : PEP005 synergizes with other latency reversing agents in reactivating latent HIV. (A) J-Lat A1 cells were treated with 5 ng/ml PMA, 6 nM PEP005, 500 nM SAHA, 2 μM JQ1, 2 μM GSK343, or 10 μM Prostratin alone or in combination with 6 nM PEP005 for 24 hours and the percentage of GFP expressing cells was determined using flow cytometry. **, p<0.01; *, p<0.05 compared with control treatment; **, p<0.01; *, p<0.05 compared with PEP005 treatment alone. (B) The U1 cells were treated with 5 ng/ml PMA, 6 nM PEP005, 500 nM SAHA, 2 μM JQ1, or 2 μM GSK343, alone or in combination with 6 nM PEP005 for 24 hours and the HIV transcription was evaluated using RT-qPCR. Numbers indicate fold-increase over the control. **, p<0.01; *, p<0.05 compared with control treatment; **, p<0.01; *, p<0.05 compared with PEP005 treatment alone. (C and D) PEP005 synergizes with other LRAs to significantly increase GFP or HIV-1 mRNA expression in J-Lat A1 (C) or U1 (D) cell lines. The Bliss independence model was utilized for calculation of synergy for LRA combinations [35]. Dotted horizontal line signifies pure additive effect (Δfaxy=0). Synergy is defined as Δfaxy>0 while Δfaxy<0 indicates antagonism. Statistical significance was determined using a one tailed ratio t-test comparing predicted and observed drug combination effects. *p<0.05; **p<0.01. -
FIG. 3 : PEP005 activates PKCδ/θ-IκBα/ε-NF-κB signaling. (A) J-Lat A1 cells or PBMCs isolated from peripheral blood of healthy HIV-negative individuals were treated with 12 nM of PEP005 for up to 6 hours. Western blot analysis was performed to detect the expression of PKC isoforms, IκB isoforms, as well as expression of NF-κB/p65. (B) Quantitation of phosphorylation of IκBα or IκBε in J-Lat A1 cells after 2 hr treatment with PEP005 in panel A. Relative band intensities from three independent experiments in J-Lat A1 cells as determined using ImageJ (NIH) are shown in the bar graph. * p<0.05. (C) Quantitation of PKCδ/θ S643/S676 phosphorylation after 2 hr treatment of PEP005 in panel A. Relative band intensities from three independent experiments in J-Lat A1 cells (Left panel) or PBMCs (Right panel) as determined using ImageJ (NIH) are shown in the bar graph. * p<0.05, ** p<0.01. -
FIG. 4 : PEP005-induced HIV reactivation is mediated through PKCδ/θ-IκBα/ε-NF-κB signaling. (A) PEP005-induced HIV reactivation is suppressed by inhibition of the PKCδ/θ. J-Lat A1 cells were treated with 6 nM of PEP005 in the presence of 1, 2 or 5 μM of PKC inhibitor (PKCδ/θ inhibitor; Milipore/Calbiochem (539649)) and evaluated for GFP expression by RT-qPCR. (B) NF-κB inhibition partially suppresses PEP005-induced HIV reactivation in J-Lat A1 cells. J-Lat A1 cells were treated with 6 nM of PEP005 in the presence of 2.5 μM Bay 11-7082, an NF-κB inhibitor, and were evaluated for GFP expression by RT-qPCR. (C) J-Lat A1 cells were treated with PEP005 or alternatively with PKC inhibitor alone or in the presence of PEP005 and the relative binding of NF-κB to the HIV LTR was determined using ChIP-qPCR. -
FIG. 5 : Expression of T cell activation markers in PEP005-treated primary CD4+ T cells. (A) CD4+ T cells were isolated from uninfected control subjects and treated with 12 nM PEP005 for 24 hrs. Total RNA was extracted, and gene expression of CD38, CD69, CD25, or HLA-DR was analyzed by RT-qPCR. (B-F) PBMCs isolated from peripheral blood of healthy HIV-negative donors were treated for 24 or 72 hours with 12 nM of PEP005, and the expression of CD38 (B), CD69 (C, D), and HLA-DR (E, F) was evaluated using flow cytometry after co-staining with the CD3 T cell marker. -
FIG. 6 : PEP005 does not induce expression of pro-inflammatory cytokines in primary CD4+ T cells from peripheral blood of HIV-negative donors. CD4+ T cells were isolated from healthy donors and treated with 6 or 12 nM of PEP005 for 24 or 72 hours, and the relative expression of TNF-α (A), IFN-γ (B), IL-2 (C), and IL-6 (D) was quantified using RT-qPCR and normalized to GAPDH internal control. -
FIG. 7 : PEP005 causes minimal cytotoxicity and T cell proliferation. To evaluate the impact of PEP005 on the cell viability, U1 cells (A), J-Lat A1 cells (B) and CD4+ T cells (C) were treated with 12 nM of PEP005 for 24 hours, the cell viability was examined with the MTT assay and the S-phase cell cycle progression was evaluated after BrdU incorporation using BrdU ELISA. 25-50 μM Etoposide or 100-150 μM Doxorubicin (Doxo) served as positive controls for MTT and BrdU assays respectively [48-50]. Statistical analysis was performed in comparison with controls. **, p<0.01. -
FIG. 8 : PEP005 induces full-length HIV transcripts in primary CD4+ T cells from HIV infected individuals on suppressive ART. Primary CD4+ T cells were isolated from peripheral blood of HIV infected individuals on suppressive ART and treated with 6 or 12 nM PEP005, 200 ng/ml PMA plus 2 μM Ionomycin, or DMSO for 6 hours. Induction of HIV transcription was measured using RT-qPCR for the 5′ LTR region or Poly A region of the virus. **, p<0.01. -
FIG. 9 : PEP005 and JQ1 synergistically induceHIV transcription 6 hrs after treatment in primary CD4+ T cells from HIV infected individuals on suppressive ART. Primary CD4+ T cells were isolated from the peripheral blood of HIV positive individuals on suppressive ART and treated with 12 nM PEP005 alone or in combination with JQ1 for 6 hours, and HIV transcription was quantified using RT-qPCR for the 5′ LTR region or Poly A region of the virus. The amount of cDNA was only enough to amplify either Poly A region or LTR region during PCR indonor 1 anddonor 9. -
FIG. 10 : PEP005 and JQ1 synergistically induceHIV transcription 48 hrs after treatment in primary CD4+ T cells from HIV infected individuals on suppressive ART. Primary CD4+ T cells were isolated from the peripheral blood of HIV positive individuals on suppressive ART and treated with 12 nM PEP005 alone or in combination with JQ1 for 48 hours, and HIV transcription was quantified using RT-qPCR for the 5′ LTR region or Poly A region of the virus. The amount of cDNA was only enough to amplify LTR region during PCR indonor 9. -
FIG. 11 : PEP005 and JQ1 synergistically induce HIV transcription in primary CD4+ T cells from HIV infected individuals on suppressive ART. Primary CD4+ T cells were isolated from the peripheral blood of HIV positive individuals on suppressive ART and treated with 12 nM PEP005 alone or in combination with JQ1 for 6 or 48 hours, and HIV transcription was quantified using RT-qPCR for the 5′ LTR region or Poly A region of the virus. (A) Copy number of HIV RNA in one μg total RNA in the primary CD4+ T cells after reactivation with 12 nM PEP005. *, p<0.05. (B) PEP005 synergizes with JQ1 to significantly increase HIV mRNA expression in primary CD4+ T cells isolated from patients under suppressive ART. The Bliss independence model was utilized for calculation of synergy for LRA combinations [35]. Dotted horizontal line signifies pure additive effect (Δfaxy=0). For these analyses, we included all data for which every parameter for the synergy analysis was available and excluded individual cases where the parameters were not met. Synergy is defined as Δfaxy>0 while Δfaxy<0 indicates antagonism. Statistical significance was determined using a one tailed paired ratio t-test comparing predicted and observed drug combination effects. *p<0.05; **p<0.01. -
FIG. 12 : PEP005 down-modulates the expression of CD4, CCR5 and CXCR4 and inhibits HIV infection of primary CD4+ T cells ex vivo. (A) PEP005 inhibits expression of HIV receptors/co-receptors. The CD4+ T cells were isolated from the peripheral blood of HIV-negative uninfected controls and treated with 12 nM PEP005 for 72 hours. The expression of CD4, CCR5 and CXCR4 genes was evaluated using RT-qPCR after normalizing for GAPDH. (B) and (C) PEP005 inhibits HIV infection of primary CD4+ T cells ex vivo. Primary CD4+ T cells were pre-treated with 12 or 24 nM PEP005 overnight and infected with the virus. The CD4+ T cells were incubated for 5 days without PEP005. The cells were collected for RT-qPCR targeting the HIV LTR region (B), or supernatants were collected for p24 ELISA (C). *, p<0.05; **, p<0.01. - Stable latent viral reservoirs in HIV infected individuals are rapidly reactivated following the interruption of anti-retroviral therapy (ART). Despite an early initiation of ART, viral reservoirs are established and persist as demonstrated in the case of the Mississippi baby and from recent studies of the SIV model of AIDS. Therefore, new strategies are needed for the eradication of the latent HIV reservoirs. The present inventors have discovered that ingenol-3-angelate (PEP005), a member of a new class of anti-cancer ingenol compounds, effectively reactivated HIV from latency in primary CD4+ T cells from HIV infected individuals receiving ART. Importantly, a combination of PEP005 and JQ1, a p-TEFb agonist, reactivated HIV from latency at a level that was on average 7.5-fold higher compared to PEP005 alone. As such, the potency of synergistic effects of PEP005 and JQ1 provides novel opportunities for advancing HIV eradication strategies. In summary, ingenols represent a new group of compounds for combating HIV latency.
- Unless specifically indicated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs. In addition, any method or material similar or equivalent to a method or material described herein can be used in the practice of the present invention. For purposes of the present invention, the following terms are defined.
- The terms “a,” “an,” or “the” as used herein not only include aspects with one member, but also include aspects with more than one member. For instance, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and reference to “the agent” includes reference to one or more agents known to those skilled in the art, and so forth.
- The terms “subject”, “patient” or “individual” are used herein interchangeably to include a human or animal. For example, the animal subject may be a mammal, a primate (e.g., a monkey), a livestock animal (e.g., a horse, a cow, a sheep, a pig, or a goat), a companion animal (e.g., a dog, a cat), a laboratory test animal (e.g., a mouse, a rat, a guinea pig, a bird), an animal of veterinary significance, or an animal of economic significance.
- A “therapeutically effective amount” includes an amount or quantity effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
- As used herein, the term “administering” includes oral administration, topical contact, administration as a suppository, intravenous, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal, or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc. One skilled in the art will know of additional methods for administering an effective amount of the compounds described herein for reactivating a latent viral infection caused by a virus such as HIV.
- By “co-administer” it is meant that a first compound described herein is administered at the same time, just prior to, or just after the administration of a second compound described herein, e.g., the sequential or simultaneous administration of ingenol-3-angelate (PEP005) and one or more latency reactivation agents. For example, two or more compounds can be co-administered by administering a pharmaceutical composition adapted for oral administration that contains the two or more compounds. As another example, two or more compounds can be co-administered by administering one compound and then administering the other compound. In some instances, the co-administered compounds are administered by the same route. In other instances, the co-administered compounds are administered via different routes. For example, one compound can be administered orally, and the other compound can be administered, e.g., sequentially or simultaneously, via intravenous or intramuscular injection.
- The terms “anti-retroviral therapy” or “ART” include administering one or more compounds simultaneously or sequentially to reduce or suppress the replication of a virus within an infected individual. This can be achieved, for example, by administering various classes of anti-retroviral compounds to an infected individual that inhibit various steps of the replication cycle. These classes of compounds include, but are not limited to, fusion inhibitors, nucleoside reverse transcriptase inhibitors, integrase inhibitors, protease inhibitors, and combinations thereof. For example, anti-retroviral therapy can be used to treat viral infections caused by human immunodeficiency virus (HIV), cytomegalovirus (CMV), or adenovirus. One non-limiting goal of this therapy in HIV, for example, is to decrease the viral levels and increase the amount of CD4+ T cells in infected individuals.
- The term “latent virus” includes a state of the viral life cycle where the viral genome has integrated into the chromosomal DNA of the infected cell, but replication of the genome and viral proliferation in the infected cell is dormant. In some instances, the dormant viral replication impedes the treatment of viral infection with anti-retroviral therapy because anti-retroviral therapy targets various stages of the viral replication cycle. For example, latent HIV virus cannot be treated using anti-retroviral therapy, and is known problem inhibiting the functionality of anti-retroviral treatment techniques.
- The term “latent reservoir” includes a cell or group of cells that are infected with a latent virus such as, for example, latent HIV.
- The term “reactivating a latent virus” includes administering a compound or combination of compounds to induce a latent virus infected cell into an active replication and proliferation state. The term includes, inter alia, administering one or more latency reactivation agents described herein.
- The terms “latency reactivation agent” or “latency reversing agent” include a compound or combination of compounds capable of reactivating a latent virus. For example, the terms include a single compound that is capable of inducing reactivation of a latent virus. As another example, the terms also include a combination of two or more compounds that are capable of synergistically or additively inducing reactivation of a latent virus. Latency reactivation agents are useful for treating a variety of latent viral infections including, but not limited to, latent HIV, CMV, and adenoviral infections. Latency reactivation agents include, but are not limited to, ingenol-3-angelate (PEP005), as well as any compound belonging to a class selected from a positive transcription elongation factor b activator, a histone methyltransferase (HMT) inhibitor, a histone deacetylase (HDAC) inhibitor, a DNA methyltransferase inhibitor, an NF-κB activator, an Akt/HEXIM-1 modulator, a Jak/Stat pathway modulator, a diterpene compound (e.g., an ingenol derivative, a phorbol ester), a macrolide lactone, a diacylglycerol (DAG) lactone, a protein kinase C (PKC) activator, and combinations thereof.
- The term “effective amount” includes a dosage sufficient to produce a desired result with respect to the indicated disorder, condition, or mental state. The desired result may comprise a subjective or objective improvement in the recipient of the dosage. In one non-limiting example, an effective amount of PEP005 and JQ1 includes an amount sufficient to reactivate latent HIV expression. Also, for example, an effective amount includes an amount sufficient to suppress HIV infection of primary CD4+ T cells. Thus, an effective amount can be an amount that down-modulates cell surface expression of HIV co-receptors comprising CD4, CCR5, and/or CXCR4. The effective amount will vary with the type of subject being treated, the compound or combination of compounds applied, and/or the amount of anti-retroviral treatments administered to the subject.
- The phrase “enhancing a therapeutic benefit” includes any of a number of subjective or objective factors indicating a beneficial response or improvement of the condition being treated as discussed herein. For example, enhancing the therapeutic benefit comprises administering two or more compounds to produce a desired clinical result greater than any one of the compounds administered separately. Also, for example, enhancing the therapeutic benefit comprises administering two or more latency reactivating agents to produce the desired clinical result greater than any one of the latency reactivating agents administered separately. In yet another example, enhancing the therapeutic benefit comprises administerting two or more latency reactivation agents in combination with a viral therapy vaccine to more effectively combat and kill reactivated virally infected cells.
- The term “pharmaceutically acceptable” includes a substance which does not interfere with the effectiveness or the biological activity of the active ingredients and which is not toxic to the hosts in the amounts used, and which hosts may be either humans or animals to which it is to be administered.
- The term “low to minimal cytotoxicity” includes measuring the toxic effect of an administered compound or compounds in host cells based on the amount of the compound(s) administered. Thus, low to minimal cytotoxicity can be an amount that is not toxic to the cells or at a level of toxicity that the majority of cells are unaffected.
- The term “global T cell activation” includes inducing a large scale immune response in a host organism. In some instances, global T cell activation could trigger a fatal systemic immune response.
- The terms “synergistic” or “synergistically” include an enhanced therapeutic benefit or effect where a combination of two or more compounds produces a result that exceeds their expected additive effect. For example, synergistic latency reactivation agents enhance latent viral reactivation to an extent that exceeds their expected additive effect.
- The term “viral therapy vaccine” includes compositions that are administered to a subject that increase the subject's response to a viral infection. In some instances, a viral therapy vaccine can be administered to a subject to increase the subject's immune response to a viral infection. For example, a viral therapy vaccine can be administered to a subject to increase the subject's ability to combat and kill virally infected cells that have been reactivated from their latent state.
- The term “cytotoxic T lymphocytes (CTL) booster” includes compositions comprising proteins, DNA, RNA, and/or other biologic or non-biologic matter that can be used to improve CTL response and proliferation to combat and kill virally infected cells that have been reactivated from their latent state.
- The present invention provides compositions comprising ingenol-3-angelate (PEP005) and one or more additional latency reactivation agents. The present invention also provides methods for reactivating a latent virus in a subject infected with the virus, the method comprising administering to the subject an effective amount of ingenol-3-angelate (PEP005) optionally in combination with one or more additional latency reactivation agents. In particular embodiments, a combination of ingenol-3-angelate (PEP005) with one or more additional latency reactivation agents advantageously provides a synergistic effect at inducing reactivation of a latent virus such as HIV. The present invention further provides kits comprising ingenol-3-angelate (PEP005) and one or more additional latency reactivation agents.
- 1. Compositions
- In certain aspects, the present invention provides a composition comprising ingenol-3-angelate (PEP005) and one or more additional latency reactivation agents.
- In some embodiments, the ingenol-3-angelate (PEP005) is present in an amount that is effective to enhance a therapeutic benefit of the one or more additional latency reactivation agents. In particular embodiments, the therapeutic benefit comprises reactivation of a latent virus in a subject infected with the virus. In certain instances, the latent virus is present as viral reservoirs in virus-infected cells (e.g., HIV-infected resting CD4+ T cells).
- In some embodiments, the virus is selected from the group consisting of a human immunodeficiency virus (HIV), a cytomegalovirus (CMV), adenoviruses, papovaviruses, herpesviruses, varicella-zoster virus, Epstein-Barr virus, pox viruses, vaccinia virus, hepatitis B virus, rhinoviruses, hepatitis A virus, poliovirus, rubellavirus, hepatitis C virus, arboviruses, rabiesvirus, influenza viruses A and B, measles virus, mumps virus, and HTLV I and II. In particular embodiments, the virus is selected from the group consisting of a human immunodeficiency virus (HIV), a cytomegalovirus (CMV), and an adenovirus.
- In some embodiments, the one or more additional latency reactivation agents is selected from the group consisting of a positive transcription elongation factor b activator, a histone methyltransferase (HMT) inhibitor, a histone deacetylase (HDAC) inhibitor, a DNA methyltransferase inhibitor, an NF-κB activator, an Akt/HEXIM-1 modulator, a Jak/Stat pathway modulator, a diterpene compound (e.g., an ingenol derivative, a phorbol ester), a macrolide lactone, a diacylglycerol (DAG) lactone, a protein kinase C (PKC) activator, and combinations thereof. Non-limiting examples of other latency reactivation agents include juglone (5HN, 5-hydroxynaphthalene-1,4-dione), disulfiram, AV6 (4-3′,4′-dichloroanilino-6-methoxyquinoline), and combinations thereof.
- Non-limiting examples of positive transcription elongation factor b activators include JQ1, hexamethylene bisacetamide (HMBA), and combinations thereof.
- Non-limiting examples of histone methyltransferase (HMT) inhibitors include GSK343, BIX01294, chaetocin, 3-deazaneplanocin A (DZNep), and combinations thereof.
- Non-limiting examples of histone deacetylase (HDAC) inhibitors include vorinostat (suberanilohydroxamic acid; SAHA), suberoyl bis-hydroxamic acid (SBHA), trichostatin A (TsA), scriptaid, oxamflatin, givinostat (ITF2357), belinostat (PXD101), droxinostat, CG05/CG06, valproic acid (VPA), sodium butyrate, apicidin, and combinations thereof.
- Non-limiting examples of DNA methyltransferase inhibitors include decitabine, azacitidine, and combinations thereof.
- Non-limiting examples of NF-κB activators include 12-deoxyphorbol-13-acetate (prostratin), phorbol myristate acetate (PMA), TNFα, and combinations ther Non-limiting examples of the Akt/HEXIM-1 modulator is hexamethylene bisacetamide (HMBA).
- A non-limiting example of a Jak/Stat pathway modulator includes IL-7.
- Non-limiting examples of diterpene compounds include an ingenol derivative, a phorbol ester, SJ23B, gnidimacrin, and combinations thereof. In some embodiments, the ingenol derivative is an ester of the diterpene ingenol and an organic acid. In certain embodiments, the organic acid is a saturated or unsaturated optionally substituted C1-C22 organic acid. In some instances, the ingenol derivative is selected from the group consisting of ingenol-3-trans-cinnamate (ING A), ingenol-3-hexanoate (ING B), ingenol-3-dodecanoate (ING C), 3-(2,4,6-dodecatrienoyl)-ingenol, 3-(2,4,6,8-tetradecatetranoyl)-ingenol, 20-hydroxy-ingenol-3-angelate, 20-O-acetyl-ingenol-3-angelate, and combinations thereof. In other instances, the ingenol derivative is any one of the compounds described in US 2015/0030638 and US 2003/0166613, the disclosures of which are hereby incorporated by reference in their entirety for all purposes. Non-limiting examples of phorbol esters include phorbol myristate acetate (PMA), 12-deoxyphorbol-13-acetate (prostratin), 12-deoxyphorbol 13-phenylacetate (DPP), and combinations thereof.
- A non-limiting example of a macrolide lactone includes bryostatin.
- In particular embodiments, the one or more additional latency reactivation agents is a positive transcription elongation factor b activator. In certain instances, the positive transcription elongation factor b activator is JQ1, i.e., the composition comprises a combination of ingenol-3-angelate (PEP005) and JQ1.
- In particular embodiments, the one or more additional latency reactivation agents is a histone methyltransferase (HMT) inhibitor. In certain instances, the histone methyltransferase (HMT) inhibitor is GSK343, i.e., the composition comprises a combination of ingenol-3-angelate (PEP005) and GSK343.
- In particular embodiments, the one or more additional latency reactivation agents is a histone deacetylase (HDAC) inhibitor. In certain instances, the histone deacetylase (HDAC) inhibitor is vorinostat (suberanilohydroxamic acid; SAHA), i.e., the composition comprises a combination of ingenol-3-angelate (PEP005) and SAHA.
- In some embodiments, the composition further comprises a viral therapy vaccine. In particular embodiments, the viral therapy vaccine is present in an amount effective to enhance a therapeutic benefit of the latency reactivation agents. In certain embodiments, the therapeutic benefit comprises increasing a subject's immune respone (e.g., cytotoxic T lymphocyte (CTL) response) by activating the proliferation of CTLs in the subject to more effectively combat and kill reactivated virally infected cells. In certain instances, the activated CTLs target viral proteins or fragments thereof that are presented on the surface of reactivated virally infected cells. Non-limiting examples of viral therapy vaccines include CTL boosters. Non-limiting examples of CTL boosters include viral proteins that are presented on the surface of infected cells, such as B Gag, Nef, Rev, Tat, and/or Env peptides.
- In some embodiments, the composition further comprises a pharmaceutically acceptable excipient or diluent.
- 2. Methods
- In certain other aspects, the present invention provides a method for reactivating a latent virus in a subject infected with the virus, the method comprising administering to the subject an effective amount of ingenol-3-angelate (PEP005).
- In some embodiments, the effective amount of the ingenol-3-angelate (PEP005) is an amount that is capable of inducing RNA transcription from the latent virus in an infected cell (e.g., CD4+ T cell) from the subject. In certain instances, the latent virus is present as viral reservoirs in virus-infected cells (e.g., HIV-infected resting CD4+ T cells).
- In some embodiments, the effective amount of the ingenol-3-angelate (PEP005) is an amount that displays low to minimal cytotoxicity without inducing global T cell activation (e.g., in the absence of major global T cell activation).
- In some embodiments, the effective amount of the ingenol-3-angelate (PEP005) is an amount that suppresses or prevents the reactivated latent virus from infecting uninfected CD4+ T cells in the subject. In certain embodiments, the effective amount of the ingenol-3-angelate (PEP005) down-modulates the expression of cell surface receptors that are known to mediate viral (e.g., HIV) attachment and entry, thereby preventing the spread of viral infection to uninfected bystander CD4+ T cells.
- In some embodiments, the ingenol-3-angelate (PEP005) is administered orally, topically, intravenously, intraperitoneally, intramuscularly, intralesionally, intrathecally, intranasally, subcutaneously, parenterally, or transmucosally.
- In particular embodiments, the method of the present invention further comprises administering one or more additional latency reactivation agents selected from the group consisting of a positive transcription elongation factor b activator, a histone methyltransferase (HMT) inhibitor, a histone deacetylase (HDAC) inhibitor, a DNA methyltransferase inhibitor, an NF-κB activator, an Akt/HEXIM-1 modulator, a Jak/Stat pathway modulator, a diterpene compound (e.g., an ingenol derivative, a phorbol ester), a macrolide lactone, a diacylglycerol (DAG) lactone, a protein kinase C (PKC) activator, and combinations thereof. Non-limiting examples of other latency reactivation agents include juglone (5HN, 5-hydroxynaphthalene-1,4-dione), disulfiram, AV6 (4-3′,4′-dichloroanilino-6-methoxyquinoline), and combinations thereof.
- Non-limiting examples of positive transcription elongation factor b activators include JQ1, hexamethylene bisacetamide (HMBA), and combinations thereof.
- Non-limiting examples of histone methyltransferase (HMT) inhibitors include GSK343, BIX01294, chaetocin, 3-deazaneplanocin A (DZNep), and combinations thereof.
- Non-limiting examples of histone deacetylase (HDAC) inhibitors include vorinostat (suberanilohydroxamic acid; SAHA), suberoyl bis-hydroxamic acid (SBHA), trichostatin A (TsA), scriptaid, oxamflatin, givinostat (ITF2357), belinostat (PXD101), droxinostat, CG05/CG06, valproic acid (VPA), sodium butyrate, apicidin, and combinations thereof.
- Non-limiting examples of DNA methyltransferase inhibitors include decitabine, azacitidine, and combinations thereof.
- Non-limiting examples of NF-κB activators include 12-deoxyphorbol-13-acetate (prostratin), phorbol myristate acetate (PMA), TNFα, and combinations ther Non-limiting examples of the Akt/HEXIM-1 modulator is hexamethylene bisacetamide (HMBA).
- A non-limiting example of a Jak/Stat pathway modulator includes IL-7.
- Non-limiting examples of diterpene compounds include an ingenol derivative, a phorbol ester, SJ23B, gnidimacrin, and combinations thereof. In some embodiments, the ingenol derivative is an ester of the diterpene ingenol and an organic acid. In certain embodiments, the organic acid is a saturated or unsaturated optionally substituted C1-C22 organic acid. In some instances, the ingenol derivative is selected from the group consisting of ingenol-3-trans-cinnamate (ING A), ingenol-3-hexanoate (ING B), ingenol-3-dodecanoate (ING C), 3-(2,4,6-dodecatrienoyl)-ingenol, 3-(2,4,6,8-tetradecatetranoyl)-ingenol, 20-hydroxy-ingenol-3-angelate, 20-O-acetyl-ingenol-3-angelate, and combinations thereof. In other instances, the ingenol derivative is any one of the compounds described in US 2015/0030638 and US 2003/0166613, the disclosures of which are hereby incorporated by reference in their entirety for all purposes. Non-limiting examples of phorbol esters include phorbol myristate acetate (PMA), 12-deoxyphorbol-13-acetate (prostratin), 12-deoxyphorbol 13-phenylacetate (DPP), and combinations thereof.
- A non-limiting example of a macrolide lactone includes bryostatin.
- In particular embodiments, the method of the present invention further comprises administering one or more additional latency reactivation agents comprising a positive transcription elongation factor b activator. In certain instances, the positive transcription elongation factor b activator is JQ1, i.e., the method comprises co-administering a combination of ingenol-3-angelate (PEP005) and JQ1.
- In particular embodiments, the method of the present invention further comprises administering one or more additional latency reactivation agents comprising a histone methyltransferase (HMT) inhibitor. In certain instances, the positive transcription elongation factor b activator is GSK343, i.e., the method comprises co-administering a combination of ingenol-3-angelate (PEP005) and GSK343.
- In particular embodiments, the method of the present invention further comprises administering one or more additional latency reactivation agents comprising a histone deacetylase (HDAC) inhibitor. In certain instances, the positive transcription elongation factor b activator is vorinostat (suberanilohydroxamic acid; SAHA), i.e., the method comprises co-administering a combination of ingenol-3-angelate (PEP005) and SAHA.
- In preferred embodiments, a combination of the ingenol-3-angelate (PEP005) and the one or more additional latency reactivation agents (e.g., JQ1, GSK343, and/or SAHA) is capable of synergistically inducing reactivation of the latent virus.
- In certain embodiments, the one or more additional latency reactivation agents is each independently administered orally, topically, intravenously, intraperitoneally, intramuscularly, intralesionally, intrathecally, intranasally, subcutaneously, parenterally, or transmucosally.
- In certain embodiments, the method comprises administering an effective amount of ingenol-3-angelate (PEP005) alone or with one or more additional latency reactivation agents for reactivating a latent virus in a virally infected cell in a subject infected with the virus in combination with administering an effective amount of a viral therapy vaccine for increasing a subject's immune respone (e.g., CTL response) to more effectively combat and kill the reactivated virally infected cell.
- In some embodiments, the effective amount of the viral therapy vaccine is an amount that is capable of increasing the subject's immune respone (e.g., cytotoxic T lymphocyte (CTL) response) by activating the proliferation of CTLs in the subject to more effectively combat and kill reactivated virally infected cells. In certain instances, the activated CTLs target viral proteins or fragments thereof that are presented on the surface of reactivated virally infected cells.
- In some embodiments, the effective amount of the viral therapy vaccine is an amount that displays killing of virally infected cells that have been reactivated from their latent state.
- Non-limiting examples of viral therapy vaccines include cytotoxic T lymphocytes (CTL) boosters. Non-limiting examples of CTL boosters include viral proteins that are presented on the surface of infected cells, such as B Gag, Nef, Rev, Tat, and/or Env peptides.
- In some embodiments, the method comprises co-administering a combination of ingenol-3-angelate (PEP005), JQ1, and a cytotoxic T lymphocyte (CTL) booster. In other embodiments, the method comprises co-administering a combination of ingenol-3-angelate (PEP005), GSK343, and a CTL booster. In yet other embodiments, the method comprises co-administering a combination of ingenol-3-angelate (PEP005), vorinostat (suberanilohydroxamic acid; SAHA), and a CTL booster.
- In particular embodiments, a combination of ingenol-3-angelate (PEP005) with one or more additional latency reactivation agents (e.g., JQ1, GSK343, and/or SAHA) and a viral therapy vaccine is capable of synergistically inducing reactivation of the latent virus and increasing an immune response to reactivated virally infected cells.
- In other embodiments, the viral therapy vaccine is administered before ingenol-3-angelate (PEP005) and/or the one or more additional latency reactivation agents is administered.
- In certain embodiments, the viral therapy vaccine is administered orally, topically, intravenously, intraperitoneally, intramuscularly, intralesionally, intrathecally, intranasally, subcutaneously, parenterally, or transmucosally.
- In some embodiments, the virus is selected from the group consisting of a human immunodeficiency virus (HIV), a cytomegalovirus (CMV), adenoviruses, papovaviruses, herpesviruses, varicella-zoster virus, Epstein-Barr virus, pox viruses, vaccinia virus, hepatitis B virus, rhinoviruses, hepatitis A virus, poliovirus, rubellavirus, hepatitis C virus, arboviruses, rabiesvirus, influenza viruses A and B, measles virus, mumps virus, and HTLV I and II. In particular embodiments, the virus is selected from the group consisting of a human immunodeficiency virus (HIV), a cytomegalovirus (CMV), and an adenovirus.
- In some embodiments, the subject is a human. In other embodiments, the subject is receiving anti-retroviral therapy (ART), e.g., highly active anti-retroviral therapy (HAART), suppressive anti-retroviral therapy, and the like.
- 3. Kits
- In yet other aspects, the present invention provides a kit comprising: (a) ingenol-3-angelate (PEP005); and (b) one or more additional latency reactivation agents.
- In some embodiments, the kit further comprises a label with instructions for administering the ingenol-3-angelate (PEP005) and/or the one or more additional latency reactivation agents.
- In certain embodiments, the one or more additional latency reactivation agents is selected from the group consisting of a positive transcription elongation factor b activator, a histone methyltransferase (HMT) inhibitor, a histone deacetylase (HDAC) inhibitor, a DNA methyltransferase inhibitor, an NF-κB activator, an Akt/HEXIM-1 modulator, a Jak/Stat pathway modulator, a diterpene compound (e.g., an ingenol derivative, a phorbol ester), a macrolide lactone, a diacylglycerol (DAG) lactone, a protein kinase C (PKC) activator, and combinations thereof.
- In particular embodiments, the one or more additional latency reactivation agents is a positive transcription elongation factor b activator. In certain instances, the positive transcription elongation factor b activator is JQ1, i.e., the kit comprises a combination of ingenol-3-angelate (PEP005) and JQ1.
- In particular embodiments, the one or more additional latency reactivation agents is a histone methyltransferase (HMT) inhibitor. In certain instances, the positive transcription elongation factor b activator is GSK343, i.e., the kit comprises a combination of ingenol-3-angelate (PEP005) and GSK343.
- In particular embodiments, the one or more additional latency reactivation agents is a histone deacetylase (HDAC) inhibitor. In certain instances, the positive transcription elongation factor b activator is vorinostat (suberanilohydroxamic acid; SAHA), i.e., the kit comprises a combination of ingenol-3-angelate (PEP005) and SAHA.
- In certain embodiments, the kit further comprises a viral therapy vaccine. Non-limiting examples of viral therapy vaccines include cytotoxic T lymphocytes (CTL) boosters. Non-limiting examples of CTL boosters include viral proteins that are presented on the surface of infected cells, such as B Gag, Nef, Rev, Tat, and/or Env peptides.
- In some embodiments, the kit comprises a combination of ingenol-3-angelate (PEP005), JQ1, and a cytotoxic T lymphocytes (CTL) booster. In other embodiments, the kit comprises a combination of ingenol-3-angelate (PEP005), GSK343, and a CTL booster. In yet other embodiments, the kit comprises a combination of ingenol-3-angelate (PEP005), vorinostat (suberanilohydroxamic acid; SAHA), and a CTL booster.
- The present invention provides compositions and kits comprising ingenol-3-angelate (PEP005) as a latency reactivation agent and one or more additional latency reactivation agents. The present invention also provides methods for reactivating a latent virus by administering an effective amount of ingenol-3-angelate (PEP005) as a latency reactivation agent alone or in combination with one or more additional latency reactivation agents.
- Non-limiting examples of additional latency reactivation agents include positive transcription elongation factor b activators, histone methyltransferase (HMT) inhibitors, histone deacetylase (HDAC) inhibitors, DNA methyltransferase inhibitors, NF-κB activators, Akt/HEXIM-1 modulators, Jak/Stat pathway modulators, diterpene compounds (e.g., ingenol derivatives, phorbol esters), macrolide lactones, diacylglycerol (DAG) lactones, protein kinase C (PKC) activators, juglone (5HN, 5-hydroxynaphthalene-1,4-dione), disulfiram, AV6 (4-3′,4′-dichloroanilino-6-methoxyquinoline), pharmaceutically acceptable salts thereof, derivatives thereof, and combinations thereof.
- 1. Ingenol-3-angelate (PEP005)
- Ingenol-3-angelate, also known as ingenol mebutate, PEP005, or PICATO®, is a natural product found in the plant Euphorbia peplus. Ingenol-3-angelate is an ester of the diterpene ingenol and angelic acid. The full IUPAC chemical name of ingenol-3-angelate is (1aR,2S,5R,5aS,6S,8aS,9R,1 OaR)-5,5a-Dihydroxy-4-(hydroxymethyl)-1,1,7,9-tetramethyl-11-oxo-1a,2,5,5a,6,9,10,10a-octahydro-1H-2,8a-methanocyclopenta[a]cyclpropa[e][10]annulen-6-yl (2Z)-2-methylbut-2-enoate. The structure of ingenol-3-angelate is shown below:
- 2. Positive Transcription Elongation Factor b Activators
- Positive transcription elongation factor b, also known as P-TEFb, plays an essential role in the regulation of transcription by RNA polymerase II (Pol II) in eukaryotes. For the HIV virus, P-TEFb is targeted by the HIV Tat protein which bypasses normal cellular P-TEFb control and directly brings P-TEFb to the promoter proximal paused polymerase in the HIV genome. In certain embodiments, activators of P-TEFb are capable of binding to, stimulating, increasing, activating, facilitating, enhancing activation or enzymatic activity, sensitizing or upregulating the activity or expression of P-TEFb. In certain other embodiments, activators of P-TEFb are capable of increasing, enhancing, or upregulating the expression of an mRNA that encodes P-TEFb. Non-limiting examples positive transcription elongation factor b activators include JQ1, hexamethylene bisacetamide (HMBA), and combinations thereof.
- In particular embodiments, the positive transcription elongation factor b activator is JQ1. The full IUPAC chemical name of JQ1 is (S)-tert-butyl 2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate. JQ1 is an inhibitor of the bromo and extra terminal (BET) family of bromodomain proteins. BET proteins are key mediators for the assembly of the P-TEFb complex. The structure of JQ1 is shown below:
- 3. Histone Methyltransferase (HMT) Inhibitors
- Histone methyltransferases, or HMTs, are histone-modifying enzymes that catalyze the transfer of methyl groups to lysine and arginine residues of histone proteins. By modifying the methylation of histone proteins, HMTs play an important biological role in the epigenetic modification of chromatin that directly impacts cellular activities including gene expression, DNA methylation, and genomic stability. Methylation of histone protein H3 at
lysines 9 and 27 has been implicated in HIV latency (see, Shirakawa et al., Trends Microbiol, 21(6):277-285 (2013)). In certain embodiments, inhibitors of HMTs are capable of binding to, inhibiting, partially or totally blocking stimulation or enzymatic activity, decreasing, preventing, delaying activation, inactivating, desensitizing, or down regulating the activity or expression of HMTs. In certain other embodiments, inhibitors of HMTs are capable of decreasing, down regulating, or inhibiting the expression of an mRNA that encodes HMT. Non-limiting examples of HMT inhibitors include GSK343, BIX01294, chaetocin, 3-deazaneplanocin A (DZNep), G9a, and combinations thereof. - In particular embodiments, the HMT inhibitor is GSK343. GSK343 is an inhibitor of EZH2, which is a histone H3-lysine 27 methyltransferase. Inhibition of EZH2 has been implicated in reactivating latent HIV-infected cells. The chemical structure of GSK343 is shown below:
- 4. Histone Deacetylase (HDAC) Inhibitors
- Histone deacetylases, also known as HDACs, are enzymes that remove acetyl groups from N-acetylated lysine amino acids on histone proteins. This process allows the histone proteins to more tightly wrap around DNA molecules. HDAC inhibitors have been shown to induce reactivation of virally infected latent cells, including herpes simples virus and HIV (see, Danaher et al., J Neurovirol, 11(3):306-17 (2005) and Shirakawa et al., Trends Microbiol, 21(6):277-85 (2013)). In certain embodiments, inhibitors of HDACs are capable of binding to, inhibiting, partially or totally blocking stimulation or enzymatic activity, decreasing, preventing, delaying activation, inactivating, desensitizing, or down regulating the activity or expression of HDACs. In certain other embodiments, inhibitors of HDACs are capable of decreasing, down regulating, or inhibiting the expression of an mRNA that encodes HDAC. Exemplary HDAC inhibitors include, but are not limited to, vorinostat, suberoyl bis-hydroxamic acid (SBHA), trichrostatin A (TsA), scriptaid, oxamflatin, givinostat (ITF2357), belinostat (PXD101), droxinostat, CG05/CGO6, valproic acid (VPA), sodium butyrate, apicidin, and combinations thereof.
- In particular embodiments, the HDAC inhibitor is vorinostat. Vorinostat is also known as suberanilohydroxamic acid, SAHA, or ZOLINZA®. The full IUPAC chemical name for SAHA is N-Hydroxy-N′-phenyloctanediamide. The chemical structure of vorinostat is shown below:
- 5. DNA Methyltransferase Inhibitors
- DNA methyltransferases are enzymes that transfer a methyl group to DNA. DNA methylation typically acts to suppress gene transcription, and methylation patterns play an important role in epigenetics. As such, inhibitors of DNA methyltransferas can directly alter cellular gene expression. Indeed, DNA methyltransfase inhibitors, in altering the gene expression pattern, can induce reactivation of HIV infected cells (see, Blazkova et al., PLoS Pathog, 5(8):e1000554 (2009)). In certain embodiments, inhibitors of DNA methyltransferases are capable of binding to, inhibiting, partially or totally blocking stimulation or enzymatic activity, decreasing, preventing, delaying activation, inactivating, desensitizing, or down regulating the activity or expression of DNA methyltransferases. In certain other embodiments, inhibitors of DNA methyltransferases are capable of decreasing, down regulating, or inhibiting the expression of an mRNA that encodes DNA methyltransferase. Non-limiting examples of DNA methyltransferases include decitabine, azacitidine, and combinations thereof.
- 6. NF-κB Activators
- NF-κB is a protein complex that controls, among other things, transcription of cellular DNA; thus, it is a transcription factor. In some instances, activated NF-κB binds to DNA-binding sites where expression of specific genes is turned on. Several viruses, including HIV, have binding sites of NF-κB that control the expression of viral genes. NF-κB activation has been implicated in the reactivation of infected cells from their latent state (see, Hiscott et al., J. Clin. Invest., 107(2):143-151 (2001)). In certain embodiments, activators of NF-κB are capable of binding to, stimulating, increasing, activating, facilitating, enhancing activation or enzymatic activity, sensitizing or upregulating the activity or expression of NF-κB or members of the NF-κB signaling pathway. In certain other embodiments, activators of NF-κB are capable of increasing, enhancing, or upregulating the expression of an mRNA that encodes NF-κB or members of the NF-κB signaling pathway. Non-limiting examples of NF-κB activators include 12-deoxyphorbol-13-acetate (prostratin), phorbol myristate acetate (PMA), TNFα, and combinations thereof.
- 7. Akt/HEXIM-1 Modulators
- Non-phosphorylated HEXIM-1, in combination with other cellular factors including 7SK snRNA, forms a complex with positive transcription elongation factor b (P-TEFb) that sequesters P-TEFb and represses PTEFb activity. When HEXIM-1 is phosphorylated via the phosphoinositide 3-kinase/RAC-alpha serine/threonine-protein kinase (PI3K/Akt) pathway, the protein complex sequestering P-TEFb is released, thereby activating P-TEFb. For the HIV virus, activation of the PI3K/Akt pathway and phosphorylation of HEXIM-1 leading to the release of active P-TEFb has been implicated in the reactivation of HIV infected cells (see, Contreras et al. PLOS Pathog., 3(10):e146 (2007)). In certain embodiments, modulators of Akt/HEXIM-1 are capable of binding to, stimulating, increasing, activating, facilitating, enhancing activation or enzymatic activity, sensitizing or upregulating the activity or expression of members of the PI3K/Akt pathway. In certain other embodiments, modulators of Akt/HEXIM-1 are capable of increasing, enhancing, or upregulating the expression of an mRNA that encodes members of the PI3K/Akt pathway. Non-limiting examples of Akt/HEXIM-1 modulators include hexamethylene bisacetamide (HMBA).
- 8. Jak/Stat Pathway Modulators
- The Jak/Stat pathway is a transcription regulation pathway that transmits information from chemical signals outside the cell to gene promoters on the DNA in the cell nucleus. The pathway is made up of three components, a cellular membrane receptor, a Janus kinase (Jak), and a signal transducer and activator of transcription (Stat). The receptor is activated by a signal from a chemical messenger, thereby activating the kinase function of Jak, which then autophosphorylates itself. The STAT protein then binds to the phosphorylated receptor, where STAT is phosphorylated by JAK. The phosphorylated STAT protein binds to another phosphorylated STAT protein (dimerizes) and translocates into the cell nucleus. In the nucleus, the dimer binds to DNA and promotes transcription of genes responsive to STAT. An activated Jak/Stat pathway has been associated with actively replicating retroviral infections such as HIV (see, Kohler et al., JLB, 73(3) 407-416 (2003)). In certain embodiments, modulators of the Jak/Stat pathway are capable of binding to, stimulating, increasing, activating, facilitating, enhancing activation or enzymatic activity, sensitizing or upregulating the activity or expression of members of the Jak/Stat pathway. In certain other embodiments, modulators of the Jak/Stat pathway are capable of increasing, enhancing, or upregulating the expression of an mRNA that encodes members of the Jak/Stat pathway. Non-limiting examples of Jak/Stat pathway modulators include IL-7.
- 9. Diterpene Compounds
- Diterpenes are a class of organic compounds based off of four isoprene units which are derived from genanylgeraniol pyrophosphate, and most diterpenes are of fungal or plant origin. In some instances, diterpenes have been shown to activate the protein kinase C (PKC) pathway. In certain embodiments, diterpenes are capable modulating the PKC pathway by binding to, stimulating, increasing, activating, facilitating, enhancing activation or enzymatic activity, sensitizing or upregulating the activity or expression of the members of the PKC family. In certain other embodiments, diterpenes are capable modulating the PKC pathway by increasing, enhancing, or upregulating the expression of an mRNA that encodes members of the PKC family. Non-limiting examples of diterpenes include ingenol derivatives, phorbol esters, SJ23B, gnidimacrin, and combinations thereof.
- In some embodiments, the ingenol derivative is an ester of the diterpene ingenol and an organic acid. In certain embodiments, the organic acid is a saturated or unsaturated optionally substituted C1-C22 organic acid. In some instances, the ingenol derivative is selected from the group consisting of ingenol-3-trans-cinnamate (ING A), ingenol-3-hexanoate (ING B), ingenol-3-dodecanoate (ING C), 3-(2,4,6-dodecatrienoyl)-ingenol, 3-(2,4,6,8-tetradecatetranoyl)-ingenol, 20-hydroxy-ingenol-3-angelate, 20-O-acetyl-ingenol-3-angelate, and combinations thereof. In other instances, the ingenol derivative is any one of the compounds described in US 2015/0030638 and US 2003/0166613, the disclosures of which are hereby incorporated by reference in their entirety for all purposes.
- Phorbol esters are tetracyclic members of the diterpene family. Phorbol is the parent diterpene of phorbol esters. Biologically, phorbol esters mimic the action of diacylglycerol, an activator of protein kinase C (PKC). In certain embodiments, phorbol esters are capable modulating the PKC pathway by binding to, stimulating, increasing, activating, facilitating, enhancing activation or enzymatic activity, sensitizing or upregulating the activity or expression of the members of the PKC family. In certain other embodiments, phorbol esters are capable modulating the PKC pathway by increasing, enhancing, or upregulating the expression of an mRNA that encodes members of the PKC family. Non-limiting examples of phorbol esters include phorbol myristate acetate (PMA), 12-deoxyphorbol-13-acetate (prostratin), 12-deoxyphorbol 13-phenylacetate (DPP), and combinations thereof.
- 10. Macrolide Lactones
- Macrolide lactones are organic compounds that contain a large macrocyclic lactone ring. The lactone rings of macrolide lactones are usually 14, 15, or 16-membered. In some instances, macrolide lactones such as bryostatin and analogs thereof have been shown to activate the protein kinase C (PKC) pathway and can induce HIV reactivation in latently infected cells as described in DeChristopher et al., Nature Chemistry, 4, 705-710 (2012). In certain embodiments, macrolide lactones are capable of modulating the PKC pathway by binding to, stimulating, increasing, activating, facilitating, enhancing activation or enzymatic activity, sensitizing or upregulating the activity or expression of members of the PKC family. In certain other embodiments, macrolide lactones are capable modulating the PKC pathway by increasing, enhancing, or upregulating the expression of an mRNA that encodes members of the PKC pathway. Non-limiting examples of macrolide lactones include bryostatin.
- 11. Diacylglycerol (DAG) Lactones
- Diacylglycerol (DAG) lactones are effective modulators of critical cellular signaling pathways. Diacylglycerol (DAG) is a known activator of protein kinase C (PKC) and the PKC pathway, and DAG lactones are structural mimetics of DAG. As such, DAG lactones are also capable of activating the PKC pathway and can induce HIV reactivation in latently infected cells. In certain embodiments, DAG lactones are capable of modulating the PKC pathway by binding to, stimulating, increasing, activating, facilitating, enhancing activation or enzymatic activity, sensitizing or upregulating the activity or expression of members of the PKC family. In certain other embodiments, DAG lactones are capable modulating the PKC pathway by increasing, enhancing, or upregulating the expression of an mRNA that encodes members of the PKC pathway. Non-limiting examples of DAG lactones are described in Hamer et al. (J. Virology, 77(19):10227-10236 (2003)).
- 12. Protein Kinase C (PKC) Activators
- Protein kinase C, also known as PKC, is a family of protein kinase enzymes that are involved in controlling the function of many other proteins and cellular processes by phosphorylating the hydroxyl groups of serine and threonine amino acid residues of these proteins. Activation of PKC has many downstream effects, one of them being NF-κB activation, which can lead to HIV reactivation in latently infected cells. PKC activity is activated by, among other things, diacylglycerol (DAG) or DAG mimetics. In certain embodiments, activators of PKC are capable of binding to, stimulating, increasing, activating, facilitating, enhancing activation or enzymatic activity, sensitizing or upregulating the activity or expression of PKC. In certain other embodiments, activators of PKC are capable of increasing, enhancing, or upregulating the expression of an mRNA that encodes PKC. Non-limiting examples of PKC activators include diterpenes, phorbol esters, macrolide lactones, and DAG lactones such as those compounds described herein.
- In certain aspects, the present invention provides compositions and kits comprising ingenol-3-angelate (PEP005) alone or with one or more additional latency reactivation agents to reactivate latent virally infected cells in combination with a viral therapy vaccine to increase a subject's immune respone (e.g., cytotoxic T lymphocyte (CTL) response) to more effectively combat and kill the reactivated virally infected cells. In other aspects, the present invention provides methods for reactivating a latent virus in a virally infected cell and increasing a subject's immune respone (e.g., CTL response) to more effectively combat and kill the reactivated virally infected cells comprising administering ingenol-3-angelate (PEP005) as a latency reactivation agent alone or with one or more additional latency reactivation agents in combination a viral therapy vaccine.
- Non-limiting examples of viral therapy vaccines include cytotoxic T lymphocytes (CTL) boosters. Non-limiting examples of cytotoxic T lymphocytes (CTL) boosters include viral proteins that are presented on the surface of infected cells. In HIV, those proteins include B Gag, Nef, Rev, Tat, Env, Pol, Vpr, Vpu, and Vif peptides. Other non-limiting examples of CTL boosters include viral DNA, RNA, and other biologic or non-biologic material.
- Cytotoxic T lymphocytes (CTLs) are part of the adaptive immune response, and play a critical role in fighting viral infections. CTLs recognize and bind infected cells using T-cell receptors (TCR). TCRs contain a highly variable binding region that allow them to recognize a large range of antigens. TCRs bind to the major histocompatibility complex I (MHC I) of infected cells presenting an appropriate antigen. TCRs binding is highly specific, so only a small number of CTLs will be able to recognize a particular antigen. Once an antigen is recognized by CTLs binding to the MHC I complex of the infected cell, they activate to induce cellular death. Activated CTLs proliferate to fight the detected infection.
- For the HIV virus, CTL response is limited by viral mutational rate. The high mutational rate in the proteins expressed on the cellular MHC complex creates ‘escape mutations’ that can avoid detection of activated CTLs, making the viral infection difficult to irradicate. The use of a viral therapy vaccine to induce and boost CTL response to better combat the infection, particularly in latently reactivated virally infected cells, is provided herein. In some embodiments, viral therapy vaccines stimulate CTL response to more effectively combat and kill the reactivated virally infected cells. In certain embodiments, CTLs are stimulated with a mixture of consensus HIV peptides known to be exhibited on the MHC I complex of infected cells.
- The compounds described herein are useful in the manufacture of a pharmaceutical composition or a medicament for reactivating latent viral reservoirs in an infected subject. In certain aspects, a pharmaceutical composition or medicament can be administered to a subject for the treatment of a viral infection such as an HIV infection that leads to the reduction or elimination of the virus from latent reservoirs in infected resting cells such as central and/or transitional memory CD4+ T cells. In certain other aspects, a pharmaceutical composition or medicament can be administered to a subject for the treatment of a viral infection such as an HIV infection that leads to the reactivation of virally infected cells from latent reservoirs and activation of the subject's immune system to combat and kill the recently reactivated virally infected cells.
- Pharmaceutical compositions or medicaments for use in the present invention can be formulated by standard techniques or methods well-known in the art of pharmacy using one or more physiologically acceptable carriers or excipients. Suitable pharmaceutical carriers are described herein and in, e.g., “Remington's Pharmaceutical Sciences” by E. W. Martin. Compounds and agents of the present invention and their physiologically acceptable salts and solvates can be formulated for administration by any suitable route, including, but not limited to, orally, topically, nasally, rectally, pulmonary, parenterally (e.g., intravenously, subcutaneously, intramuscularly, etc.), and combinations thereof. In some embodiments, the therapeutic agent is dissolved in a liquid, for example, water. The most suitable route of administration in any given case will depend in part on the nature, severity, and optionally, the stage of the viral infection. Co-administration of a plurality or combination of compounds may be by the same or different route of administration or together in the same pharmaceutical formulation.
- The pharmaceutical compositions of the present invention can include ingenol-3-angelate (PEP005) in combination with positive transcription elongation factor b activators, histone methyltransferase (HMT) inhibitors, histone deacetylase (HDAC) inhibitors, DNA methyltransferase inhibitors, NF-κB activators, Akt/HEXIM-1 modulators, Jak/Stat pathway modulators, diterpene compounds (e.g., ingenol derivatives, phorbol esters), macrolide lactones, diacylglycerol (DAG) lactones, protein kinase C (PKC) activators, and combinations thereof, or any pharmaceutically acceptable salts thereof, as an active ingredient and a pharmaceutically acceptable carrier and/or excipient or diluent. In some embodiments, the pharmaceutical composition can include ingenol-3-angelate (PEP005) in combination with JQ1, GSK343, SAHA, and combinations thereof. In other embodiments, the pharmaceutical composition can include ingenol-3-angelate (PEP005) in combination with one or more latency reactivation agents (e.g., JQ1, GSK343, and/or SAHA) and one or more viral therapy vaccines (e.g., a CTL booster). A pharmaceutical composition of the invention may optionally contain additional therapeutic ingredients, e.g., one or more compounds for use in anti-retroviral therapy.
- The compounds of the present invention can be combined as the active ingredient in intimate admixture with a suitable pharmaceutical carrier and/or excipient according to conventional pharmaceutical compounding techniques. Any carrier and/or excipient suitable for the form of preparation desired for administration is contemplated for use with the compounds disclosed herein.
- In certain embodiments, the pharmaceutical compositions are suitable for systemic administration. Systemic administration includes enteral administration (e.g., absorption of the compound through the gastrointestinal tract) or parenteral administration (e.g., injection, infusion, or implantation). In some embodiments, the compositions may be administered via a syringe or intravenously.
- In some embodiments, the present invention provides a pharmaceutical composition including ingenol-3-angelate (PEP005) in combination with a positive transcription elongation factor b activator, such as JQ1, and a pharmaceutically acceptable excipient. In some embodiments, the present invention provides a pharmaceutical composition including ingenol-3-angelate (PEP005) in combination with a histone methyltransferase (HMT) inhibitor, such as GSK343, and a pharmaceutically acceptable excipient. In some embodiments, the present invention provides a pharmaceutical composition including ingenol-3-angelate (PEP005) and a histone deacetylase (HDAC) inhibitor, such as SAHA, and a pharmaceutically acceptable excipient. In some embodiments, the present invention provides a pharmaceutical composition including ingenol-3-angelate (PEP005) in combination with one or more latency reactivation agents (e.g., JQ1, GSK343, and/or SAHA), one or more viral therapy vaccines (e.g., a CTL booster), and a pharmaceutically acceptable excipient. In some of these embodiments, the pharmaceutically acceptable excipient includes a salt or a diluent.
- For oral administration, a pharmaceutical composition or a medicament can take the form of, e.g., a tablet or a capsule prepared by conventional means with a pharmaceutically acceptable excipient. Preferred are tablets and gelatin capsules comprising the active ingredient(s), together with (a) diluents or fillers, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose (e.g., ethyl cellulose, microcrystalline cellulose), glycine, pectin, polyacrylates and/or calcium hydrogen phosphate, calcium sulfate, (b) lubricants, e.g., silica, anhydrous colloidal silica, talcum, stearic acid, its magnesium or calcium salt (e.g., magnesium stearate or calcium stearate), metallic stearates, colloidal silicon dioxide, hydrogenated vegetable oil, corn starch, sodium benzoate, sodium acetate and/or polyethyleneglycol; for tablets also (c) binders, e.g., magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone and/or hydroxypropyl methylcellulose; if desired (d) disintegrants, e.g., starches (e.g., potato starch or sodium starch), glycolate, agar, alginic acid or its sodium salt, or effervescent mixtures; (e) wetting agents, e.g., sodium lauryl sulfate, and/or (f) absorbents, colorants, flavors and sweeteners. In some embodiments, the tablet contains a mixture of hydroxypropyl methylcellulose, polyethyleneglycol 6000 and titanium dioxide. Tablets may be either film coated or enteric coated according to methods known in the art.
- Liquid preparations for oral administration can take the form of, for example, solutions, syrups, or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives, for example, suspending agents, for example, sorbitol syrup, cellulose derivatives, or hydrogenated edible fats; emulsifying agents, for example, lecithin or acacia; non-aqueous vehicles, for example, almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils; and preservatives, for example, methyl or propyl-p-hydroxybenzoates or sorbic acid. The preparations can also contain buffer salts, flavoring, coloring, and/or sweetening agents as appropriate. If desired, preparations for oral administration can be suitably formulated to give controlled release of the active compound.
- Typical formulations for topical administration include creams, ointments, sprays, lotions, and patches. The pharmaceutical composition can, however, be formulated for any type of administration, e.g., intradermal, subdermal, intravenous, intramuscular, intranasal, intracerebral, intratracheal, intraarterial, intraperitoneal, intravesical, intrapleural, intracoronary or intratumoral injection, with a syringe or other devices. Formulation for administration by inhalation (e.g., aerosol), or for oral, rectal, or vaginal administration is also contemplated.
- Compositions for pulmonary administration include, but are not limited to, dry powder compositions consisting of the powder of a compound described herein, or a salt thereof, and the powder of a suitable carrier and/or lubricant. The compositions for pulmonary administration can be inhaled from any suitable dry powder inhaler device known to a person skilled in the art. In certain instances, the compositions may be conveniently delivered in the form of an aerosol spray from pressurized packs or a nebulizer, with the use of a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound(s) and a suitable powder base, for example, lactose or starch.
- The compounds can also be formulated in rectal compositions, for example, suppositories or retention enemas, for example, containing conventional suppository bases, for example, cocoa butter or other glycerides.
- The compositions set forth herein can be formulated for parenteral administration by injection, for example by bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, for example, in ampoules or in multi-dose containers, with an added preservative. Injectable compositions are preferably aqueous isotonic solutions or suspensions, and suppositories are preferably prepared from fatty emulsions or suspensions. The compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers. Alternatively, the compound(s) can be in powder form for reconstitution with a suitable vehicle, for example, sterile pyrogen-free water, before use. In addition, they may also contain other therapeutically valuable substances. The compositions are prepared according to conventional mixing, granulating or coating methods, respectively, and contain about 0.1 to 75%, preferably about 1 to 50%, of the compound(s).
- Furthermore, the compound(s) can be formulated as a depot preparation. Such long-acting formulations can be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, one or more of the compounds described herein can be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
- In some embodiments, the compounds are prepared with a polysaccharide such as chitosan or derivatives thereof (e.g., chitosan succinate, chitosan phthalate, etc.), pectin and derivatives thereof (e.g., amidated pectin, calcium pectinate, etc.), chondroitin and derivatives thereof (e.g., chondroitin sulfate), and alginates.
- In some embodiments, the compounds are loaded onto polymeric nanoparticles that can target the site of infection. Examples of nanoparticles include biodegradable nanoparticles, pH-sensitive nanoparticles (e.g., comprising Eudragit® S100), trimethylchitosan nanoparticles, polymeric nanoparticles (e.g., comprising PLGA, PEG-PLGA and/or PEG-PCL), and mannose-grafted polymeric nanoparticles. See, e.g., Coco et al., Int. J. Pharm. 440:3-12 (2013).
- The compounds can be encapsulated in a controlled drug-delivery system such as a pressure controlled delivery capsule (see, e.g., Takaya et al., J. Control Rel., 50:111-122 (1998)), a colon targeted delivery system, a osmotic controlled drug delivery system, and the like. The pressure controlled delivery capsule can contain an ethylcellulose membrane. The colon target delivery system can contain a tablet core containing lactulose which is over coated with an acid soluble material, e.g., Eudragit E®, and then overcoated with an enteric material, e.g., Eudragit L®. The osmotic controlled drug delivery system can be a single or more osmotic unit encapsulated with a hard gelatin capsule (e.g., capsule osmotic pump; commercially available from, e.g., Alzet, Cupertino, Calif.). Typically, the osmotic unit contains an osmotic push layer and a drug layer, both surrounded by a semipermeable membrane. For a broad overview of delivery systems, see, Banga, A. J., THERAPEUTIC PEPTIDES AND PROTEINS: FORMULATION, PROCESSING, AND DELIVERY SYSTEMS, Technomic Publishing Company, Inc., Lancaster, Pa. (1995), incorporated herein by reference.
- Polymers can be used for ion-controlled release of compositions of the present invention. Various degradable and non-degradable polymeric matrices for use in controlled drug delivery are known in the art (see, Langer R., Accounts Chem. Res., 26:537-542 (1993)). For example, the block copolymer, polaxamer 407, exists as a viscous yet mobile liquid at low temperatures but forms a semisolid gel at body temperature. In other embodiments, liposomes are used for controlled release as well as drug targeting of the lipid-capsulated drug (Betageri et al., LIPOSOME DRUG DELIVERY SYSTEMS, Technomic Publishing Co., Inc., Lancaster, Pa. (1993)).
- In some embodiments, the compositions further include a pharmaceutical surfactant. In other embodiments, the compositions further include a cryoprotectant. Non-limiting examples of cryoprotectants include glucose, sucrose, trehalose, lactose, sodium glutamate, PVP, cyclodextrin, 2-hydroxypropyl-β-cyclodextrin (HPI3CD) glycerol, maltose, mannitol, saccharose, and mixtures thereof.
- Pharmaceutical compositions or medicaments can be administered to a subject at a therapeutically effective dose to reactivate latent infections by viruses, as described herein. In some embodiments, the pharmaceutical composition or medicament is administered to a subject in an amount sufficient to elicit an effective therapeutic response in the subject. In certain embodiments, the pharmaceutical composition or medicament can be administered to a subject at a therapeutically effective dose to reactivate virally infected cells from latent reservoirs and increase the subject's immune system to combat and kill reactivated virally infected cells.
- In some embodiments, the methods of administration comprise administering ingenol-3-angelate (PEP005) alone or in combination with one or more additional latency reactivation agents including positive transcription elongation factor b activators, histone methyltransferase (HMT) inhibitors, histone deacetylase (HDAC) inhibitors, DNA methyltransferase inhibitors, NF-κB activators, Akt/HEXIM-1 modulators, Jak/Stat pathway modulators, diterpene compounds (e.g., ingenol derivatives, phorbol esters), macrolide lactones, diacylglycerol (DAG) lactones, protein kinase C (PKC) activators, and combinations thereof to a patient in need thereof. In some embodiments, the methods of administration can include PEP005 in combination with JQ1, GSK343, SAHA, and combinations thereof.
- In some embodiments, ingenol-3-angelate (PEP005) is administered to a patient infected with a virus, such as HIV, to reactivate a latent viral infection.
- In some embodiments, ingenol-3-angelate (PEP005) is administered in combination with a positive transcription elongation factor b activator, such as JQ1, to a patient infected with a virus, such as HIV, to reactivate a latent viral infection.
- In some embodiments, ingenol-3-angelate (PEP005) is administered in combination with a histone methyltransferase (HMT) inhibitor, such as GSK343, to a patient infected with a virus, such as HIV, to reactivate a latent viral infection.
- In some embodiments, ingenol-3-angelate (PEP005) is administered in combination with a histone deacetylase (HDAC) inhibitor, such as SAHA, to a patient infected with a virus, such as HIV, to reactivate a latent viral infection.
- In some embodiments, ingenol-3-angelate (PEP005) is administered in combination with a DNA methyltransfase inhibitor to a patient infected with a virus, such as HIV, to reactivate a latent viral infection.
- In some embodiments, ingenol-3-angelate (PEP005) is administered in combination with an NF-κB activator to a patient infected with a virus, such as HIV, to reactivate a latent viral infection.
- In some embodiments, ingenol-3-angelate (PEP005) is administered in combination with a Akt/HEXIM-1 modulator to a patient infected with a virus, such as HIV, to reactivate a latent viral infection.
- In some embodiments, ingenol-3-angelate (PEP005) is administered in combination with a Jak/Stat pathway modulator to a patient infected with a virus, such as HIV, to reactivate a latent viral infection.
- In some embodiments, ingenol-3-angelate (PEP005) is administered in combination with a diterpene compound to a patient infected with a virus, such as HIV, to reactivate a latent viral infection.
- In some embodiments, ingenol-3-angelate (PEP005) is administered in combination with an ingenol derivative to a patient infected with a virus, such as HIV, to reactivate a latent viral infection.
- In some embodiments, ingenol-3-angelate (PEP005) is administered in combination with a phorbol ester to a patient infected with a virus, such as HIV, to reactivate a latent viral infection.
- In some embodiments, ingenol-3-angelate (PEP005) is administered in combination with a macrolide lactone to a patient infected with a virus, such as HIV, to reactivate a latent viral infection.
- In some embodiments, ingenol-3-angelate (PEP005) is administered in combination with a diacylglycerol lactone to a patient infected with a virus, such as HIV, to reactivate a latent viral infection.
- In some embodiments, ingenol-3-angelate (PEP005) is administered in combination with a protein kinase C activator, such as a diterpene compound, to a patient infected with a virus, such as HIV, to reactivate a latent viral infection.
- In some embodiments, ingenol-3-angelate (PEP005) is administered with a viral therapy vaccine and optionally one or more latency reactivation agents to a patient infected with a virus, such as HIV, to reactivate a latent viral infection (i.e., latent virally infected cells) and to increase the patient's immune system to combat and kill the reactivated virally infected cells.
- The formulations of the present invention are useful in the manufacture of a pharmaceutical composition or a medicament. A pharmaceutical composition or medicament can be administered to a subject in need thereof, e.g. a patient infected with a viral infection, such as, but not limited to HIV, CMV, and adenovirus.
- In certain methods of reactivating a latent virus, set forth herein, the methods comprise first administering ingenol-3-angelate (PEP005), to a patient infected with a latent virus, and then administering a positive transcription elongation factor b activator, such as JQ1, to the patient. In certain methods of reactivating a latent virus, set forth herein, the methods comprise first administering a positive transcription elongation factor b activator, such as JQ1, to a patient infected with a latent virus, and then administering ingenol-3-angelate (PEP005) to the patient. In certain other methods of reactivating a latent virus, ingenol-3-angelate (PEP005) and a positive transcription elongation factor b activator, such as JQ1, are administered simultaneously to a patient infected with a latent virus.
- In certain methods of reactivating a latent virus, set forth herein, the methods comprise first administering ingenol-3-angelate (PEP005), to a patient infected with a latent virus, and then administering a histone methyltransferase (HMT) inhibitor, such as GSK343, to the patient. In certain methods of reactivating a latent virus, set forth herein, the methods comprise first administering a histone methyltransferase (HMT) inhibitor, such as GSK343, to a patient infected with a latent virus, and then administering ingenol-3-angelate (PEP005) to the patient. In certain other methods of reactivating a latent virus, ingenol-3-angelate (PEP005) and a histone methyltransferase (HMT) inhibitor, such as GSK343, are administered simultaneously to a patient infected with a latent virus.
- In certain methods of reactivating a latent virus, set forth herein, the methods comprise first administering ingenol-3-angelate (PEP005), to a patient infected with a latent virus, and then administering a histone deacetylase (HDAC) inhibitor, such as SAHA, to the patient. In certain methods of reactivating a latent virus, set forth herein, the methods comprise first administering a histone deacetylase (HDAC) inhibitor, such as SAHA, to a patient infected with a latent virus, and then administering ingenol-3-angelate (PEP005) to the patient. In certain other methods of reactivating a latent virus, ingenol-3-angelate (PEP005) and a histone deacetylase (HDAC) inhibitor, such as SAHA, are administered simultaneously to a patient infected with a latent virus.
- In certain methods of reactivating a latent virus and combating and killing reactivated virally infected cells, set forth herein, the methods comprise first administering a viral therapy vaccine to a patient infected with a latent virus, and then administering ingenol-3-angelate (PEP005) to the patient. In certain other methods of reactivating a latent virus and combating and killing reactivated virally infected cells, set forth herein, the methods comprise first administering a viral therapy vaccine to a patient infected with a latent virus, and then administering ingenol-3-angelate (PEP005) as well as one or more latency reactivating agents to the patient. In certain other methods of reactivating a latent virus and combating and killing reactivated virally infected cells, set forth herein, the methods comprise simultaneously administering a viral therapy vaccine, the ingenol-3-angelate (PEP005), and optionally one or more latency reactivating agents to a patient infected with a latent virus.
- In some embodiments, the present invention provides a method of delivering an effective amount of ingenol-3-angelate (PEP005) alone or in combination with one or more additional latency reactivation agents including positive transcription elongation factor b activators, histone methyltransferase (HMT) inhibitors, histone deacetylase (HDAC) inhibitors, DNA methyltransferase inhibitors, NF-κB activators, Akt/HEXIM-1 modulators, Jak/Stat pathway modulators, diterpene compounds (e.g., ingenol derivatives, phorbol esters), macrolide lactones, diacylglycerol (DAG) lactones, protein kinase C (PKC) activators, and combinations thereof to a patient in need thereof.
- In some embodiments, the present invention provides a method of delivering an effective amount of a viral therapy vaccine and ingenol-3-angelate (PEP005) alone or in combination with one or more additional latency reactivation agents described herein.
- Pharmaceutical compositions or medicaments can be administered to a subject at a therapeutically effective dose to reactivate latent viral infections as described herein. The pharmaceutical composition or medicament is administered to a subject in an amount sufficient to elicit an effective therapeutic response in the subject.
- The dosage of compounds administered is dependent on the subject's body weight, age, individual condition, surface area or volume of the area to be treated, and/or on the form of administration. The size of the dose will also be determined by the existence, nature, and extent of any adverse effects that accompany the administration of a particular compound in a particular subject. Typically, a dosage of the active compounds is a dosage that is sufficient to achieve the desired effect. Optimal dosing schedules can be calculated from measurements of compound accumulation in the body of a subject. In general, dosage may be given once or more daily, weekly, or monthly. Persons of ordinary skill in the art can easily determine optimum dosages, dosing methodologies, and repetition rates.
- In some embodiments, a unit dosage for oral administration to a subject (e.g., human) of about 50 to about 70 kg may contain between about 1 and about 500 mg, about 5 and about 500 mg, about 5 and about 250 mg, about 25 to about 250 mg, about 100 and about 1000 mg, about 200 and about 2000 mg, about 500 and about 5000 mg, or about 1000 and about 2000 mg of the compound(s). In particular embodiments, a unit dosage for oral administration to a subject (e.g., human) of about 50 to about 70 kg may contain about 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 75 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1250 mg, 1500 mg, 2000 mg, 2500 mg, 3000 mg, or more of the compound(s).
- In some embodiments, a unit dosage for intravenous administration to a subject (e.g., human) of about 50 to about 70 kg may contain between about 0.1 and about 100 mg, about 0.5 and about 100 mg, about 0.5 and about 50 mg, about 0.5 and about 25 mg, about 0.5 and about 10 mg, about 0.25 to about 50 mg, about 0.25 to about 25 mg, about 0.1 to about 50 mg, about 0.1 to about 25 mg, or about 0.1 to about 10 mg of the compound(s). In particular embodiments, a unit dosage for intravenous administration to a subject (e.g., human) of about 50 to about 70 kg may contain about 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 75 mg, 100 mg, or more of the compound(s).
- Exemplary doses of the compositions described herein include milligram or microgram amounts of the composition per kilogram of subject weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 10 micrograms per kilogram to about 5 milligrams per kilogram, about 10 microgram per kilogram to about 500 micrograms per kilogram, or about 1 microgram per kilogram to about 100 micrograms per kilogram. It is furthermore understood that appropriate doses of a composition depend upon the potency of the composition with respect to the desired effect to be achieved. When one or more of these compositions is to be administered to a mammal, a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular subject will depend upon a variety of factors including the activity of the specific composition employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.
- In some embodiments, a pharmaceutical composition or medicament of the present invention is administered orally, e.g., in a dose in the range of from about 1 to about 1000 micrograms (μg) of compound per kg of subject body weight, from about 1 to about 500 μg/kg body weight, from about 10 to about 1000 μg/kg body weight, from about 10 to about 500 μg/kg body weight, from about 50 to about 1000 μg/kg body weight, from about 50 to about 500 μg/kg body weight, from about 100 to about 1000 μg/kg body weight, or from about 100 to about 500 μg/kg body weight. In particular embodiments, the dose is about 10, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 μg/kg body weight. The dose can be administered once per day or divided into sub-doses and administered in multiple doses, e.g., twice, three times, or four times per day. However, as will be appreciated by a skilled artisan, oral compositions described herein may be administered in different amounts and at different times.
- In some embodiments, a pharmaceutical composition or medicament of the present invention is administered intravenously, e.g., in a dose in the range of from about 1 to about 500 micrograms (μg) of compound per kg of subject body weight, from about 1 to about 200 μg/kg body weight, from about 1 to about 100 μg/kg body weight, from about 10 to about 500 μg/kg body weight, from about 10 to about 200 μg/kg body weight, from about 10 to about 100 μg/kg body weight, or from about 10 to about 80 μg/kg body weight. In particular embodiments, the dose is about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, or 500 μg/kg body weight. The dose can be administered once per day or divided into sub-doses and administered in multiple doses, e.g., twice, three times, or four times per day. However, as will be appreciated by a skilled artisan, intravenous compositions described herein may be administered in different amounts and at different times.
- In certain embodiments, ingenol-3-angelate (PEP005) is administered orally at a dose between about 100 to about 500 μg/kg body weight. In other embodiments, ingenol-3-angelate (PEP005) is administered intravenously at a dose between about 10 to about 80 μg/kg body weight. In particular embodiments, ingenol-3-angelate (PEP005) is adminstered in combination with one or more additional latency reactivation agents described herein. In some embodiments, each compound may be administered at a dose of the individual compounds that is effective for reactivating a latent virus. In other embodiments, each compound may be administered at a lower dose relative to the dose of the individual compounds that is effective for reactivating a latent virus. Without being bound to any particular theory, it has been found that the administration of a lower dose of one or more additional latency reactivation agents with a lower dose of ingenol-3-angelate (PEP005) is advantageous because such a combination of compounds reduces or eliminates the side-effects associated with the administration of the additional latency reactivation agents at the higher concentrations that are effective for reactivating a latent virus when they are used alone.
- In some embodiments, the lower dose of ingenol-3-angelate (PEP005) when used in combination with one or more additional latency reactivation agents is about 100 μg/kg body weight or less for oral administration (e.g., about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 μg/kg body weight) or about 10 μg/kg body weight of less for intravenous administration (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μg/kg body weight). In other embodiments, the lower dose of the one or more additional latency reactivation agents is reduced by about 25% to about 35% (e.g., by about 25%, 30%, or 35%) compared to the dose of the additional latency reactivation agent alone.
- In some embodiments, the compounds are administered one or more times a day, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times a day.
- In some embodiments, the compounds are administered for about 1 to about 31 days, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. In some embodiments, the compounds are administered for at least 1 day. In other embodiments, the compounds are administered for one or more weeks, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more weeks. In yet other embodiments, the compounds are administered for one or more months, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months.
- To achieve the desired therapeutic effect, compounds may be administered for multiple days at the therapeutically effective daily dose. Thus, therapeutically effective administration of compounds to treat a pertinent condition or disease described herein in a subject requires periodic (e.g., daily or twice daily) administration that continues for a period ranging from three days to two weeks or longer. While consecutive daily doses are a preferred route to achieve a therapeutically effective dose, a therapeutically beneficial effect can be achieved even if the agents are not administered daily, so long as the administration is repeated frequently enough to maintain a therapeutically effective concentration of the agents in the subject. For example, one can administer the agents every day, every other day, or, if higher dose ranges are employed and tolerated by the subject, twice a week.
- Optimum dosages, toxicity, and therapeutic efficacy of such compounds may vary depending on the relative potency of individual compounds and can be determined by standard pharmaceutical procedures in experimental animals, for example, by determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio, LD50/ED50. Agents that exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side-effects can be used, care should be taken to design a delivery system that targets such compounds to the affected site to minimize potential damage to normal cells and, thereby, reduce side-effects.
- The data obtained from, for example, animal studies can be used to formulate a dosage range for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration.
- A dose can be formulated in animal models to achieve a concentration range that includes the IC50 (the concentration of the agent that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in stool or an enteric tissue sample can be measured, for example, by high performance liquid chromatography (HPLC). In general, the dose equivalent of compounds is from about 1 ng/kg to about 100 mg/kg for a typical subject.
- The dosage of a pharmaceutical composition of the present invention can be monitored and adjusted throughout treatment, depending on severity of symptoms, frequency of recurrence, and/or the physiological response to the therapeutic regimen. Those of skill in the art commonly engage in such adjustments in therapeutic regimens.
- In some embodiments, a pharmaceutical composition or medicament is administered to a patient at a therapeutically effective dose to induce reactivation of latent virally infected cells. The pharmaceutical composition or medicament is administered to a patient in an amount sufficient to elicit an effective therapeutic response in the patient. An effective therapeutic response is a response that at least partially reactivates latent virally infected cells. An amount adequate to accomplish this is defined as a “therapeutically effective dose.”
- Single or multiple administrations of the compositions are administered depending on the dosage and frequency as required and tolerated by the patient. In any event, the composition should provide a sufficient quantity of the compounds of the invention to effectively treat the patient. Generally, the dose is sufficient to induce viral reactivation from latent virally infected cells without producing unacceptable toxicity or side-effects to the patient.
- A wide variety of kits and systems can be prepared according to the present invention, depending upon the intended user of the kit and system and the particular needs of the user. In some embodiments, the present invention provides a kit that includes ingenol-3-angelate (PEP005) and one or more additional latency reactivation agents selected from positive transcription elongation factor b activators, histone methyltransferase (HMT) inhibitors, histone deacetylase (HDAC) inhibitors, DNA methyltransferase inhibitors, NF-κB activators, Akt/HEXIM-1 modulators, Jak/Stat pathway modulators, diterpene compounds (e.g., ingenol derivatives, phorbol esters), macrolide lactones, diacylglycerol (DAG) lactones, protein kinase C (PKC) activators, and combinations thereof. In certain embodiments, the kit includes ingenol-3-angelate (PEP005) and a compound selected from JQ1, GSK343, SAHA, and combinations thereof. In certain embodiments, the kit includes ingenol-3-angelate (PEP005) and JQ1. In other embodiments, the kit includes ingenol-3-angelate (PEP005) and GSK343. In yet other embodiments, the kit includes ingenol-3-angelate (PEP005) and SAHA. In other embodiments, the kit includes ingenol-3-angelate (PEP005), one or more additional latency reactivation agents (e.g., JQ1, GSK343, and/or SAHA), and one or more viral therapy vaccines (e.g., CTL booster).
- Some of the kits described herein include a label describing a method of administering ingenol-3-angelate (PEP005) and/or one or more additional latency reactivation agents described herein. Some of the kits described herein include a label describing a method of reactivating a latent virus, e.g., HIV, CMV, or adenovirus.
- The compositions of the present invention, including but not limited to compositions including ingenol-3-angelate (PEP005), one or more additional latency reactivation agents, and optionally a viral therapy vaccine described herein may, if desired, be presented in a bottle, jar, vial, ampoule, tube, or other container-closure system approved by the Food and Drug Administration (FDA) or other regulatory body, which may provide one or more dosages containing the compounds. The package or dispenser may also be accompanied by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, the notice indicating approval by the agency. In certain aspects, the kit may include a formulation or composition as described herein, a container closure system including the formulation or a dosage unit form including the formulation, and a notice or instructions describing a method of use as described herein.
- In some embodiments, the kit includes a container which is compartmentalized for holding the various elements of a formulation (e.g., the dry ingredients and the liquid ingredients) or composition, instructions for making the formulation or composition, and instructions for administering the formulation or composition for reactivating a latent virus such as, e.g., HIV, CMV, adenovirus, or combinations thereof.
- In certain embodiments, the kit may include the pharmaceutical preparation in dehydrated or dry form, with instructions for its rehydration (or reconstitution) and administration.
- Kits with unit doses of the compounds described herein, e.g. in oral, rectal, transdermal, or injectable doses (e.g., for intramuscular, intravenous, or subcutaneous injection), are provided. In such kits, an informational package insert describing the use and attendant benefits of the composition for reactivating a latent virus may be included in addition to the containers containing the unit doses.
- Some embodiments of the present invention include packages that include ingenol-3-angelate (PEP005) and one or more additional latency reactivation agents.
- Some other embodiments of the present invention include packages that include ingenol-3-angelate (PEP005), one or more additional latency reactivation agents (e.g., JQ1, GSK343, and/or SAHA), and one or more viral therapy vaccines (e.g., CTL booster).
- The following example is offered to illustrate, but not to limit, the claimed invention.
- Although anti-retroviral therapy (ART) is highly effective in suppressing HIV replication, it fails to eradicate the virus from HIV-infected individuals. Stable latent HIV reservoirs are rapidly established early after HIV infection. Therefore, effective strategies for eradication of the HIV reservoirs are urgently needed. This example demonstrates that ingenol-3-angelate (PEP005), the only active component in a previously FDA approved drug (PICATO) for the topical treatment of precancerous actinic keratosis, can effectively reactivate latent HIV in vitro and ex vivo with relatively low cellular toxicity. Biochemical analysis showed that PEP005 reactivated latent HIV through the induction of the pS643/S676-PKCδ/θ-IκBα/ε-NF-κB signaling pathway. Importantly, PEP005 alone was sufficient to induce expression of fully elongated and processed HIV RNAs in primary CD4+ T cells from HIV infected individuals receiving suppressive ART. Furthermore, PEP005 and the P-TEFb agonist, JQ1, exhibited synergism in reactivation of latent HIV with a combined effect that is 7.5-fold higher than the effect of PEP005 alone. Conversely, PEP005 suppressed HIV infection of primary CD4+ T cells through down-modulation of cell surface expression of HIV co-receptors. As such, this example demonstrates that PEP005 is a safe and effective compound for HIV eradication strategies.
- Anti-retroviral therapy (ART) is effective in suppressing HIV replication but it fails to eliminate latent viral reservoirs in HIV infected resting CD4+ T cells which, in blood, consist mainly of central and transitional memory CD4+ T cells [1-4]. Current ART options do not eradicate HIV from infected cells. In addition, these cells are invisible to the virus-specific immune responses in the setting of viral latency [5,6]. The viral reservoir is rapidly seeded and HIV latency might be established immediately after virus infection [7,8]. Despite initiation of ART in infants within hours of birth to HIV infected mothers, stable viral reservoirs were established and viral rebound occurred when therapy was interrupted [9]. In the simian immunodeficiency virus (SIV) model of AIDS, stable viral reservoirs are established within 2.5 days of infection [10]. The viral reactivation was detected in rhesus macaques following therapy interruption despite the initiation of ART at 3 days post SIV infection [10,11]. Collectively, these studies demonstrate that a very early initiation of ART may not be sufficient to prevent nor eliminate latent virus reservoirs [9,11,12]. It has been observed that the morbidity of HIV persistence in HIV-positive individuals on long-term ART includes drug toxicities and a higher risk of developing complications including dyslipidemia, cardiovascular disease and insulin resistance [13-15]. Therefore, a therapeutic cure of HIV is urgently needed that leads to viral eradication and experimental strategies for directly targeting HIV latent reservoirs are warranted.
- Recent studies have explored an experimental strategy for viral eradication of HIV infected CD4+ T cells by activating HIV transcription and viral antigen expression from the latent viral reservoirs in the presence of ART [6]. This would lead to the detection and clearance of infected cells by the virus-specific host immune responses while the ART prevents new rounds of infection. Cytopathic effects of the viral reactivation would further increase the clearance of the latent viral reservoir. This “shock and kill” strategy was applied in a pilot clinical trial using the histone deacetylase (HDAC) inhibitor, vorinostat, in patients receiving suppressive ART [16-18]. The findings from these studies showed some promise but failed to result in significant clearance of residual HIV reservoirs. Potential mechanisms of this failure include the modest induction of HIV by this earlier generation of latency reversing agents (LRAs) used singly and due to immune defects in clearance of infected cells in spite of the reactivation of viral expression [19,20]. These studies demonstrate an urgent need for the development of new strategies both for disrupting HIV latency and facilitating elimination of infected cells after HIV expression is reactivated.
- Several cell signaling pathways are critical for the establishment and maintenance of HIV latency [6,21,22]. Disruption of one or more of these pathways could lead to effective reactivation of HIV from latency. Various compounds have been tested for the disruption of HIV latency, and those inducing HIV reactivation from the viral long terminal repeat (LTR) through the stimulation of the protein kinase C (PKC)-NF-κB pathway showed high potency. These include phorbol esters (PMA and prostratin) and non-phorbol ester diterpenes (bryostatin and gnidimacrin) that induce NF-κB nuclear translocation and activation through the PKC pathway [22,23]. Some of these compounds effectively induce latent HIV reactivation in vitro at picomolar levels [24,25]. The LRAs, functioning through the PKC-NF-κB signaling, are able to reactivate latent HIV across a broad range of HIV latency models [20]. A recent study showed that LRAs stimulating PKC-NF-κB signaling may be most effective in inducing complete transcription of HIV from resting CD4+ T cells of HIV infected individuals on suppressive ART [26]. Moreover, these compounds cause down-modulation of the expression of cell surface receptors, CD4, CXCR4 or CCR5, and protect cells against HIV infection [22]. Therefore, LRAs that activate PKC-NF-κB signaling are potential candidates for HIV cure studies. We previously reported that an ingenol ester, ingenol-3-hexanoate or IngB, is an excellent candidate for the reactivation of HIV from latency [24]. The modified ingenol-3-hexanoate was originally isolated from an Amazonian plant, Euphorbia tirucalli. It exerts low toxicity in CD4+ T cells and does not induce global T-cell activation. It caused reactivation of latent HIV at nanomolar levels [24]. However, since IngB induces expression and activation of both NF-κB and CyclinT1/CDK9, and stimulates IFNγ expression in primary CD4+ T cells, further search for new ingenol compounds with better HIV reactivation potential and lower cellular toxicity is needed [24,27,28].
- Among previously identified ingenol compounds, ingenol-3-angelate (PEP005) is currently approved for clinical use. A recently FDA approved drug, PICATO, for the topical treatment of precancerous actinic keratosis contains ingenol-3-angelate as an active component [29]. A prior study suggested that ingenol-3-angelate could induce HIV expression from the U1 monocyte cell line harboring HIV genome [30]. In the current study, we report that PEP005 can effectively reactivate latent HIV through the activation of the pS643/S676-PKCδ/θ-IκBα/ε-NF-κB pathway in an HIV latency model in vitro but does not induce or increase NF-κB protein production by itself. It also reactivated full-length HIV transcription based on an assay targeting the poly A tail region of HIV transcripts in cells from ART-suppressed HIV-positive individuals, while exerting minimal toxicity and effects on T cell activation ex vivo [26]. Importantly, the effect of PEP005 was synergistic with JQ1, a p-TEFb activator, and the combination was highly potent in reactivating latent HIV expression both in vitro and ex vivo. Our findings identify this anti-cancer drug, PEP005, as having a distinct mechanism of molecular signaling and as a compound for advancing HIV eradication strategies.
- Cell Culture:
- J-Lat A1 cells (harboring a single copy of latent HIV LTR and one copy of green fluorescent protein gene under the HIV LTR control) or U1 cells (harboring two latent HIV genomes with defective Tat gene) were cultured in RPMI1640 medium with 10% fetal bovine serum (FBS) and 1% Pen/strep in a 37° C. incubator containing 5% CO2 [24,31]. Both of the cell lines were obtained from NIH AIDS Reagent Program. For reactivation of HIV LTR, cells were treated with PMA (Sigma), JQ1 (Biovision), Prostatin (Sigma), SAHA (Santa Cruz), TNF-α (BD), GSK343 (Sigma), or PEP005 (Tocris Bioscience) for 24 h. HIV reactivation was quantified by GFP expression using flow cytometry and the data were analyzed using FlowJo Software for J-Lat A1 cells, or by quantitative RT-PCR (RT-qPCR) for J-Lat A1 and U1 cells. Cell viability was evaluated using Live/Dead dye (Life Technologies) by flow cytometry. So far, there is not a single in vitro cell culture model available that captures all the features of HIV latency. However, PKC agonists seem to reactivate latent HIV in all the cell culture models of viral latency. Similar to other J-Lat cell lines, the J-Lat A1 clone has been widely used by researchers for HIV latency studies. The T-cell-derived J-Lat A1 cells harbor one copy of a construct containing a Tat and a green fluorescent protein (GFP) gene framed by the 5′ and 3′ HIV LTR [32]. The J-Lat A1 cells also contain the TAR loop located within the R region of HIV LTR (nt+1 to +60), which provides an opportunity for Tat-TAR interactions for the modulation of transcription and transcriptional elongation. Consequently, the J-Lat A1 cell model is a suitable cell culture model to investigate latency reversal by PKC agonists.
- Primary CD4+ T Cell Isolation and Detection of T Cell Activation Markers:
- Peripheral blood samples were collected from 13 HIV-infected individuals receiving suppressive ART for >3 years except for one patient. All subjects had suppression of plasma viremia for more than 6 months (Average 5.84 years). At the time of the study enrollment, CD4+ T cell counts in peripheral blood samples ranged from 264 to 1100 cells/mm3 (Average 639 cell/mm3) and plasma viral loads were <20 copies per ml as measured by qPCR (Table 1). Isolation of the peripheral blood mononuclear cells (PBMC) and purification of CD4+ T cells using the EasySep kit (STEMCELL Technologies Inc. Vancouver, BC, Canada) were performed as previously described [24]. The purified CD4+ T cells were plated at a density of 1×106 cells/ml and treated with 200 ng/ml PMA plus 2 μM Ionomycin, 6-12 nM PEP005, 2 μM of JQ1, or 12 nM PEP005 plus 2 μM JQ1 for 6 hrs or 48 hrs and the cells were collected for RNA purification. To measure changes in the cell activation status of CD4+ and CD8+ T cell subsets, PBMCs were isolated from uninfected controls and 2×106 cells were incubated with DMSO, 200 ng/ml PMA plus 2 M Ionomycin, 6-12 nM PEP005 for 24 hrs or 72 hrs, and immunostained with anti-CD3, anti-CD38, anti-CD69, or anti-HLA-DR antibodies (Biolegend) for 20 min at 4° C. Cells were fixed in 1% PFA and analyzed by flow cytometry (FlowJo software from TreeStar). In addition, PBMCs from HIV-negative uninfected controls were similarly treated for 24 or 72 hrs and cells were collected for cytokine analysis using RT-qPCR.
-
TABLE 1 Characteristics of HIV-positive individuals receiving HAART Time under Viral CD4 ART Patients Age Sex load count ART (years) 2040 45 M <20 347 Tenofovir/Emtricitabine/ 4 Raltegravir 2041 56 M <20 1095 Emtricitabine/tenofovir/ 7 efavirenz 2044 60 F <20 1100 Emtricitabine/tenofovir/ 8 efavirenz 2050 48 M <20 757 Emtricitabine/tenofovir/ 8 efavirenz 2051 56 M <20 368 Emtricitabine/tenofovir/ 5 efavirenz 2052 35 M <20 264 Lopinavir/ Ritonavir 4 2053 50 M <20 260 Atazanavir/ Ritonavir 6 2054 53 M <20 477 Emtricitabine/tenofovir/ 12 efavirenz 2055 45 M <20 736 Tenofovir/Emtricitabine/ 6 Atazanavir/Ritonavir 2056 63 M <20 510 Nevimpine/Tenofovir/ 5 Emtricitabine 2057 55 M <20 775 Tenofovir/Emtricitabine/ 3 Atazanavir/Ritonavir 2060 57 M <20 813 Atazanavir/Ritonavir/ 6 Tenofovir/Emtricitabine 2061 57 M <20 805 Elvitegravir/Cobicistat/ 2 Emtricitabine/Tenofovir - Cell Viability and Proliferation Measurements:
- Cells were placed in 96-well plates and incubated for 24 or 72 h hrs with compounds. Cell viability was measured using MTT assay (Roche Laboratories), and cell proliferation/S-phase progression was determined by using BrdU ELISA kit (Cell signaling, #1863).
- Immunoblot Analysis:
- One million J-Lat A1 cells or PBMCs from HIV-negative uninfected controls were incubated with 12 nM PEP005 for 6 hrs. Whole cell protein extracts were prepared with RIPA buffer containing proteinase inhibitors and phosphatase inhibitors (Sigma). Expression of the isoforms of PKC protein or NF-κB/p65 was evaluated using the PKC Isoform Sampler Antibody Kit (Cell Signaling, 9960S) or anti-NF-κB/p65 (Abcam). The level of phosphorylation of PKC was determined using anti-Phospho-Ser664-PKCS, anti-Phospho-Ser643/676 PKCS or anti-Phospho-T538-PKCθ (Millipore), and the level of phosphorylation of IκB was determined using p-IκBα (Ser32), p-IκBβ (Thr19/Ser23) or p-IκBε (Ser18/22) antibodies (Millipore).
- HIV RNA Quantification in Patient Samples:
- Total RNA was extracted using the Qiagen RNeasy Kit that included a DNA digestion step. Quantitative RT-PCR was performed using Taqman Fast Virus 1-Step Master Mix (Applied Biosystems) in a ViiA7 real-time PCR system (Applied Biosystems). Transcripts containing the U5 region of the 5′ LTR were amplified using well-conserved primers (HXB2 559-543, 626-643) and fluorescent probe (HXB2 559-584) [24,33]. Transcripts containing the U3 to R region of the 3′ LTR including a Poly A tail were amplified in a one-step RT-PCR procedure related to a recently published assay with primers and probe binding sites as follows: gccctcagatgctrcatataa (SEQ ID NO:8) (HXB2 9496-9516), ttttttttttttttttttttttttttgaag (SEQ ID NO:1) (9632-9636+poly T) and FAM-tgcctgtactgggtctctctggttag-MGB (SEQ ID NO:2) (HXB2 9529-9554) [26,34]. External HIV RNA standards were prepared from in vitro transcripts quantified by spectrophotometry. HIV RNA copy numbers were normalized to RNA input.
- Quantitative analysis of synergy of latency reversing agent combinations: We adapted the Bliss independence model as implemented by Laird et al. to test for synergy when PEP005 was combined with other latency reversing agents [35,36]. For drugs x and y, we used the equation faxyP=fax+fay−(fax)(fay), where faxyP represents the predicted fraction affected by the combination of drug x and drug y given the observed effects of drug x (fax) and drug y (fay) used individually and faxy,O=the observed effect when x and y were tested together. Calculation of fax for U1 cells and patient derived T cells followed the approach of Laird for intracellular HIV RNA: fax=(HIV RNA copies with drug x background copies with DMSO)/(HIV RNA copies with PMA−background copies with DMSO). For these analyses, we included data for which every parameter for the synergy analysis was available and excluded individual cases where some of the parameters were not available. In cases where one or more experimental drug conditions resulted in RNA expression exceeding the PMA condition, we imputed the highest HIV RNA value in that experiment+1 to represent the denominator for calculation of fax. The calculation of fax for J-Lat A1 cells used the % GFP positive cells in place of intracellular HIV RNA. With this model, Δfaxy=faxyO (the observed fraction affected by the drug combination)−faxyP (the predicted fraction affected by the drug combination) provides an indication of synergy (A faxy>0), additive effect (Bliss independence) (A faxy=0) or antagonism (A faxy<0). Calculations analyzing synergy were done using Python. Statistical significance was determined using a one tailed ratio t-test executed in the statistical package “R”.
- Chromatin Immunoprecipitation (ChIP):
- ChIP assay was performed as previously described [24,37]. Briefly, 1×106 J-Lat A1 cells were incubated with PEP005, or PKCδ/inhibitor (PKCδi, Millipore/Calbiochem), fixed in 1% formaldehyde then resuspended in lysis buffer containing 1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.1 (ChIP Assay Kit, Millipore) and protease inhibitor cocktail (Sigma-Aldrich). Lysates were sonicated to obtain DNA fragments of 200-1500 bp. The immune complex was retrieved by incubating for 45 min with 50 μl of protein A/G-agarose beads saturated with BSA/salmon sperm DNA. Following the washes, the chromatin was eluted and reverse cross-linked overnight. DNA was extracted (Qiagen PCR purification kit) and quantitative real-time PCR was performed using Agilent Brilliant Ultra-Fast SYBR Green QPCR reagent using the 7500 real-time PCR System. The upstream primer sequence was 5′-AGCTTGCTACAAGGGACTTTCC-3′ (SEQ ID NO:3), and the downstream primer sequence was 5′-ACCCAGTACAGGCAAAAAGCAG-3′ (SEQ ID NO:4).
- Real-Time PCR Analysis of GFP or HIV Gene Expression:
- Total RNA was isolated from J-Lat A1 cells or U1 cells using the RNeasy Kit (Qiagen) followed by digestion with DNase I (Invitrogen). First strand cDNA was synthesized using Superscript II (Invitrogen). Real-time PCR (TaqMan) was performed on a
ViiA 7 detector using the following primer/probe set: for J-Lat A1 cells, primer 1: 5′-GGAGCGACCATCTTCTTCA-3′(SEQ ID NO:5), primer 2: 5′-AGGGTGTCGCCCTCGAA-3′ (SEQ ID NO:6),probe 5′-FAM CTACAAGACCC GCGCCGAGGTG TAMRA-3′ (SEQ ID NO:7), forU1 cells HIV 5′ LTR primers/probe were used (see above) [24]. The GAPDH primer/probe set was purchased from Applied Biosystems. - Pre-Treatment of Primary CD4+ T Cells with PEP005 and HIV-1 Infection of Primary CD4+ T Cells:
- Primary CD4+ T cells were isolated from peripheral blood samples of healthy HIV-negative donors and were pre-treated with PEP005 overnight. The CD4+ T cells were infected with HIV-1 (HIV-1 IIIB expanded in Jurkat T cells, 100 ng p24-gag) through spinoculation as described previously [38]. Following an exposure of 24 hrs to HIV, the cells were washed. Cell supernatants were collected and HIV p24 levels were measured by ELISA. The cells were also collected for RNA extraction and measurement of viral transcripts by RT-qPCR.
- Statistical Analysis:
- Means and standard errors (SE) were calculated for all data points from at least 3 independent experiments in triplicates. Statistical significance was determined using the two-way Student t test, where p value <0.05 considered significant.
- In order to determine the potential of PEP005 to induce HIV expression, J-Lat A1 cells, an established HIV latency lymphocyte cell culture model in vitro [24,32,39], were treated with increasing concentrations (2-40 nM) of PEP005 (
FIG. 1A ). PEP005 induced HIV expression in a dose-dependent manner and in the absence of any apparent cellular toxicity. A 7-fold increase in the reactivation of HIV latency was detected in J-Lat A1 cells at 20 nM of PEP005 as compared to untreated controls (FIGS. 1B and 1C ). The effect of PEP005 on HIV expression was evident even at the 2 nM level. Compared to other compounds known to reactivate HIV from latency, PEP005 appeared to be more potent than SAHA (a histone deacetylase inhibitor), JQ1 (a BET bromodomain inhibitor) and GSK343 (an inhibitor of EZH2) (FIG. 1D ). At the 10 nM level, PEP005-induced HIV reactivation was similar to that induced by PMA and more potent than 2 μM Prostratin (p=0.012). Interestingly, although EZH2 was shown to be critical for establishment of HIV latency through tri-methylation of H3K27 and inhibition of EZH2 by 3-deazaneplanocin A (DZNep) resulted in reactivation of latent HIV in vitro [40], the specific EZH2 inhibitor, GSK343 (recently developed by GSK) [41,42], failed to induce HIV expression in J-Lat A1 cells (FIG. 1D ). Taken together, these data show that PEP005 is highly potent in reactivating latent HIV in vitro. - The Effect of PEP005 on Reactivation of Latent HIV is Potently Enhanced by Combination with JQ1 in Both J-Lat A1 Cells and U1 Cells In Vitro.
- Several molecular pathways are involved in the establishment and maintenance of HIV latency [6,21]. In order to optimally reactivate latent HIV expression, we utilized combinations of latency reversing agents (LRAs) in the J-Lat A1 cells and U1 cells in vitro [43]. Several compounds were selected including the HDAC inhibitor, SAHA (500 nM); the BET bromodomain inhibitor, JQ1 (2 μM); the EZH2 inhibitor, GSK343 (2 μM); and the PKC agonist Prostratin (10 μM), in combination with PEP005 (6 nM). A lower concentration of PEP005 was used in these assays since PEP005 is very potent in reactivating latent HIV expression (
FIG. 1D ) and the combined effects of it with other LRAs would be difficult to distinguish at a higher concentration of PEP005 [24]. PEP005 induced reactivation of latent HIV and was highly effective in combination with JQ1 in J-Lat A1 cells (FIG. 2A ). Surprisingly, GSK343 alone could not effectively induce HIV expression but was able to enhance the magnitude of latent HIV reactivation by PEP005. These findings indicate that pre-disruption of H3K27Me3-mediated chromatin repression may be required for enhanced latent HIV reactivation. Interestingly, Prostratin also showed a trend for enhancing effect of PEP005 on latent HIV reactivation. However, this was not statistically significant. - To determine whether the combined effects of PEP005 on latent HIV reactivation occurs in other latent HIV-infected immune cell types, we examined HIV transcription in U1 cells, a well-studied promonocyte cell line that harbors two complete HIV genomes with Tat gene mutations and is used as an HIV latency cell culture model [31]. While PEP005 alone induced about 25-fold increase in latent HIV reactivation compared to controls, addition of JQ1 boosted induction of HIV transcription to more than 250-fold increase (
FIG. 2B ). We found a combined effect of PEP005 on reactivation of HIV latency with GSK343, as well as with SAHA in U1 cells. We previously reported a similar pattern of synergy when J-Lat A1 cells were treated with IngB and JQ1 [24]. - Our data indicated that PEP005 synergistically reactivates latent HIV in both J-Lat A1 cells and U1 cells. To assess whether these combined effects meet criteria for drug synergy, we compared the experimentally observed combined effects in J-Lat A1 cells and U1 cells to the effects predicted under the Bliss independence model for combined drug effects ([35] and
FIGS. 2C and 2D ). This model assumes that if two compounds act through different mechanisms, their effects are merely additive in the absence of synergistic interactions. In contrast, effects of combinations that are greater or lesser than the idealized Bliss independence prediction imply synergy or antagonism, respectively [31]. We found that PEP005 demonstrates significant synergism with JQ1 or EZH2 inhibitors to induce GFP expression in vitro (FIG. 2C ). HDAC inhibitor SAHA and another PKC agonist Prostratin did not exhibit synergy with PEP005. In U1 cells, PEP005 synergized significantly with JQ1 or SAHA to induce HIV mRNA expression in vitro (FIG. 2D ), but GSK343 did not exhibit synergy with PEP005. Taken together, PEP005 exerted a synergistic effect with JQ1 on reactivation of HIV from latency in both J-Lat A1 and U1 cell lines. - Although it was shown that PEP005 can activate the PKC-NF-κB pathway, it is not known at which step of the PKC-NF-κB pathway is modulated during the reactivation of latent HIV. Therefore, we examined protein expression of several components of the PKC pathway in J-Lat A1 cells by Western blot analysis using antibodies specific for four PKC super families, including PKCμ/D, PKCα, PKCδ, and PKCθ. There was no significant induction of expression of these PKC proteins except for a modest up-regulation of PKCδ that was detected at 1 hr following 12 nM PEP005 treatment. Moreover, rapid phosphorylation of Ser643/Ser676 in PKCδ/θ was induced by PEP005 with greater than a two-fold increase as early as 30 minutes post treatment (
FIGS. 3A-C ). These findings were further validated in PEP005 treated PBMCs from healthy donors. To investigate the involvement of the up-stream kinases in PKC-NF-κB signaling, Western blot analysis was performed using anti-phospho-IκB antibodies. Our data showed that PEP005 treatment induced phosphorylation of IκBα and IκBε, but not of IκBβ (FIGS. 3A-B ). Interestingly, expression of NF-κB/p65 did not change in the presence of PEP005 (FIG. 3A ). This is clearly different from the effects of IngB treatment which involved an increased expression of NF-κB/p65 protein [24]. To further confirm the role of PKC-NF-κB signaling in reactivation of HIV latency, J-Lat A1 cells were treated with a PKCδ/θ inhibitor (FIG. 4A ). Our data showed that inhibition of PKCδ/θ resulted in a reduction of latent HIV reactivation by more than 65%. The addition of the NF-κB inhibitor, Bay-11-7082, to J-Lat A1 cells resulted in an approximately 50% reduction in PEP005-induced disruption of HIV latency (FIG. 4B ). To determine whether PEP005 reactivates latent HIV by promoting NFκB/p65 binding to the HIV LTR, ChIP-qPCR assays were performed with J-Lat A1 cells treated with 12 nM of PEP005 with or without PKCδ/θ inhibitor. PEP005 treatment resulted in a 6-fold increase in NF-κB/p65 binding to HIV LTR region (FIG. 4C ). This increase was reduced by more than 70% following the addition of PKC inhibitor. Collectively, our findings indicate that PEP005-induced reactivation of latent HIV most likely occurs through the PKCδ/θ-NF-κB signaling pathway. However, this does not exclude a possibility that other PKC isoforms are also potentially involved. - To be clinically applicable, effective LRAs should be highly potent, minimally cytotoxic and able to penetrate anatomic sanctuaries and immune cell types without inducing global T cell activation [22]. Therefore, we sought to examine the effects of PEP005 on T cell activation and cytotoxicity. Evaluation of the expression of T cell activation biomarkers by RT-qPCR showed that PEP005 treatment did not cause any significant change in the expression of CD38, CD25, or HLA-DR in purified primary CD4+ T cells (
FIG. 5A ). However, there was an increased expression of CD69 in CD4+ T cells. Flow cytometric analysis of global T cells for the expression of CD38, CD69, or HLA-DR further supported the gene expression data. There was no significant change in the expression of CD38 (24 hr) and HLA-DR in CD4+ T cells (FIGS. 5B, 5E and 5F ). However, there was an increase in expression of CD69, an inducible glycoprotein that is expressed early during T lymphocytes activation (FIGS. 5C and 5D ). These findings provide additional evidence that PEP005 reverses HIV latency through PKC-NF-κB signaling in vivo. Since the expression of CD69 is dependent on NF-κB binding to its promoter region, it is understandable that PEP005 induced PKC-NF-κB signaling would up-regulate CD69 expression in CD4+ T cells [44]. - We examined the potential side effects of PEP005 on the inflammatory cytokine expression as a possible consequence of increased CD69 production. It is well recognized that acutely or chronically HIV infected individuals seem to express higher levels of inflammatory cytokines including IL-6 and TNF-α in the peripheral blood [45-47]. Therefore, it is important that the agents for disrupting HIV latency do not exacerbate the unresolved chronic immune activation and inflammatory cytokine expression during HIV eradication interventions. To address this question, we examined CD4+ T cells purified from PBMCs of healthy HIV-negative donors ex vivo for pro-inflammatory cytokine expression following stimulation with PEP005 for 24 or 72 hours. The expression levels of TNF-α, IFN-γ, IL-2, and IL-6 cytokines were determined by RT-qPCR (
FIGS. 6A-D ). There was no significant increase in the expression of these cytokines, except for TNF-α that showed a tendency towards an up-regulation. However, the increase was not statistically significant. - In order to further evaluate the effects of PEP005 on T cell activation, we determined the ability of PEP005 to induce T cell proliferation and cytotoxicity using BrdU incorporation and MTT assays. PEP005 induced minimal levels of cellular toxicity in both J-Lat A1 and U1 cell lines, as well as in primary CD4+ T cells from peripheral blood samples of healthy HIV-negative donors (
FIG. 7 ). Importantly, PEP005 treatment did not induce any significant increase in the proportion of cells in S-phase with J-Lat A1 cells, U1 cells or primary CD4+ T cells. In summary, despite the increased CD69 expression, minimal to no induction was found for the expression of pro-inflammatory cytokines and no significant impact was seen on cell cycling, indicating that PEP005 is a potential LRA candidate for evaluation in vivo. - PEP005 Induces Latent HIV Expression in Primary CD4+ T Cells Ex Vivo from Individuals with Suppressive ART.
- We examined the ability of PEP005 to induce latent HIV expression in primary CD4+ T cells from HIV infected individuals under suppressive ART. The viral loads were below 20 copies/ml of plasma and the CD4+ T cell counts ranged from 260 to 1100 (Table 1.). Two different doses (6 nM and 12 nM) of PEP005 were added for 6 hrs to purified primary CD4+ T cells from the peripheral blood of these individuals. The level of HIV mRNA expression was measured by RT-qPCR with primers/probe specific for the
HIV 5′ LTR region. The magnitude of induction of HIV RNA transcription by PMA we observed in this study differs somewhat from earlier studies [51] because of differences in experimental design (shorter incubation times, PMA concentration) as well as the inherent variability of responses of latently infected primary CD4+ T cells to LRAs. At 6 nM, PEP005 induced an increase in HIV transcription in 2 out of 7 individuals; at 12 nM, HIV transcription was observed in 5 out of 7 individuals (FIG. 8 ). To verify the capacity of PEP005 to reactivate latent HIV to produce full-length transcripts, RT-qPCR was performed using the primers/probe targeting HIV 3′ polyadenylation (poly A) region [26]. After 6 hours of PEP005 treatment, 5 of the 7 donors had 2 fold or >2 fold increase in full-length HIV transcripts, while 5 of the 6 donors showed 2 or >2 fold increase after 12 nM of treatment. These results indicate that PEP005 is effective in reactivating transcription of HIV from latently infected cells ex vivo (FIG. 8 ). A higher dose of PEP005 is expected to induce more potent reactivation of latent HIV from patient samples. - HIV Induction in Primary CD4+ T Cells from HIV Infected Individuals on ART is Enhanced by the Combination of PEP005 with JQ1.
- Our data showed that PEP005 used in combination with JQ1 resulted in synergistic reactivation of HIV expression in both the J-Lat A1 cells and the U1 cell models of HIV latency in vitro (
FIG. 2 ). This prompted us to examine whether the combination of JQ1 and PEP005 would significantly enhance the ex vivo induction of latent HIV expression in primary CD4+ T cells from HIV infected individuals on suppressive ART. HIV transcription was measured by RT-qPCR following the treatment of cells with 12 nM PEP005 alone, 2 μM JQ1 alone, or 12 nM PEP005 plus 2 μM JQ1 for 6 hrs or 48 hrs. After 6 hrs of stimulation, a strong combined effect of PEP005 and JQ1 induced transcription of HIV RNA using 5′ LTR assay was seen in CD4+ T cell samples from all individuals except one. When transcripts were measured with assay for the HIV poly A region, an enhanced combination effect was seen in 6 of 8 HIV infected individuals. Indonor 1 anddonor 9, the amount of cDNA was only sufficient to measure HIV RNA with either the Poly A region or LTR region assay (FIG. 9 ). The combination treatment at 48 hrs was similarly more potent compared to PEP005 treatment alone in 6 of 7 patient samples when assessed by assay ofHIV 5′ LTR region (up to 10 fold increase) and in all 6 patient samples by assay of the Poly A region of the HIV genome (1.6 to 14 fold increase). Indonor 9, the amount of cDNA was only sufficient for the Poly A region assay (FIG. 10 ). When analyzed as HIV RNA copy number in 1 μg of total RNA from CD4+ T cells, PEP005 significantly induced latent HIV reactivation compared with control treatment after 6 hr incubation, and combined treatment of PEP005 with JQ1 further significantly induced full-length latent HIV reactivation compared with PEP005 treatment alone after 6 or 48 hr incubation (*, p<0.05; ** p<0.01,FIG. 11A ). Applying the Bliss independence model for combined drug effects, criteria for synergistic ex vivo reactivation of full length latent HIV transcription by PEP005 and JQ1 were met with the caveat that this assessment does not determine whether these viral transcripts were translated into viral proteins of viral particles (FIG. 11B ). Taken together, PEP005 exerted a synergistic effect with JQ1 on reactivation of HIV transcription from latency in both cell line models and from CD4+ T cells obtained from patients on suppressive ART that included expression of fully elongated and processed HIV RNAs. - Recent studies have shown that Prostratin and analogs down-modulate HIV receptor/co-receptor expression, which could have protective effects against the viral infection [52,53]. Conversely, SAHA, known to induce latent HIV expression in HIV infected individuals receiving ART, was reported to increase susceptibility of naive CD4+ T cells to HIV acquisition [54]. Diterpene compounds are known to inhibit expression of HIV receptors/co-receptors including CD4, CCR5, and CXCR4, which are important for the viral attachment and entry into immune cells [27,52,53,55,56]. We sought to examine the effect of PEP005 on the cell surface expression of HIV receptors and co-receptors in CD4+ T cells. Primary CD4+ T cells from peripheral blood samples of healthy HIV-negative donors were treated with 12 nM PEP005 and evaluated for the expression levels of CD4, CCR5, and CCXR4 using RT-qPCR. Our data showed that PEP005 treatment caused a significant reduction in the expression of all these HIV receptors/co-receptors, indicating that PEP005 may not pose the risk of increasing susceptibility of CD4+ T cells to HIV infection during its reactivation of HIV latency (
FIG. 12A ). Instead, PEP005 contributed to suppression of propagating HIV infection of CD4+ T cells following the reactivation of latent HIV. To investigate the potentially protective effects of PEP005, primary CD4+ T cells were infected with HIV with or without pre-treatment of 12 or 24 nM PEP005 for 24 hrs. The virological outcome in the CD4+ T cell cultures in vitro was monitored for 5 days. We observed that PEP005 dampened HIV gene expression as well as decreased the level of viral replication as determined at 3 and 5 days after HIV infection of primary CD4+ T cells (FIGS. 12B and 12C ). These findings demonstrate that PEP005 prevents HIV infection of naive CD4+ T cells through down-modulation of HIV co-receptor expression (CD4, CXCR4 and CCR5), which is beneficial during the process of latent HIV reactivation. - Since the discovery of stable, latent viral reservoirs in HIV-infected individuals in spite of long-term suppressive ART, several studies have been performed to better understand the mechanisms of the establishment of HIV latency as well as to identify crucial steps for the maintenance of the viral latency [5,6,21]. The identification of memory CD4+ T cells, mostly central memory CD4+ T cells, as the major viral reservoir has provided clues for designing novel strategies for HIV eradication [5,57]. Accordingly, a “shock and kill” strategy was proposed to eradicate latent HIV reservoirs [6]. Diterpene compounds are among the potential candidates for disrupting HIV latency. They are more potent than several other LRAs in inducing latent HIV expression in multiple HIV latency models including in vitro J-Lat cell lines, primary CD4+ T cell based cell cultures, and resting CD4+ T cells from the peripheral blood of HIV infected individuals [20,24]. Diterpenes can down-modulate the expression of cell surface receptors that are known to mediate HIV attachment and entry and can potentially prevent the spread of viral infection to bystander CD4+ T cells [27,52-54]. Therefore, it is useful to identify diterpene compounds suitable for HIV cure studies that could reactivate HIV from the latent reservoirs with low to minimal cytotoxicity while preventing new viral infections of CD4+ T cells.
- The data from this study show, for the first time, that PEP005, a diterpene compound, can effectively reactivate latent HIV expression in vitro and ex vivo. It has been reported that an ingenol ester, IngB, is also able to reactivate HIV from latency [24,27]. Both of these compounds showed efficacy in reactivation of HIV latency at low nanomolar levels without exerting apparent cytotoxicity. IngB could induce increased levels of protein expression of NF-κB, P-TEFb, and IFNγ [24,27], while PEP005 does not increase NF-κB protein expression levels. Activation of NF-κB and P-TEFb is essential for HIV reactivation from latency. However, excessive induction of these proteins may potentially be problematic because NF-κB is a master regulator for multiple signaling pathways, and P-TEFb is a general activator of transcriptional elongation, that also contributes to multiple signaling pathways [58,59]. Further studies are warranted to investigate whether IngB or PEP005 alone can induce protein expression of NF-κB or P-TEFb in resting CD4+ T cells, and not just in proliferating CD4+ T cells. Thirdly, IngB induced IFNγ protein expression [24,27], while PEP005 barely caused any increase in this or other inflammatory cytokine expression.
- Although PEP005 shares a similar core structure with IngB, its C3 side chain structure is different and quite distinct from IngB (
FIG. 1 ). Substitution of the C3-ester by different aliphatic and aromatic side chains confer markedly different biological properties [60,61], so it is likely that differences in molecular mechanisms of action and different T cell activation properties between PEP005 and Ingenol B are conferred by the structural differences of the C3-R. Consequently, PEP005 may offer several advantages over IngB. High levels of ingenols, up to 20 μM, induced NF-κB-independent cell death in Jurkat cells [62]. This concentration is about 1000-fold higher than the PEP005 concentrations used in this study, indicating that low concentrations of PEP005 capable of HIV reactivation may trigger signaling pathways differently and result in its low-to-minimal cytotoxicity. Therefore, the use of PEP005 at low concentrations for reactivation of latent HIV is important to minimize cytotoxic effects. - In the recently FDA-approved drug PICATO, PEP005 is the only active component with PKC agonist activity. Pharmacokinetic studies of PEP005 have been performed in small animals in vivo. Similar to the safety of the topical application of PICATO, the systemic (intravenous) use of PEP005 in small animals (mini pig and rat model) was reported to be relatively safe, with the maximum nonlethal dose >73 μg/kg (See, Assessment report of PICATO to European Medicines Agency, Sep. 20, 2012). These data support the use of PEP005 in HIV cure studies.
- Since multiple molecular signaling pathways are involved in establishment and maintenance of HIV latency, a single LRA may not be adequate to achieve disruption of the multi-pronged regulatory mechanisms promoting HIV latency. SAHA, an HDAC inhibitor, shows some ability to reactivate HIV. However, the efficacy of SAHA varies among HIV infected individuals indicating that SAHA alone may not be sufficient for effective reactivation of latent HIV for all HIV infected individuals [16,17,63]. Therefore, a combination of compounds should be explored to induce viral expression from latently infected cells [43]. Our data show that PEP005 alone potently reactivates latent HIV expression in vitro. However, a combination of PEP005 and JQ1 was substantially more effective in inducing latent HIV in J-Lat A1 cells or U1 cells and in ex vivo primary CD4+ T cells from infected patients receiving suppressive ART. This could be attributed to potentiating effects of the PKC-NF-kB pathway on p-TEFb mechanism for reactivating. The range of drug concentrations that provides the best drug synergy using PEP005 can be determined [64].
- Synergistic reactivation of latent HIV is also achieved by combining JQ1 with other ingenol compounds, as it has been observed that JQ1 boosted the reversal of HIV latency by IngB in J-Lat A1 cells [24]. However, it is not known whether this combined treatment leads to a synergistic effect on HIV expression in patient cells under ART ex vivo. Recently, Sillicano and colleagues showed that PKC agonists, such as Prostratin or
Bryostatin 1, had a synergistic effect with JQ1 on the reactivation latent HIV ex vivo [35]. It was intriguing to note that GSK343, an EZH2 inhibitor, had no capacity to reactivate latent HIV by itself, but displayed increased potency in combination with PEP005 in J-Lat A1 cells. These observations are complemented by a study showing that GSK343 boosts SAHA or JQ1 reactivation of latent HIV in a primary resting T-cell model of HIV latency [65]. These findings indicate that pre-disruption of methylation of histone tails may facilitate efficient reactivation of latent HIV in combination with a second LRA. - PEP005 displayed potent activity to reactivate HIV from latency, producing polyadenylated viral transcripts while exerting minimal to low cytotoxicity and in the absence of major global T cell activation. Until now, only Bryostatin and PMA have been shown to stimulate comparable transcription of polyadenylated HIV RNA in primary CD4+ T cells isolated from HIV infected individuals on suppressive ART [26]. Considering that PEP005 had a similar capacity as PMA to reactivate latent HIV both in vitro and ex vivo (
FIG. 1D andFIG. 8 ), and that PEP005 could induce GFP protein expression in J-Lat A1 cells (FIG. 1 ), PEP005 is useful for inducing HIV virus expression in vivo. There was an indication that PEP005 up-regulated TNFα to some extent, but these effects were not significantly different compared to controls. Similar to what is found with other PKC agonists, PEP005-induced NF-kB activation leads to increased expression of CD69 since its promoter contains multiple NF-κB binding sites [44]. However, it was interesting to note that cell proliferation was not significantly altered in J-Lat A1, U1 cells or primary CD4+ T cells, and inflammatory cytokine expression was not significantly enhanced by PEP005. These findings indicate that modulation of some of the T-cell activation markers may not necessarily lead to global T-cell activation. It has also been proposed that a low level of T cell activation may be required for efficient reactivation of HIV latency [26]. The ability of PEP005 to reduce cell surface expression of CD4, CCR5, and CXCR4 on T cells directly impacts susceptibility of primary CD4+ T cells to in vitro HIV infection. This supports the concept that reactivation of latent HIV by PEP005 may block viral spread to uninfected bystander CD4+ T cells, making it an attractive compound for advancing to clinical HIV cure studies. This study demonstrates that ingenols, including PEP005, represent a new group of compounds for combating HIV latency for viral eradication. -
- 1. Finzi D, Hermankova M, Pierson T, Carruth L M, Buck C, et al. (1997) Identification of a reservoir for HIV-1 in patients on highly active antiretroviral therapy. Science 278: 1295-1300.
- 2. Siliciano J D, Kajdas J, Finzi D, Quinn T C, Chadwick K, et al. (2003) Long-term follow-up studies confirm the stability of the latent reservoir for HIV-1 in resting CD4+ T cells. Nat Med 9: 727-728.
- 3. Wong J K, Hezareh M, Gunthard H F, Havlir D V, Ignacio C C, et al. (1997) Recovery of replication-competent HIV despite prolonged suppression of plasma viremia. Science 278: 1291-1295.
- 4. Chun T W, Stuyver L, Mizell S B, Ehler L A, Mican J A, et al. (1997) Presence of an inducible HIV-1 latent reservoir during highly active antiretroviral therapy. Proc Natl Acad Sci USA 94: 13193-13197.
- 5. Chomont N, El-Far M, Ancuta P, Trautmann L, Procopio F A, et al. (2009) HIV reservoir size and persistence are driven by T cell survival and homeostatic proliferation. Nat Med 15: 893-900.
- 6. Richman D D, Margolis D M, Delaney M, Greene W C, Hazuda D, et al. (2009) The challenge of finding a cure for HIV infection. Science 323: 1304-1307.
- 7. Lerner P, Guadalupe M, Donovan R, Hung J, Flamm J, et al. (2011) The gut mucosal viral reservoir in HIV-infected patients is not the major source of rebound plasma viremia following interruption of highly active antiretroviral therapy. J Virol 85: 4772-4782.
- 8. Guadalupe M, Reay E, Sankaran S, Prindiville T, Flamm J, et al. (2003) Severe CD4+ T-cell depletion in gut lymphoid tissue during primary human
immunodeficiency virus type 1 infection and substantial delay in restoration following highly active antiretroviral therapy. J Virol 77: 11708-11717. - 9. Giacomet V, Trabattoni D, Zanchetta N, Biasin M, Gismondo M, et al. (2014) No cure of HIV infection in a child despite early treatment and apparent viral clearance. Lancet 384: 1320.
- 10. Hirao L A, Grishina I, Bourry O, Hu W K, Somrit M, et al. (2014) Early mucosal sensing of SIV infection by paneth cells induces IL-1beta production and initiates gut epithelial disruption. PLoS Pathog 10: e1004311.
- 11. Whitney J B, Hill A L, Sanisetty S, Penaloza-MacMaster P, Liu J, et al. (2014) Rapid seeding of the viral reservoir prior to SIV viraemia in rhesus monkeys. Nature.
- 12. Persaud D, Gay H, Ziemniak C, Chen Y H, Piatak M, Jr., et al. (2013) Absence of detectable HIV-1 viremia after treatment cessation in an infant. N Engl J Med 369: 1828-1835.
- 13. Shalit P (2014) Management of dyslipidemia in patients with human immunodeficiency virus.
Rev Cardiovasc Med 15 Suppl 1: S38-46. - 14. Freitas P, Carvalho D, Santos A C, Madureira A J, Martinez E, et al. (2014) Adipokines, hormones related to body composition, and insulin resistance in HIV fat redistribution syndrome. BMC Infect Dis 14: 347.
- 15. Nasi M, Pinti M, De Biasi S, Gibellini L, Ferraro D, et al. (2014) Aging with HIV infection: A journey to the center of inflammAIDS, immunosenescence and neuroHIV. Immunol Lett.
- 16. Archin N M, Liberty A L, Kashuba A D, Choudhary S K, Kuruc J D, et al. (2012) Administration of vorinostat disrupts HIV-1 latency in patients on antiretroviral therapy. Nature 487: 482-485.
- 17. Elliott J H, Wightman F, Solomon A, Ghneim K, Ahlers J, et al. (2014) Activation of HIV transcription with short-course vorinostat in HIV-infected patients on suppressive antiretroviral therapy. PLoS Pathog 10: e1004473.
- 18. Rasmussen T A, Schmeltz Sogaard O, Brinkmann C, Wightman F, Lewin S R, et al. (2013) Comparison of HDAC inhibitors in clinical development: effect on HIV production in latently infected cells and T-cell activation. Hum Vaccin Immunother 9: 993-1001.
- 19. Shan L, Deng K, Shroff N S, Durand C M, Rabi S A, et al. (2012) Stimulation of HIV-1-specific cytolytic T lymphocytes facilitates elimination of latent viral reservoir after virus reactivation. Immunity 36: 491-501.
- 20. Spina C A, Anderson J, Archin N M, Bosque A, Chan J, et al. (2013) An in-depth comparison of latent HIV-1 reactivation in multiple cell model systems and resting CD4+ T cells from aviremic patients. PLoS Pathog 9: e1003834.
- 21. Jiang G, Espeseth A, Hazuda D J, Margolis D M (2007) c-Myc and Spl contribute to proviral latency by recruiting
histone deacetylase 1 to the humanimmunodeficiency virus type 1 promoter. J Virol 81: 10914-10923. - 22. Jiang G, Dandekar S (2014) Targeting NF-kappaB signaling with protein kinase C agonists as an emerging strategy for combating HIV latency. AIDS Res Hum Retroviruses.
- 23. Beans E J, Fournogerakis D, Gauntlett C, Heumann L V, Kramer R, et al. (2013) Highly potent, synthetically accessible prostratin analogs induce latent HIV expression in vitro and ex vivo. Proc Natl Acad Sci USA 110: 11698-11703.
- 24. Jiang G, Mendes E A, Kaiser P, Sankaran-Walters S, Tang Y, et al. (2014) Reactivation of HIV latency by a newly modified Ingenol derivative via protein kinase Cdelta-NF-kappaB signaling. AIDS.
- 25. Huang L, Ho P, Yu J, Zhu L, Lee K H, et al. (2011) Picomolar dichotomous activity of gnidimacrin against HIV-1. PLoS One 6: e26677.
- 26. Bullen C K, Laird G M, Durand C M, Siliciano J D, Siliciano R F (2014) New ex vivo approaches distinguish effective and ineffective single agents for reversing HIV-1 latency in vivo. Nat Med 20: 425-429.
- 27. Abreu C M, Price S L, Shirk E N, Cunha R D, Pianowski L F, et al. (2014) Dual role of novel ingenol derivatives from Euphorbia tirucalli in HIV replication: inhibition of de novo infection and activation of viral LTR. PLoS One 9: e97257.
- 28. Pandelo Jose D, Bartholomeeusen K, da Cunha R D, Abreu C M, Glinski J, et al. (2014) Reactivation of latent HIV-1 by new semi-synthetic ingenol esters. Virology 462-463C: 328-339.
- 29. Fidler B, Goldberg T (2014) Ingenol mebutate gel (picato): a novel agent for the treatment of actinic keratoses. P T 39: 40-46.
- 30. Warrilow D, Gardner J, Darnell G A, Suhrbier A, Harrich D (2006)
HIV type 1 inhibition by protein kinase C modulatory compounds. AIDS Res Hum Retroviruses 22: 854-864. - 31. Emiliani S, Fischle W, Ott M, Van Lint C, Amella C A, et al. (1998) Mutations in the tat gene are responsible for human
immunodeficiency virus type 1 postintegration latency in the U1 cell line. J Virol 72: 1666-1670. - 32. Jordan A, Bisgrove D, Verdin E (2003) HIV reproducibly establishes a latent infection after acute infection of T cells in vitro. EMBO J 22: 1868-1877.
- 33. Kumar A M, Borodowsky I, Fernandez B, Gonzalez L, Kumar M (2007) Human
immunodeficiency virus type 1 RNA Levels in different regions of human brain: quantification using real-time reverse transcriptase-polymerase chain reaction. J Neurovirol 13: 210-224. - 34. Shan L, Rabi S A, Laird G M, Eisele E E, Zhang H, et al. (2013) A novel PCR assay for quantification ofHIV-1 RNA. J Virol 87: 6521-6525.
- 35. Laird G M, Bullen C K, Rosenbloom D I, Martin A R, Hill A L, et al. (2015) Ex vivo analysis identifies effective HIV-1 latency-reversing drug combinations. J Clin Invest.
- 36. Bliss C I (1939) The toxicity of poisons applied jointly. Annals of Applied Biology 26: 585-615.
- 37. Jiang G, Sancar A (2006) Recruitment of DNA damage checkpoint proteins to damage in transcribed and nontranscribed sequences. Mol Cell Biol 26: 39-49.
- 38. Tang Y, George A, Nouvet F, Sweet S, Emeagwali N, et al. (2014) Infection of female primary lower genital tract epithelial cells after natural pseudotyping of HIV-1: possible implications for sexual transmission of HIV-1. PLoS One 9: e101367.
- 39. Lavigne M, Eskeland R, Azebi S, Saint-Andre V, Jang S M, et al. (2009) Interaction of HP1 and Brg1/Brm with the globular domain of histone H3 is required for HP1-mediated repression. PLoS Genet 5: e1000769.
- 40. Friedman J, Cho W K, Chu C K, Keedy K S, Archin N M, et al. (2011) Epigenetic silencing of HIV-1 by the histone H3 lysine 27 methyltransferase enhancer of
Zeste 2. J Virol 85: 9078-9089. - 41. Verma S K, Tian X, LaFrance L V, Duquenne C, Suarez D P, et al. (2012) Identification of Potent, Selective, Cell-Active Inhibitors of the Histone Lysine Methyltransferase EZH2. ACS Med Chem Lett 3: 1091-1096.
- 42. Amatangelo M D, Garipov A, Li H, Conejo-Garcia J R, Speicher D W, et al. (2013) Three-dimensional culture sensitizes epithelial ovarian cancer cells to EZH2 methyltransferase inhibition. Cell Cycle 12: 2113-2119.
- 43. Margolis D M, Hazuda D J (2013) Combined approaches for HIV cure. Curr Opin HIV AIDS 8: 230-235.
- 44. Lopez-Cabrera M, Munoz E, Blazquez M V, Ursa M A, Santis A G, et al. (1995) Transcriptional regulation of the gene encoding the human C-type lectin leukocyte receptor AIM/CD69 and functional characterization of its tumor necrosis factor-alpha-responsive elements. J Biol Chem 270: 21545-21551.
- 45. Stacey A R, Norris P J, Qin L, Haygreen E A, Taylor E, et al. (2009) Induction of a striking systemic cytokine cascade prior to peak viremia in acute human
immunodeficiency virus type 1 infection, in contrast to more modest and delayed responses in acute hepatitis B and C virus infections. J Virol 83: 3719-3733. - 46. Macal M, Sankaran S, Chun T W, Reay E, Flamm J, et al. (2008) Effective CD4+ T-cell restoration in gut-associated lymphoid tissue of HIV-infected patients is associated with enhanced Thl7 cells and polyfunctional HIV-specific T-cell responses. Mucosal Immunol 1: 475-488.
- 47. Guadalupe M, Sankaran S, George M D, Reay E, Verhoeven D, et al. (2006) Viral suppression and immune restoration in the gastrointestinal mucosa of human immunodeficiency virus type 1-infected patients initiating therapy during primary or chronic infection. J Virol 80: 8236-8247.
- 48. Don A S, Martinez-Lamenca C, Webb W R, Proia R L, Roberts E, et al. (2007) Essential requirement for
sphingosine kinase 2 in a sphingolipid apoptosis pathway activated by FTY720 analogues. J Biol Chem 282: 15833-15842. - 49. Ferreira P M, Lima D J, Debiasi B W, Soares B M, Machado Kda C, et al. (2013) Antiproliferative activity of Rhinella marina and Rhaebo guttatus venom extracts from Southern Amazon. Toxicon 72: 43-51.
- 50. Johnson T M, Meade K, Pathak N, Marques M R, Attardi L D (2008) Knockin mice expressing a chimeric p53 protein reveal mechanistic differences in how p53 triggers apoptosis and senescence. Proc Natl Acad Sci USA 105: 1215-1220.
- 51. Ho Y C, Shan L, Hosmane N N, Wang J, Laskey S B, et al. (2013) Replication-competent noninduced proviruses in the latent reservoir increase barrier to HIV-1 cure. Cell 155: 540-551.
- 52. Kulkosky J, Culnan D M, Roman J, Dornadula G, Schnell M, et al. (2001) Prostratin: activation of latent HIV-1 expression suggests a potential inductive adjuvant therapy for HAART. Blood 98: 3006-3015.
- 53. Mehla R, Bivalkar-Mehla S, Zhang R, Handy I, Albrecht H, et al. (2010) Bryostatin modulates latent HIV-1 infection via PKC and AMPK signaling but inhibits acute infection in a receptor independent manner. PLoS One 5: el 11160.
- 54. Lucera M, Tilton C A, Mao H, Dobrowolski C, Tabler C, et al. (2014) The histone deacetylase inhibitor vorinostat (SAHA) increases the susceptibility of uninfected CD4+ T cells to HIV by increasing the kinetics and efficiency of post-entry viral events. J Virol.
- 55. Kalinina O V, Pfeifer N, Lengauer T (2013) Modelling binding between CCR5 and CXCR4 receptors and their ligands suggests the surface electrostatic potential of the co-receptor to be a key player in the HIV-1 tropism. Retrovirology 10: 130.
- 56. Bedoya L M, Marquez N, Martinez N, Gutierrez-Eisman S, Alvarez A, et al. (2009) SJ23B, a jatrophane diterpene activates classical PKCs and displays strong activity against HIV in vitro. Biochem Pharmacol 77: 965-978.
- 57. Soriano-Sarabia N, Bateson R E, Dahl N P, Crooks A M, Kuruc J D, et al. (2014) The quantitation of replication-competent HIV-1 in populations of resting CD4+ T cells. J Virol.
- 58. Baldwin A S, Jr. (2001) Series introduction: the transcription factor NF-kappaB and human disease. J Clin Invest 107: 3-6.
- 59. Zhou Q, Li T, Price D H (2012) RNA polymerase II elongation control. Annu Rev Biochem 81: 119-143.
- 60. Liang X, Grue-Sorensen G, Mansson K, Vedso P, Soor A, et al. (2013) Syntheses, biological evaluation and SAR of ingenol mebutate analogues for treatment of actinic keratosis and non-melanoma skin cancer. Bioorg Med Chem Lett 23: 5624-5629.
- 61. Sorg B, Schmidt R, Hecker E (1987) Structure/activity relationships of polyfunctional diterpenes of the ingenane type. I. Tumor-promoting activity of homologous, aliphatic 3-esters of ingenol and of
delta 7,8-isoingenol-3-tetradecanoate. Carcinogenesis 8: 1-4. - 62. Blanco-Molina M, Tron G C, Macho A, Lucena C, Calzado M A, et al. (2001) Ingenol esters induce apoptosis in Jurkat cells through an AP-1 and NF-kappaB independent pathway. Chem Biol 8: 767-778.
- 63. Blazkova J, Chun T W, Belay B W, Murray D, Justement J S, et al. (2012) Effect of histone deacetylase inhibitors on HIV production in latently infected, resting CD4(+) T cells from infected individuals receiving effective antiretroviral therapy. J Infect Dis 206: 765-769.
- 64. Chou T C (2006) Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol Rev 58: 621-681.
- 65. Tripathy M K, McManamy M E, Burch B D, Archin N M, Margolis D M (2015) H3K27 demethylation at the proviral promoter sensitizes latent HIV to the effects of vorinostat in ex-vivo cultures of resting CD4+ T cells. J Virol.
- Although the forgoing invention has been described in some detail by way of illustration and example for clarity and understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain variations, changes, modifications and substitutions of equivalents may be made thereto without necessarily departing from the spirit and scope of this invention. As a result, the embodiments described herein are subject to various modifications, changes and the like, with the scope of this invention being determined solely by reference to the claims appended hereto. Those of skill in the art will readily recognize a variety of non-critical parameters that could be changed, altered or modified to yield essentially similar results. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference. Where a conflict exists between the instant application and a reference provided herein, the instant application shall dominate.
Claims (21)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US16/506,841 US20200163924A1 (en) | 2014-08-08 | 2019-07-09 | Compositions and methods for reactivating latent viral infections |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201462035333P | 2014-08-08 | 2014-08-08 | |
PCT/US2015/042716 WO2016022358A1 (en) | 2014-08-08 | 2015-07-29 | Compositions and methods for reactivating latent viral infections |
US15/426,437 US10376486B2 (en) | 2014-08-08 | 2017-02-07 | Compositions and methods for reactivating latent viral infections |
US16/506,841 US20200163924A1 (en) | 2014-08-08 | 2019-07-09 | Compositions and methods for reactivating latent viral infections |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US15/426,437 Continuation US10376486B2 (en) | 2014-08-08 | 2017-02-07 | Compositions and methods for reactivating latent viral infections |
Publications (1)
Publication Number | Publication Date |
---|---|
US20200163924A1 true US20200163924A1 (en) | 2020-05-28 |
Family
ID=55264351
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US15/426,437 Active US10376486B2 (en) | 2014-08-08 | 2017-02-07 | Compositions and methods for reactivating latent viral infections |
US16/506,841 Abandoned US20200163924A1 (en) | 2014-08-08 | 2019-07-09 | Compositions and methods for reactivating latent viral infections |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US15/426,437 Active US10376486B2 (en) | 2014-08-08 | 2017-02-07 | Compositions and methods for reactivating latent viral infections |
Country Status (2)
Country | Link |
---|---|
US (2) | US10376486B2 (en) |
WO (1) | WO2016022358A1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2024064391A3 (en) * | 2022-09-22 | 2024-05-02 | K-Gen Therapeutics, Inc. | Use of protein kinase c (pkc) activating compounds as an adjuvant |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2016022358A1 (en) | 2014-08-08 | 2016-02-11 | The Regents Of The University Of California | Compositions and methods for reactivating latent viral infections |
US9817109B2 (en) | 2015-02-27 | 2017-11-14 | Texas Instruments Incorporated | Gesture recognition using frequency modulated continuous wave (FMCW) radar with low angle resolution |
WO2019245993A1 (en) * | 2018-06-19 | 2019-12-26 | Nantcell, Inc. | Hiv treatment compositions and methods |
Family Cites Families (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AUPQ801700A0 (en) | 2000-06-07 | 2000-06-29 | Peplin Research Pty Ltd | Enzyme and viral activation |
CN103119160B (en) * | 2010-05-14 | 2016-06-01 | 达那-法伯癌症研究所 | For regulating compositions and the method for metabolism |
EP2736528A4 (en) * | 2011-07-29 | 2015-03-18 | Philadelphia Children Hospital | Compositions and methods for the treatment of hiv |
CA2864995C (en) | 2012-03-02 | 2016-12-06 | Amazonia Fitomedicamentos Ltda. | Ingenol derivatives in the reactivation of latent hiv virus |
US20140079636A1 (en) * | 2012-04-16 | 2014-03-20 | Dinesh U. Chimmanamada | Targeted therapeutics |
CN105102000B (en) * | 2012-11-01 | 2021-10-22 | 无限药品公司 | Cancer therapy using PI3 kinase subtype modulators |
WO2016022358A1 (en) | 2014-08-08 | 2016-02-11 | The Regents Of The University Of California | Compositions and methods for reactivating latent viral infections |
-
2015
- 2015-07-29 WO PCT/US2015/042716 patent/WO2016022358A1/en active Application Filing
-
2017
- 2017-02-07 US US15/426,437 patent/US10376486B2/en active Active
-
2019
- 2019-07-09 US US16/506,841 patent/US20200163924A1/en not_active Abandoned
Non-Patent Citations (3)
Title |
---|
Kim et al., "Histone deacetylase inhibitors: molecular mechanisms of action and clinical trials as anti-cancer drugs," Am J Transl Res 3(2):166-179 (Year: 2011) * |
Michels et al., "Binding of the 7SK snRNA turns the HEXIM1 protein into a P-TEFb (CDK/cyclin T) inhibitor," The EMBO Journal 23: 2608-2619 (Year: 2004) * |
Palermo et al., "RNA Polymerase II Stalling Promoters Nucleosome Occlusion and pTEFb Recruitment to Drive Immortalization by Epstein-Barr Virus," PLoS Pathogens, 7(10): e1002334 (Year: 2011) * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2024064391A3 (en) * | 2022-09-22 | 2024-05-02 | K-Gen Therapeutics, Inc. | Use of protein kinase c (pkc) activating compounds as an adjuvant |
Also Published As
Publication number | Publication date |
---|---|
US20170258750A1 (en) | 2017-09-14 |
US10376486B2 (en) | 2019-08-13 |
WO2016022358A1 (en) | 2016-02-11 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Jiang et al. | Synergistic reactivation of latent HIV expression by ingenol-3-angelate, PEP005, targeted NF-kB signaling in combination with JQ1 induced p-TEFb activation | |
US20200163924A1 (en) | Compositions and methods for reactivating latent viral infections | |
Abner et al. | HIV “shock and kill” therapy: In need of revision | |
Tong et al. | Merimepodib, an IMPDH inhibitor, suppresses replication of Zika virus and other emerging viral pathogens | |
Xing et al. | Targeting HIV latency: pharmacologic strategies toward eradication | |
Chibber et al. | Advances in the possible treatment of COVID-19: A review. | |
Andersen et al. | Natural product-derived compounds in HIV suppression, remission, and eradication strategies | |
US20160095850A1 (en) | Methods and compositions for treatment of hiv infection | |
CA2179627C (en) | Mixtures of dideoxy-nucleosides and hydroxycarbamide for inhibiting retroviral spread | |
US9943536B2 (en) | Reactivation of HIV-1 gene expression to treat persistent HIV infection | |
EA028605B1 (en) | Combination of oseltamivir and a mek inhibitor for the treatment of viral diseases | |
WO2016196471A1 (en) | Methods and compositions for treatment of hiv infection | |
Knights | A critical review of the evidence concerning the HIV latency reversing effect of disulfiram, the possible explanations for its inability to reduce the size of the latent reservoir in vivo, and the caveats associated with its use in practice | |
Massanella et al. | Attacking the HIV reservoir from the immune and viral perspective | |
Lim et al. | Synergistic reactivation of latent HIV-1 provirus by PKA activator dibutyryl-cAMP in combination with an HDAC inhibitor | |
US20230011398A1 (en) | Combination therapy approach to eliminate hiv infections | |
Nandi et al. | Repurposing of chemotherapeutics to combat COVID-19 | |
US20180169108A1 (en) | Drug Combinations For the Treatment of HIV | |
Zaongo et al. | Selective elimination of host cells harboring replication-competent human immunodeficiency virus reservoirs: a promising therapeutic strategy for HIV cure | |
US20200268856A1 (en) | Methods of reactivating latent human immunodeficiency virus and related compositions | |
Smee et al. | Efficacy of N-methanocarbathymidine in treating mice infected intranasally with the IHD and WR strains of vaccinia virus | |
CA3196906A1 (en) | Compositions and methods for preventing and treating coronaviruses | |
Srivatsan Padmanabhan et al. | Nitazoxanide–a potential ally in the treatment of COVID-19 | |
Romanelli et al. | Use of virostatics as a means of targeting human immunodeficiency virus infection | |
Ullah et al. | Bioluminescence imaging reveals enhanced SARS-CoV-2 clearance in mice with combinatorial regimens |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: THE REGENTS OF THE UNIVERSITY OF CALIFORNIA, CALIFORNIA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:JIANG, GUOCHUN;DANDEKAR, SATYA;SIGNING DATES FROM 20170207 TO 20170208;REEL/FRAME:050478/0864 |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: FINAL REJECTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |