WO2019241873A1 - Metal containing products and compositions to induce immunogenic cell death and uses thereof - Google Patents
Metal containing products and compositions to induce immunogenic cell death and uses thereof Download PDFInfo
- Publication number
- WO2019241873A1 WO2019241873A1 PCT/CA2019/000094 CA2019000094W WO2019241873A1 WO 2019241873 A1 WO2019241873 A1 WO 2019241873A1 CA 2019000094 W CA2019000094 W CA 2019000094W WO 2019241873 A1 WO2019241873 A1 WO 2019241873A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- agent
- copper
- icd
- combination
- cells
- Prior art date
Links
- 230000037449 immunogenic cell death Effects 0.000 title claims abstract description 105
- 239000000203 mixture Substances 0.000 title claims abstract description 22
- 229910052751 metal Inorganic materials 0.000 title claims description 106
- 239000002184 metal Substances 0.000 title claims description 106
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 266
- 239000010949 copper Substances 0.000 claims abstract description 225
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 claims abstract description 204
- 229910052802 copper Inorganic materials 0.000 claims abstract description 203
- 239000002502 liposome Substances 0.000 claims abstract description 84
- 239000003981 vehicle Substances 0.000 claims abstract description 40
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 37
- 230000028327 secretion Effects 0.000 claims abstract description 35
- 238000000034 method Methods 0.000 claims abstract description 30
- 201000011510 cancer Diseases 0.000 claims abstract description 19
- 102000000849 HMGB Proteins Human genes 0.000 claims abstract description 18
- 108010001860 HMGB Proteins Proteins 0.000 claims abstract description 18
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 18
- 201000010099 disease Diseases 0.000 claims abstract description 17
- 208000015181 infectious disease Diseases 0.000 claims abstract description 8
- 210000004027 cell Anatomy 0.000 claims description 159
- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-J 0.000 claims description 119
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 claims description 119
- 238000011282 treatment Methods 0.000 claims description 49
- 239000008194 pharmaceutical composition Substances 0.000 claims description 38
- 229910021645 metal ion Inorganic materials 0.000 claims description 34
- 238000003556 assay Methods 0.000 claims description 28
- 231100000433 cytotoxic Toxicity 0.000 claims description 21
- 230000001472 cytotoxic effect Effects 0.000 claims description 21
- 238000000338 in vitro Methods 0.000 claims description 16
- 230000001939 inductive effect Effects 0.000 claims description 16
- 239000002105 nanoparticle Substances 0.000 claims description 13
- 229920000642 polymer Polymers 0.000 claims description 13
- 230000004044 response Effects 0.000 claims description 13
- 210000001808 exosome Anatomy 0.000 claims description 12
- 238000009472 formulation Methods 0.000 claims description 11
- 239000000693 micelle Substances 0.000 claims description 10
- 230000005855 radiation Effects 0.000 claims description 10
- 239000000839 emulsion Substances 0.000 claims description 7
- 239000002071 nanotube Substances 0.000 claims description 7
- 238000002347 injection Methods 0.000 claims description 5
- 239000007924 injection Substances 0.000 claims description 5
- 230000003308 immunostimulating effect Effects 0.000 claims description 3
- 238000000099 in vitro assay Methods 0.000 claims description 3
- 238000004113 cell culture Methods 0.000 claims description 2
- 230000000536 complexating effect Effects 0.000 claims description 2
- 150000004696 coordination complex Chemical class 0.000 claims description 2
- 229960005486 vaccine Drugs 0.000 claims 2
- 210000001163 endosome Anatomy 0.000 claims 1
- 239000002246 antineoplastic agent Substances 0.000 description 85
- SDUQYLNIPVEERB-QPPQHZFASA-N gemcitabine Chemical compound O=C1N=C(N)C=CN1[C@H]1C(F)(F)[C@H](O)[C@@H](CO)O1 SDUQYLNIPVEERB-QPPQHZFASA-N 0.000 description 51
- 229960005277 gemcitabine Drugs 0.000 description 51
- 102000004082 Calreticulin Human genes 0.000 description 45
- 108090000549 Calreticulin Proteins 0.000 description 45
- GIGCDIVNDFQKRA-LTCKWSDVSA-N 4-[(2s)-2-amino-2-carboxyethyl]-n,n-bis(2-chloroethyl)benzeneamine oxide;dihydrochloride Chemical compound Cl.Cl.OC(=O)[C@@H](N)CC1=CC=C([N+]([O-])(CCCl)CCCl)C=C1 GIGCDIVNDFQKRA-LTCKWSDVSA-N 0.000 description 42
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 38
- 239000011701 zinc Substances 0.000 description 38
- 229910052725 zinc Inorganic materials 0.000 description 38
- 150000002632 lipids Chemical class 0.000 description 29
- 239000000243 solution Substances 0.000 description 29
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 28
- 238000011068 loading method Methods 0.000 description 28
- 150000002739 metals Chemical class 0.000 description 26
- STQGQHZAVUOBTE-UHFFFAOYSA-N 7-Cyan-hept-2t-en-4,6-diinsaeure Natural products C1=2C(O)=C3C(=O)C=4C(OC)=CC=CC=4C(=O)C3=C(O)C=2CC(O)(C(C)=O)CC1OC1CC(N)C(O)C(C)O1 STQGQHZAVUOBTE-UHFFFAOYSA-N 0.000 description 25
- 229960000975 daunorubicin Drugs 0.000 description 25
- STQGQHZAVUOBTE-VGBVRHCVSA-N daunorubicin 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(C)=O)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 STQGQHZAVUOBTE-VGBVRHCVSA-N 0.000 description 25
- 239000003642 reactive oxygen metabolite Substances 0.000 description 24
- 229940116901 diethyldithiocarbamate Drugs 0.000 description 23
- LMBWSYZSUOEYSN-UHFFFAOYSA-N diethyldithiocarbamic acid Chemical compound CCN(CC)C(S)=S LMBWSYZSUOEYSN-UHFFFAOYSA-N 0.000 description 23
- UHDGCWIWMRVCDJ-CCXZUQQUSA-N Cytarabine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@@H](O)[C@H](O)[C@@H](CO)O1 UHDGCWIWMRVCDJ-CCXZUQQUSA-N 0.000 description 22
- BIIVYFLTOXDAOV-YVEFUNNKSA-N alvocidib Chemical compound O[C@@H]1CN(C)CC[C@@H]1C1=C(O)C=C(O)C2=C1OC(C=1C(=CC=CC=1)Cl)=CC2=O BIIVYFLTOXDAOV-YVEFUNNKSA-N 0.000 description 21
- 229950010817 alvocidib Drugs 0.000 description 21
- 239000003814 drug Substances 0.000 description 21
- 229940079593 drug Drugs 0.000 description 19
- 229960001156 mitoxantrone Drugs 0.000 description 18
- KKZJGLLVHKMTCM-UHFFFAOYSA-N mitoxantrone Chemical compound O=C1C2=C(O)C=CC(O)=C2C(=O)C2=C1C(NCCNCCO)=CC=C2NCCNCCO KKZJGLLVHKMTCM-UHFFFAOYSA-N 0.000 description 18
- 238000010668 complexation reaction Methods 0.000 description 17
- LCOIAYJMPKXARU-VAWYXSNFSA-N salubrinal Chemical compound C=1C=CC2=CC=CN=C2C=1NC(=S)NC(C(Cl)(Cl)Cl)NC(=O)\C=C\C1=CC=CC=C1 LCOIAYJMPKXARU-VAWYXSNFSA-N 0.000 description 17
- FFWOKTFYGVYKIR-UHFFFAOYSA-N physcion Chemical compound C1=C(C)C=C2C(=O)C3=CC(OC)=CC(O)=C3C(=O)C2=C1O FFWOKTFYGVYKIR-UHFFFAOYSA-N 0.000 description 16
- QCDFBFJGMNKBDO-UHFFFAOYSA-N Clioquinol Chemical compound C1=CN=C2C(O)=C(I)C=C(Cl)C2=C1 QCDFBFJGMNKBDO-UHFFFAOYSA-N 0.000 description 15
- 239000005725 8-Hydroxyquinoline Substances 0.000 description 14
- 229950001573 abemaciclib Drugs 0.000 description 14
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 description 14
- 229960004316 cisplatin Drugs 0.000 description 14
- 229910052742 iron Inorganic materials 0.000 description 14
- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 description 14
- UZWDCWONPYILKI-UHFFFAOYSA-N n-[5-[(4-ethylpiperazin-1-yl)methyl]pyridin-2-yl]-5-fluoro-4-(7-fluoro-2-methyl-3-propan-2-ylbenzimidazol-5-yl)pyrimidin-2-amine Chemical compound C1CN(CC)CCN1CC(C=N1)=CC=C1NC1=NC=C(F)C(C=2C=C3N(C(C)C)C(C)=NC3=C(F)C=2)=N1 UZWDCWONPYILKI-UHFFFAOYSA-N 0.000 description 14
- 229960003540 oxyquinoline Drugs 0.000 description 14
- PKUBGLYEOAJPEG-UHFFFAOYSA-N physcion Natural products C1=C(C)C=C2C(=O)C3=CC(C)=CC(O)=C3C(=O)C2=C1O PKUBGLYEOAJPEG-UHFFFAOYSA-N 0.000 description 14
- MCJGNVYPOGVAJF-UHFFFAOYSA-N quinolin-8-ol Chemical compound C1=CN=C2C(O)=CC=CC2=C1 MCJGNVYPOGVAJF-UHFFFAOYSA-N 0.000 description 14
- 241000699670 Mus sp. Species 0.000 description 13
- 229910017052 cobalt Inorganic materials 0.000 description 13
- 239000010941 cobalt Substances 0.000 description 13
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 description 13
- 230000001965 increasing effect Effects 0.000 description 12
- 230000006698 induction Effects 0.000 description 12
- 230000001419 dependent effect Effects 0.000 description 11
- 230000000694 effects Effects 0.000 description 11
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 10
- 150000001875 compounds Chemical class 0.000 description 10
- 229910052720 vanadium Inorganic materials 0.000 description 10
- GPPXJZIENCGNKB-UHFFFAOYSA-N vanadium Chemical compound [V]#[V] GPPXJZIENCGNKB-UHFFFAOYSA-N 0.000 description 10
- 150000001879 copper Chemical class 0.000 description 9
- 229960000684 cytarabine Drugs 0.000 description 9
- 238000012377 drug delivery Methods 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- RHXHGRAEPCAFML-UHFFFAOYSA-N 7-cyclopentyl-n,n-dimethyl-2-[(5-piperazin-1-ylpyridin-2-yl)amino]pyrrolo[2,3-d]pyrimidine-6-carboxamide Chemical compound N1=C2N(C3CCCC3)C(C(=O)N(C)C)=CC2=CN=C1NC(N=C1)=CC=C1N1CCNCC1 RHXHGRAEPCAFML-UHFFFAOYSA-N 0.000 description 8
- UGNZSMZSJYOGNX-UHFFFAOYSA-N Isoviocristine Natural products O=C1C=C(C)C(=O)C2=CC3=CC(OC)=CC(O)=C3C(O)=C21 UGNZSMZSJYOGNX-UHFFFAOYSA-N 0.000 description 8
- WLXGUTUUWXVZNM-UHFFFAOYSA-N anthraglycoside A Natural products C1=C(C)C=C2C(=O)C3=CC(OC)=CC(O)=C3C(=O)C2=C1OC1OC(CO)C(O)C(O)C1O WLXGUTUUWXVZNM-UHFFFAOYSA-N 0.000 description 8
- 229960004390 palbociclib Drugs 0.000 description 8
- AHJRHEGDXFFMBM-UHFFFAOYSA-N palbociclib Chemical compound N1=C2N(C3CCCC3)C(=O)C(C(=O)C)=C(C)C2=CN=C1NC(N=C1)=CC=C1N1CCNCC1 AHJRHEGDXFFMBM-UHFFFAOYSA-N 0.000 description 8
- FGVVTMRZYROCTH-UHFFFAOYSA-N pyridine-2-thiol N-oxide Chemical compound [O-][N+]1=CC=CC=C1S FGVVTMRZYROCTH-UHFFFAOYSA-N 0.000 description 8
- 229960002026 pyrithione Drugs 0.000 description 8
- 229950003687 ribociclib Drugs 0.000 description 8
- 238000002255 vaccination Methods 0.000 description 8
- OCUCCJIRFHNWBP-IYEMJOQQSA-L Copper gluconate Chemical compound [Cu+2].OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O.OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O OCUCCJIRFHNWBP-IYEMJOQQSA-L 0.000 description 7
- 239000012981 Hank's balanced salt solution Substances 0.000 description 7
- 230000015572 biosynthetic process Effects 0.000 description 7
- 229940108925 copper gluconate Drugs 0.000 description 7
- 229910000365 copper sulfate Inorganic materials 0.000 description 7
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 7
- 239000000411 inducer Substances 0.000 description 7
- 239000003446 ligand Substances 0.000 description 7
- 239000002245 particle Substances 0.000 description 7
- JFJNVIPVOCESGZ-UHFFFAOYSA-N 2,3-dipyridin-2-ylpyridine Chemical class N1=CC=CC=C1C1=CC=CN=C1C1=CC=CC=N1 JFJNVIPVOCESGZ-UHFFFAOYSA-N 0.000 description 6
- UPOMZZQMFGCHGB-UHFFFAOYSA-N 4-(4-chlorophenyl)-2,6-dipyridin-2-ylpyridine Chemical compound C1=CC(Cl)=CC=C1C1=CC(C=2N=CC=CC=2)=NC(C=2N=CC=CC=2)=C1 UPOMZZQMFGCHGB-UHFFFAOYSA-N 0.000 description 6
- VWDXGKUTGQJJHJ-UHFFFAOYSA-N Catenarin Natural products C1=C(O)C=C2C(=O)C3=C(O)C(C)=CC(O)=C3C(=O)C2=C1O VWDXGKUTGQJJHJ-UHFFFAOYSA-N 0.000 description 6
- 206010009944 Colon cancer Diseases 0.000 description 6
- 239000010282 Emodin Substances 0.000 description 6
- RBLJKYCRSCQLRP-UHFFFAOYSA-N Emodin-dianthron Natural products O=C1C2=CC(C)=CC(O)=C2C(=O)C2=C1CC(=O)C=C2O RBLJKYCRSCQLRP-UHFFFAOYSA-N 0.000 description 6
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 6
- YOOXNSPYGCZLAX-UHFFFAOYSA-N Helminthosporin Natural products C1=CC(O)=C2C(=O)C3=CC(C)=CC(O)=C3C(=O)C2=C1O YOOXNSPYGCZLAX-UHFFFAOYSA-N 0.000 description 6
- 239000000232 Lipid Bilayer Substances 0.000 description 6
- 241001529936 Murinae Species 0.000 description 6
- 239000002202 Polyethylene glycol Substances 0.000 description 6
- NTGIIKCGBNGQAR-UHFFFAOYSA-N Rheoemodin Natural products C1=C(O)C=C2C(=O)C3=CC(O)=CC(O)=C3C(=O)C2=C1O NTGIIKCGBNGQAR-UHFFFAOYSA-N 0.000 description 6
- 229960005228 clioquinol Drugs 0.000 description 6
- 208000029742 colonic neoplasm Diseases 0.000 description 6
- ORTQZVOHEJQUHG-UHFFFAOYSA-L copper(II) chloride Chemical compound Cl[Cu]Cl ORTQZVOHEJQUHG-UHFFFAOYSA-L 0.000 description 6
- RHMXXJGYXNZAPX-UHFFFAOYSA-N emodin Chemical compound C1=C(O)C=C2C(=O)C3=CC(C)=CC(O)=C3C(=O)C2=C1O RHMXXJGYXNZAPX-UHFFFAOYSA-N 0.000 description 6
- VASFLQKDXBAWEL-UHFFFAOYSA-N emodin Natural products OC1=C(OC2=C(C=CC(=C2C1=O)O)O)C1=CC=C(C=C1)O VASFLQKDXBAWEL-UHFFFAOYSA-N 0.000 description 6
- 230000002209 hydrophobic effect Effects 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 229920001223 polyethylene glycol Polymers 0.000 description 6
- 241000894007 species Species 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 239000000872 buffer Substances 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- OPQARKPSCNTWTJ-UHFFFAOYSA-L copper(ii) acetate Chemical compound [Cu+2].CC([O-])=O.CC([O-])=O OPQARKPSCNTWTJ-UHFFFAOYSA-L 0.000 description 5
- 229940043378 cyclin-dependent kinase inhibitor Drugs 0.000 description 5
- 210000004443 dendritic cell Anatomy 0.000 description 5
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 5
- 239000002555 ionophore Substances 0.000 description 5
- 239000003550 marker Substances 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- 239000011859 microparticle Substances 0.000 description 5
- 229910052759 nickel Inorganic materials 0.000 description 5
- 239000002953 phosphate buffered saline Substances 0.000 description 5
- 229910052723 transition metal Inorganic materials 0.000 description 5
- 150000003624 transition metals Chemical class 0.000 description 5
- PRDFBSVERLRRMY-UHFFFAOYSA-N 2'-(4-ethoxyphenyl)-5-(4-methylpiperazin-1-yl)-2,5'-bibenzimidazole Chemical compound C1=CC(OCC)=CC=C1C1=NC2=CC=C(C=3NC4=CC(=CC=C4N=3)N3CCN(C)CC3)C=C2N1 PRDFBSVERLRRMY-UHFFFAOYSA-N 0.000 description 4
- 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 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- MJVAVZPDRWSRRC-UHFFFAOYSA-N Menadione Chemical compound C1=CC=C2C(=O)C(C)=CC(=O)C2=C1 MJVAVZPDRWSRRC-UHFFFAOYSA-N 0.000 description 4
- 229940034982 antineoplastic agent Drugs 0.000 description 4
- 230000030833 cell death Effects 0.000 description 4
- 210000000170 cell membrane Anatomy 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 4
- 229940127089 cytotoxic agent Drugs 0.000 description 4
- 238000001125 extrusion Methods 0.000 description 4
- LNTHITQWFMADLM-UHFFFAOYSA-N gallic acid Chemical compound OC(=O)C1=CC(O)=C(O)C(O)=C1 LNTHITQWFMADLM-UHFFFAOYSA-N 0.000 description 4
- 229920001477 hydrophilic polymer Polymers 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 4
- 238000011081 inoculation Methods 0.000 description 4
- 230000000236 ionophoric effect Effects 0.000 description 4
- 238000004020 luminiscence type Methods 0.000 description 4
- 229960001756 oxaliplatin Drugs 0.000 description 4
- DWAFYCQODLXJNR-BNTLRKBRSA-L oxaliplatin Chemical compound O1C(=O)C(=O)O[Pt]11N[C@@H]2CCCC[C@H]2N1 DWAFYCQODLXJNR-BNTLRKBRSA-L 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 150000003839 salts Chemical class 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- 210000004881 tumor cell Anatomy 0.000 description 4
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 description 3
- 238000008157 ELISA kit Methods 0.000 description 3
- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 description 3
- 206010021143 Hypoxia Diseases 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 239000011230 binding agent Substances 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 230000009918 complex formation Effects 0.000 description 3
- 239000002254 cytotoxic agent Substances 0.000 description 3
- 231100000599 cytotoxic agent Toxicity 0.000 description 3
- 230000003013 cytotoxicity Effects 0.000 description 3
- 231100000135 cytotoxicity Toxicity 0.000 description 3
- 230000001146 hypoxic effect Effects 0.000 description 3
- 230000028993 immune response Effects 0.000 description 3
- 210000000987 immune system Anatomy 0.000 description 3
- 238000010348 incorporation Methods 0.000 description 3
- 239000010410 layer Substances 0.000 description 3
- 239000013554 lipid monolayer Substances 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 150000003904 phospholipids Chemical class 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 230000032258 transport Effects 0.000 description 3
- QMABMHJGSFUTPF-UHFFFAOYSA-N 4-tert-butyl-2,6-bis(4-tert-butylpyridin-2-yl)pyridine Chemical compound CC(C)(C)C1=CC=NC(C=2N=C(C=C(C=2)C(C)(C)C)C=2N=CC=C(C=2)C(C)(C)C)=C1 QMABMHJGSFUTPF-UHFFFAOYSA-N 0.000 description 2
- NEZONWMXZKDMKF-JTQLQIEISA-N Alkannin Chemical compound C1=CC(O)=C2C(=O)C([C@@H](O)CC=C(C)C)=CC(=O)C2=C1O NEZONWMXZKDMKF-JTQLQIEISA-N 0.000 description 2
- ZOXJGFHDIHLPTG-UHFFFAOYSA-N Boron Chemical compound [B] ZOXJGFHDIHLPTG-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 2
- 102100037907 High mobility group protein B1 Human genes 0.000 description 2
- 101001025337 Homo sapiens High mobility group protein B1 Proteins 0.000 description 2
- 241001071917 Lithospermum Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- XYFCBTPGUUZFHI-UHFFFAOYSA-N Phosphine Chemical compound P XYFCBTPGUUZFHI-UHFFFAOYSA-N 0.000 description 2
- 229940123237 Taxane Drugs 0.000 description 2
- 239000000365 Topoisomerase I Inhibitor Substances 0.000 description 2
- 229940122803 Vinca alkaloid Drugs 0.000 description 2
- HIMJIPRMECETLJ-UHFFFAOYSA-N Wogonin Natural products COc1cc(O)c(O)c2C(=O)C=C(Oc12)c3ccccc3 HIMJIPRMECETLJ-UHFFFAOYSA-N 0.000 description 2
- 230000033289 adaptive immune response Effects 0.000 description 2
- 230000000996 additive effect Effects 0.000 description 2
- UNNKKUDWEASWDN-UHFFFAOYSA-N alkannin Natural products CC(=CCC(O)c1cc(O)c2C(=O)C=CC(=O)c2c1O)C UNNKKUDWEASWDN-UHFFFAOYSA-N 0.000 description 2
- 229940100198 alkylating agent Drugs 0.000 description 2
- 239000002168 alkylating agent Substances 0.000 description 2
- 229940045799 anthracyclines and related substance Drugs 0.000 description 2
- 230000001093 anti-cancer Effects 0.000 description 2
- 230000003432 anti-folate effect Effects 0.000 description 2
- 230000000118 anti-neoplastic effect Effects 0.000 description 2
- 229940127074 antifolate Drugs 0.000 description 2
- 229940045985 antineoplastic platinum compound Drugs 0.000 description 2
- 230000001640 apoptogenic effect Effects 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 229910052796 boron Inorganic materials 0.000 description 2
- 238000012054 celltiter-glo Methods 0.000 description 2
- 239000013522 chelant Substances 0.000 description 2
- 239000002738 chelating agent Substances 0.000 description 2
- 125000003636 chemical group Chemical group 0.000 description 2
- 235000012000 cholesterol Nutrition 0.000 description 2
- 229910000366 copper(II) sulfate Inorganic materials 0.000 description 2
- 238000002784 cytotoxicity assay Methods 0.000 description 2
- 231100000263 cytotoxicity test Toxicity 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 235000014113 dietary fatty acids Nutrition 0.000 description 2
- 229960004679 doxorubicin Drugs 0.000 description 2
- 238000005538 encapsulation Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 229930195729 fatty acid Natural products 0.000 description 2
- 239000000194 fatty acid Substances 0.000 description 2
- 150000004665 fatty acids Chemical class 0.000 description 2
- LIYGYAHYXQDGEP-UHFFFAOYSA-N firefly oxyluciferin Natural products Oc1csc(n1)-c1nc2ccc(O)cc2s1 LIYGYAHYXQDGEP-UHFFFAOYSA-N 0.000 description 2
- 239000004052 folic acid antagonist Substances 0.000 description 2
- 239000003276 histone deacetylase inhibitor Substances 0.000 description 2
- 229940121372 histone deacetylase inhibitor Drugs 0.000 description 2
- 230000002519 immonomodulatory effect Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 229940124302 mTOR inhibitor Drugs 0.000 description 2
- 210000002540 macrophage Anatomy 0.000 description 2
- 239000003628 mammalian target of rapamycin inhibitor Substances 0.000 description 2
- 229940127073 nucleoside analogue Drugs 0.000 description 2
- -1 oleoyl phosphatidylcholine Chemical compound 0.000 description 2
- JJVOROULKOMTKG-UHFFFAOYSA-N oxidized Photinus luciferin Chemical compound S1C2=CC(O)=CC=C2N=C1C1=NC(=O)CS1 JJVOROULKOMTKG-UHFFFAOYSA-N 0.000 description 2
- 125000003854 p-chlorophenyl group Chemical group [H]C1=C([H])C(*)=C([H])C([H])=C1Cl 0.000 description 2
- 150000008104 phosphatidylethanolamines Chemical class 0.000 description 2
- 150000003058 platinum compounds Chemical class 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 238000002203 pretreatment Methods 0.000 description 2
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 150000003408 sphingolipids Chemical class 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 230000002195 synergetic effect Effects 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 229940124597 therapeutic agent Drugs 0.000 description 2
- 229940121358 tyrosine kinase inhibitor Drugs 0.000 description 2
- 239000005483 tyrosine kinase inhibitor Substances 0.000 description 2
- 235000012711 vitamin K3 Nutrition 0.000 description 2
- 239000011652 vitamin K3 Substances 0.000 description 2
- 229940041603 vitamin k 3 Drugs 0.000 description 2
- XLTFNNCXVBYBSX-UHFFFAOYSA-N wogonin Chemical compound COC1=C(O)C=C(O)C(C(C=2)=O)=C1OC=2C1=CC=CC=C1 XLTFNNCXVBYBSX-UHFFFAOYSA-N 0.000 description 2
- 150000003751 zinc Chemical class 0.000 description 2
- FVXDQWZBHIXIEJ-LNDKUQBDSA-N 1,2-di-[(9Z,12Z)-octadecadienoyl]-sn-glycero-3-phosphocholine Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/C\C=C/CCCCC FVXDQWZBHIXIEJ-LNDKUQBDSA-N 0.000 description 1
- KILNVBDSWZSGLL-KXQOOQHDSA-N 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCCCC KILNVBDSWZSGLL-KXQOOQHDSA-N 0.000 description 1
- PORPENFLTBBHSG-MGBGTMOVSA-N 1,2-dihexadecanoyl-sn-glycerol-3-phosphate Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(O)=O)OC(=O)CCCCCCCCCCCCCCC PORPENFLTBBHSG-MGBGTMOVSA-N 0.000 description 1
- SNKAWJBJQDLSFF-NVKMUCNASA-N 1,2-dioleoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/CCCCCCCC SNKAWJBJQDLSFF-NVKMUCNASA-N 0.000 description 1
- NRJAVPSFFCBXDT-HUESYALOSA-N 1,2-distearoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCCCCCC NRJAVPSFFCBXDT-HUESYALOSA-N 0.000 description 1
- TZCPCKNHXULUIY-RGULYWFUSA-N 1,2-distearoyl-sn-glycero-3-phosphoserine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(=O)OC[C@H](N)C(O)=O)OC(=O)CCCCCCCCCCCCCCCCC TZCPCKNHXULUIY-RGULYWFUSA-N 0.000 description 1
- RYCNUMLMNKHWPZ-SNVBAGLBSA-N 1-acetyl-sn-glycero-3-phosphocholine Chemical compound CC(=O)OC[C@@H](O)COP([O-])(=O)OCC[N+](C)(C)C RYCNUMLMNKHWPZ-SNVBAGLBSA-N 0.000 description 1
- OFNXOACBUMGOPC-HZYVHMACSA-N 5'-hydroxystreptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](CO)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O OFNXOACBUMGOPC-HZYVHMACSA-N 0.000 description 1
- BGWLYQZDNFIFRX-UHFFFAOYSA-N 5-[3-[2-[3-(3,8-diamino-6-phenylphenanthridin-5-ium-5-yl)propylamino]ethylamino]propyl]-6-phenylphenanthridin-5-ium-3,8-diamine;dichloride Chemical compound [Cl-].[Cl-].C=1C(N)=CC=C(C2=CC=C(N)C=C2[N+]=2CCCNCCNCCC[N+]=3C4=CC(N)=CC=C4C4=CC=C(N)C=C4C=3C=3C=CC=CC=3)C=1C=2C1=CC=CC=C1 BGWLYQZDNFIFRX-UHFFFAOYSA-N 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 108010004103 Chylomicrons Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 description 1
- KDXKERNSBIXSRK-RXMQYKEDSA-N D-lysine Chemical compound NCCCC[C@@H](N)C(O)=O KDXKERNSBIXSRK-RXMQYKEDSA-N 0.000 description 1
- 229940123780 DNA topoisomerase I inhibitor Drugs 0.000 description 1
- CYCGRDQQIOGCKX-UHFFFAOYSA-N Dehydro-luciferin Natural products OC(=O)C1=CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 CYCGRDQQIOGCKX-UHFFFAOYSA-N 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
- BJGNCJDXODQBOB-UHFFFAOYSA-N Fivefly Luciferin Natural products OC(=O)C1CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 BJGNCJDXODQBOB-UHFFFAOYSA-N 0.000 description 1
- GYHNNYVSQQEPJS-UHFFFAOYSA-N Gallium Chemical compound [Ga] GYHNNYVSQQEPJS-UHFFFAOYSA-N 0.000 description 1
- ZWZWYGMENQVNFU-UHFFFAOYSA-N Glycerophosphorylserin Natural products OC(=O)C(N)COP(O)(=O)OCC(O)CO ZWZWYGMENQVNFU-UHFFFAOYSA-N 0.000 description 1
- 108010010234 HDL Lipoproteins Proteins 0.000 description 1
- 102000015779 HDL Lipoproteins Human genes 0.000 description 1
- 108010007622 LDL Lipoproteins Proteins 0.000 description 1
- 102000007330 LDL Lipoproteins Human genes 0.000 description 1
- 108090001030 Lipoproteins Proteins 0.000 description 1
- 102000004895 Lipoproteins Human genes 0.000 description 1
- DDWFXDSYGUXRAY-UHFFFAOYSA-N Luciferin Natural products CCc1c(C)c(CC2NC(=O)C(=C2C=C)C)[nH]c1Cc3[nH]c4C(=C5/NC(CC(=O)O)C(C)C5CC(=O)O)CC(=O)c4c3C DDWFXDSYGUXRAY-UHFFFAOYSA-N 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 206010057249 Phagocytosis Diseases 0.000 description 1
- BELBBZDIHDAJOR-UHFFFAOYSA-N Phenolsulfonephthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2S(=O)(=O)O1 BELBBZDIHDAJOR-UHFFFAOYSA-N 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 108010081750 Reticulin Proteins 0.000 description 1
- 239000002262 Schiff base Substances 0.000 description 1
- 150000004753 Schiff bases Chemical class 0.000 description 1
- 229930182558 Sterol Natural products 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- COYHRQWNJDJCNA-NUJDXYNKSA-N Thr-Thr-Thr Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O COYHRQWNJDJCNA-NUJDXYNKSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 108010046516 Wheat Germ Agglutinins Proteins 0.000 description 1
- CWRILEGKIAOYKP-SSDOTTSWSA-M [(2r)-3-acetyloxy-2-hydroxypropyl] 2-aminoethyl phosphate Chemical compound CC(=O)OC[C@@H](O)COP([O-])(=O)OCCN CWRILEGKIAOYKP-SSDOTTSWSA-M 0.000 description 1
- 125000002252 acyl group Chemical group 0.000 description 1
- 238000011374 additional therapy Methods 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000003042 antagnostic effect Effects 0.000 description 1
- 230000005809 anti-tumor immunity Effects 0.000 description 1
- 239000012635 anticancer drug combination Substances 0.000 description 1
- 230000030741 antigen processing and presentation Effects 0.000 description 1
- 210000000612 antigen-presenting cell Anatomy 0.000 description 1
- 239000003443 antiviral agent Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000000149 argon plasma sintering Methods 0.000 description 1
- 125000004429 atom Chemical group 0.000 description 1
- 230000003416 augmentation Effects 0.000 description 1
- 238000003705 background correction Methods 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 230000034303 cell budding Effects 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 150000004699 copper complex Chemical class 0.000 description 1
- 229910001431 copper ion Inorganic materials 0.000 description 1
- SBHDKYTVDCRMOE-JPAPVDFESA-L copper;n'-methyl-n-[(e)-[(3e)-3-[(n-methyl-c-sulfidocarbonimidoyl)hydrazinylidene]butan-2-ylidene]amino]carbamimidothioate Chemical compound [Cu+2].CN=C([S-])N\N=C(/C)\C(\C)=N\NC([S-])=NC SBHDKYTVDCRMOE-JPAPVDFESA-L 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 230000004041 dendritic cell maturation Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 239000000890 drug combination Substances 0.000 description 1
- 230000009881 electrostatic interaction Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000001723 extracellular space Anatomy 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 229910052733 gallium Inorganic materials 0.000 description 1
- 150000002327 glycerophospholipids Chemical class 0.000 description 1
- 150000002339 glycosphingolipids Chemical class 0.000 description 1
- 229910021389 graphene Inorganic materials 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 150000007857 hydrazones Chemical class 0.000 description 1
- OFNXOACBUMGOPC-UHFFFAOYSA-N hydroxystreptomycin Natural products CNC1C(O)C(O)C(CO)OC1OC1C(C=O)(O)C(CO)OC1OC1C(N=C(N)N)C(O)C(N=C(N)N)C(O)C1O OFNXOACBUMGOPC-UHFFFAOYSA-N 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 229910052738 indium Inorganic materials 0.000 description 1
- APFVFJFRJDLVQX-UHFFFAOYSA-N indium atom Chemical compound [In] APFVFJFRJDLVQX-UHFFFAOYSA-N 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- OKPOKMCPHKVCPP-UHFFFAOYSA-N isoorientaline Natural products C1=C(O)C(OC)=CC(CC2C3=CC(OC)=C(O)C=C3CCN2C)=C1 OKPOKMCPHKVCPP-UHFFFAOYSA-N 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 229940107698 malachite green Drugs 0.000 description 1
- FDZZZRQASAIRJF-UHFFFAOYSA-M malachite green Chemical compound [Cl-].C1=CC(N(C)C)=CC=C1C(C=1C=CC=CC=1)=C1C=CC(=[N+](C)C)C=C1 FDZZZRQASAIRJF-UHFFFAOYSA-M 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 231100000065 noncytotoxic Toxicity 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 239000007764 o/w emulsion Substances 0.000 description 1
- 230000009437 off-target effect Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000008823 permeabilization Effects 0.000 description 1
- 230000008782 phagocytosis Effects 0.000 description 1
- 229960003531 phenolsulfonphthalein Drugs 0.000 description 1
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 description 1
- 150000003905 phosphatidylinositols Chemical class 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229910000073 phosphorus hydride Inorganic materials 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 230000000770 proinflammatory effect Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 230000002685 pulmonary effect Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- JTQHYPFKHZLTSH-UHFFFAOYSA-N reticulin Natural products COC1CC(OC2C(CO)OC(OC3C(O)CC(OC4C(C)OC(CC4OC)OC5CCC6(C)C7CCC8(C)C(CCC8(O)C7CC=C6C5)C(C)O)OC3C)C(O)C2OC)OC(C)C1O JTQHYPFKHZLTSH-UHFFFAOYSA-N 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 150000003432 sterols Chemical class 0.000 description 1
- 235000003702 sterols Nutrition 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- 229920001059 synthetic polymer Polymers 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 150000003626 triacylglycerols Chemical class 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 230000007332 vesicle formation Effects 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
Classifications
-
- 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
-
- 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
- A61K31/13—Amines
- A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
- A61K31/136—Amines having aromatic rings, e.g. ketamine, nortriptyline having the amino group directly attached to the aromatic ring, e.g. benzeneamine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
- A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
- A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
- A61K31/444—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring heteroatom, e.g. amrinone
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/445—Non condensed piperidines, e.g. piperocaine
- A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
- A61K31/453—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a six-membered ring with oxygen as a ring hetero atom
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
-
- 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
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
- A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
- A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
- A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/24—Heavy metals; Compounds thereof
- A61K33/243—Platinum; Compounds thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/24—Heavy metals; Compounds thereof
- A61K33/34—Copper; Compounds thereof
-
- 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
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
Definitions
- This invention generally relates to cell biology and cancer treatment.
- compositions and uses thereof to induce immunogenic cell death for the treatment or prevention of cancer or other disease conditions are disclosed. Further disclosed are methods for identifying compositions with enhanced ability to induce immunogenic cell death. Additional embodiments are directed to treatment methods to induce immunogenic cell death.
- Immunogenic Cell Death is a phenomenon whereby cancer cells die in a manner that activates the immune system. Activation of the immune system can in turn elicit an immune response against the cancer. Cells infected as part of an infectious disease condition can also initiate an adaptive immune response. Without being limited by theory, the phenomenon of ICD is distinct from regular apoptosis in which cell death does not generally lead to the activation of the immune system, although mechanisms involved in apoptosis may contribute to induction of ICD- related molecular markers.
- DAMPs damage-associated molecular patterns
- ICD is often characterized by three distinct DAMPs, which are evidenced by the presence of one or more of the following molecular determinants (referred to in the literature as "markers") of ICD: (1 ) the pre-apoptotic expression of endoplasmic reticulin (ER) calreticulin (CRT). (2) the secretion of adenosine triphosphate (ATP), and (3) the secretion of nuclear high mobility group box 1 (HMGBi). It has been reported that the presence of these DAMPs during tumour cell death leads to the release of pro- inflammatory cytokines, activation of innate immune cells such as dendritic cells (DCs) and macrophages, and ultimately stimulation of the adaptive immune response.
- DCs dendritic cells
- HMGBi nuclear high mobility group box 1
- ICD inducers can be categorized as Type I or Type II inducers. It has been reported that Type I inducers induce ICD as an off-target effect, while Type II inducers typically cause primarily ER stress, which is directly related to ICD induction.
- compositions for inducing ICD for the treatment of cancer and other disease conditions seeks to address that need or to provide useful alternatives to known approaches.
- immunostimulatory pharmaceutical composition to treat, ameliorate and/or prevent a cancer or other condition by inducing Immunogenic Cell Death (ICD) comprising: copper and at least one agent, which is optionally an anti-cancer agent.
- the agent is optionally capable of complexing with copper to form a metal complex.
- the combination of the copper and the agent provide an increase in ICD, compared to the agent alone.
- the increase in the ICD response of the combination as measured by at least one of extracellular adenosine triphosphate (ATP), HMGBi release, and CRT exposure of cells in vitro may be 1 .2 to 10,000 times that of the agent alone measured under otherwise identical conditions.
- the copper, the agent, or both in certain embodiments are formulated in a liposome or other delivery vehicle known to those of skill in the art.
- the combination of the copper and the agent provide an increase in ICD, compared to the agent alone, as determined by an assay that measures at least one of ATP secretion, HMGBi release, and CRT exposure of cells.
- the copper, the agent, or both, in certain embodiments, are formulated in a liposome or other delivery vehicles known to those of skill in the art.
- a method of inducing ICD comprising administering copper and at least one agent, such as an anti cancer agent, in combination or sequentially, to a patient in need thereof.
- the combination of the copper and the agent provide an increase in ICD, compared to the agent alone, as determined by measuring at least one of adenosine triphosphate (ATP) secretion, HMGB release, and CRT exposure.
- ATP adenosine triphosphate
- the copper, the agent, or both, may be formulated in a liposome or other delivery vehicle.
- the copper and the agent are capable of inducing the ICD response with a further, second agent.
- kits comprising copper and an agent, such as an anti-cancer agent, to induce ICD, wherein a combination of the copper and the agent provide an increase in ICD, compared to the agent alone, as determined by measuring ATP secretion, HMGB
- the kit may comprise instructions to induce immunogenic cell death, e.g., dosaging or administration instructions, to a patient in need thereof.
- the kit may comprise instructions for loading the copper and/or the agent into the delivery vehicle.
- the copper and the at least one agent are capable of inducing immunogenic cell death with radiation.
- the increase in the ICD response in the presence of copper is measured by ATP secretion.
- the copper is incorporated in the same or a different liposome or other delivery vehicle as the agent.
- a further, second agent is utilized.
- a cytotoxic effect is realized, as measured by a cytotoxic assay.
- the liposome is a pre-formed liposome. That is, the liposome is prepared in solution and subsequently the agent, such as an anti-cancer agent, is loaded into the liposome by complexation with the metal.
- the agent is a therapeutic agent that can treat or prevent a disease, infection or condition.
- the agent may or may not complex with a metal.
- the agent may be an anti-cancer agent, an anti-viral agent or other therapeutic agent to treat or prevent disease, for example by inducing ICD in the presence of a metal.
- a nanoparticle or other suitable delivery vehicle is utilized instead of a liposome.
- Such alternate drug delivery vehicles are known to those of ordinary skill in the art, and may include, without limitation, a vesicle such as an exosome, a micelle, a lipid-based nanoparticle, a polymer-based nanoparticle, an emulsion or a nanotube.
- a vesicle such as an exosome, a micelle, a lipid-based nanoparticle, a polymer-based nanoparticle, an emulsion or a nanotube.
- FIGURE 1 A shows ATP release of CT26 murine colon cancer cells (referred to herein as“CT26 cells " ) treated with copper, zinc, approved chemotherapeutics, CDK inhibitors and combinations of the metal and anti-cancer agents.
- the concentration of the metal and anti- cancer agent is as indicated in the figure.
- the ATP release is indicated as a fold- increase in ATP release relative to an untreated control.
- FIGURE 1 B shows ATP release of CT26 cells treated with copper, zinc, terpyridine derivatives or Cu-dependent cytotoxics and combinations of the metal and anti-cancer agents.
- the terpyridine derivatives include 2,2';6',2"-terpyridine (TER), 4 ‘ -(4-chlorophenyl)-2, 2 " :6, 2"-terpyridine (CPT), 4,4 ⁇ 4"-tri-tert-butyl-2,2':6 , ,2"- terpyridine (TTBT).
- the Cu-dependent cytotoxics include 8-hydroxyquinoline (8HQ), clioquinol (CQ), diethyldithiocarbamate (DDC) and pyrithione (PYR).
- the concentration of the metal and anti-cancer agent is as indicated in the figure.
- the ATP release is indicated as a fold-increase in ATP release relative to an untreated control.
- FIGURE 1 C shows ATP release of CT26 cells treated with copper, zinc and other chemical compounds, as well as combinations of the metals and chemical compounds.
- the chemical compounds tested include emodin (EMD),
- epigallocathecin gallate EPGG
- P14Y physcion
- PX478 The concentration of the metal and anti-cancer agent is as indicated in the figure.
- the ATP release is indicated as a fold-increase in ATP release relative to an untreated control.
- FIGURE 2 shows the ATP and HMGBi release of CT26 cells treated with copper, zinc and an anti-cancer agent as well as combinations of the metals and the anti-cancer agent.
- the anti-cancer agents tested include 8-Hydroxyquinoline (8HQ), clioquinol (CQ), diethyldithiocarbamate (DDC) and 4"-Tri-tert-Butyl-2,2’:6 ⁇ 2"- terpyridine (TTBT).
- the concentration of the metal and anti-cancer agent is as indicated in the figure.
- the ATP release is indicated as a fold-increase in ATP release relative to an untreated control and HMGBi release is reported as ng/mL.
- FIGURE 3 shows ATP and HMGBi release, as well as CRT exposure, of CT26 cells treated with copper and 4,4',4 " -Tri-tert-Butyl-2,2':6',2"-terpyridine (TTBT) alone or in combination.
- the concentration of the metal and anti-cancer agent is as indicated in the figure.
- the ATP release is indicated as a concentration in nM.
- HMGBi release is reported as ng/mL and CRT exposure is measured as a fold- increase relative to an untreated control.
- FIGURE 4A shows the HMGBi release from CT26 cells treated with various agents, with and without copper, which can complex with the metal.
- the agents tested include daunorubicin (DNR), PX478 and salubrinal (SAL)).
- HMGBi release was measured in ng/mL.
- the concentration of copper and the anti-cancer agents is shown in the figure.
- FIGURE 4B shows the HMGB i release from CT26 cells treated with various agents, with and without copper, which cannot complex with the metal.
- the agents tested include cisplatin (CDDP), gemcitabine (GEM) and cytarabine (ARA-C).
- HMGBi release was measured in ng/mL.
- concentration of copper and the anti cancer agents is shown in the figure.
- FIGURE 5 A shows the CRT exposure from CT26 cells of various agents, with and without copper, which can complex with the metal.
- the agents tested include daunorubicin (DNR), PX478 and salubrinal (SAL). CRT exposure was measured as a fold-increase relative to an untreated control. The concentration of copper and the anti-cancer agents is shown in the figure.
- FIGURE 5B shows the CRT exposure from CT26 cells of various agents with and without copper that cannot complex with the metal.
- the agents tested include cisplatin (CDDP), gemcitabine (GEM) and cytarabine (ARA-C). CRT exposure was measured as a fold-increase relative to an untreated control. The concentration of copper and the anti-cancer agents is shown in the figure.
- FIGURE 6A shows the ATP release from CT26 cells after treatment with daunorubicin (DNR) and the metals copper, zinc or iron, alone or in combination with the anti-cancer agent.
- DNR daunorubicin
- the concentration of the anti-cancer agent and the metal is as indicated in the graph. Results are shown as a fold-increase in ATP release relative to an untreated control.
- FIGURE 6B shows the ATP release from CT26 cells after treatment with daunorubicin (DNR) and the metals manganese, cobalt and vanadium, alone or in combination with the anti-cancer agent.
- DNR daunorubicin
- the concentration of the anti-cancer agent and the metal is as indicated in the graph. Results are shown as a fold-increase in ATP release relative to an untreated control.
- FIGURE 6C shows the ATP release from CT26 cells after treatment with gemcitabine (GEM) and the metals copper, zinc or iron, alone or in combination with the anti-cancer agent.
- the concentration of gemcitabine and the metal is as indicated in the graph. Results are shown as a fold-increase in ATP release relative to an untreated control.
- FIGURE 6D shows the ATP release from CT26 cells after treatment with gemcitabine (GEM) and metals, including manganese, cobalt and vanadium, alone or in combination with the anti-cancer agent.
- the concentration of the anti-cancer agent and the metal is as indicated in the graph. Results are shown as a fold-increase in ATP release relative to an untreated control.
- FIGURE 6E shows the ATP release from CT26 cells after treatment with 4’- (4-chlorophenyl)-2,2’:6',2 '" -terpyridine (CPT) and the metals copper, zinc or iron, alone or in combination with the anti-cancer agent.
- CPT 4’- (4-chlorophenyl)-2,2’:6',2 '" -terpyridine
- the concentration of the anti cancer agent and the metal is as indicated in the graph. Results are shown as a fold- increase in ATP release relative to an untreated control.
- FIGURE 6F shows the ATP release from CT26 cells after treatment with CPT and the metals manganese, cobalt and vanadium, alone or in combination with the anti-cancer agent.
- concentration of the anti-cancer agent and the metal is as indicated in the graph. Results are shown as a fold-increase in ATP release relative to an untreated control.
- FIGURE 6G shows the ATP release from CT26 cells after treatment with 4,4',4”-tri-tert-butyl-2,2’:6 ' ,2 ' '-terpyridine (TTBT) and the metals copper, zinc or iron, alone or in combination with the anti-cancer agent.
- the concentration of the anti-cancer agent and the metal is as indicated in the graph. Results are shown as a fold-increase in ATP release relative to an untreated control.
- FIGURE 6H shows the ATP release from CT26 cells after treatment with 4,4’,4”-tri-tert-butyl-2.2 “ :6‘,2”-terpyridine (TTBT) and the metals manganese, cobalt and vanadium, alone or in combination with the anti-cancer agent.
- the concentration of the anti-cancer agent and the metal is as indicated in the graph. Results are shown as a fold-increase in ATP release relative to an untreated control.
- FIGURE 61 shows the ATP release from CT26 cells after treatment with abemaciclib (ABE) and the metals copper, zinc or iron, alone or in combination with the anti-cancer agent.
- the concentration of the anti-cancer agent and the metal is as indicated in the graph. Results are shown as a fold-increase in ATP release relative to an untreated control.
- FIGURE 6J shows the ATP release from CT26 cells after treatment with with abemaciclib (ABE) and the metals manganese, cobalt and vanadium, alone or in combination with the anti-cancer agent.
- the concentration of the anti-cancer agent and the metal is as indicated in the graph. Results are shown as a fold-increase in ATP release relative to an untreated control.
- FIGURE 7A shows the ATP release from CT26 cells after treatment with various copper salts (copper acetate, copper chloride, copper gluconate and copper sulfate), as well as cobalt, iron, manganese, nickel and zinc.
- concentration of the anti-cancer agent and the metal is as indicated in the graph. Results are shown as a fold-increase in ATP release relative to an untreated control.
- FIGURE 7B shows the ATP release from CT26 cells after treatment with 4,4'.4"-tri-tert-butyl-2,2’:6 ⁇ 2”-terpyridine (TTBT) alone and in combination with various copper salts (copper acetate, copper chloride, copper gluconate and copper sulfate), as well as cobalt, iron, manganese, nickel and zinc.
- TTBT 4,4'.4"-tri-tert-butyl-2,2’:6 ⁇ 2”-terpyridine
- FIGURE 7C shows the ATP release from CT26 cells after treatment with diethyldithiocarbamate (DDC) alone and in combination with various copper salts (copper acetate, copper chloride, copper gluconate and copper sulfate), as well as cobalt, iron, manganese, nickel and zinc.
- DDC diethyldithiocarbamate
- various copper salts copper acetate, copper chloride, copper gluconate and copper sulfate
- cobalt iron, manganese, nickel and zinc.
- results are shown as a fold-increase in ATP release relative to an untreated control.
- FIGURE 8 shows the fold-increase in reactive oxygen species (ROS) from CT26 cells after treatment with different copper salts and various metals.
- the copper salts examined include: copper sulfate, copper acetate, copper chloride and copper gluconate.
- the other metals besides copper tested for ROS were zinc, iron, manganese, cobalt and vanadium. Results are shown as a fold-increase in ROS relative to an untreated control.
- FIGURE 9A shows ROS concentrations as a function of concentration (0.25- 250 mM) of copper or zinc addition to CT26 cells. Results are shown as a fold- increase in ROS relative to an untreated control.
- FIGURE 9B shows the results of ROS studies with CT26 cells using copper or zinc in combination with daunorubicin (DNR), PX478 and salubrinal (SAL). Results are shown as a fold-increase in ROS relative to an untreated control. The metals were added at equimolar concentrations.
- FIGURE 9C shows the results of ROS studies with CT26 cells using copper or zinc in combination with cisplatin (CDDP), gemcitabine (GEM) and cytarabine (ARA-C). Results are shown as a fold-increase in ROS relative to an untreated control. The metals were added at equimolar concentrations.
- FIGURE 10A shows in vivo vaccination results with immune competent Balb/c mice that were inoculated with CT26 cells which were pre-treated with mitoxantrone (Mito), Mito and copper, cisplatin (CDDP), and CDDP and copper (Day -7).
- the results are reported as the number of tumour-free mice as a function of days post-challenge by inoculating the flank opposite to the “vaccination site " ’ with untreated CT26 cells (Day 0).
- the untreated control did not receive any pre-treatment or inoculation, serving as an“unvaccinated” control.
- FIGURE 10B shows the percentage of tumour free Balb/c mice on Day 39 post-challenge.
- the mice were inoculated on Day -7 with CT26 cells which were previously treated with mitoxantrone (Mito), Mito and copper, cisplatin (CDDP), and CDDP and copper.
- Day 0 refers to the day when the mice were challenged with untreated CT26 cells a week after“vaccination” with pre-treated cells. The untreated control did not receive any pre-treatment or inoculation, serving as an“unvaccinated” control.
- FIGURE 1 1 A shows fold-increase in extracellular ATP measured against untreated cells for the following treatments of CT26 cells: PX-478 alone, gemcitabine (Gem) alone, PX-478 and Gem, Cu(PX-478) alone, and Gem and Cu(PX-478) in combination.
- FIGURE 1 I B shows fold-increase in extracellular ATP measured against untreated cells for the following treatments of CT26 cells: PX-478 alone, cisplatin (CDDP) alone, PX-478 and CDDP, Cu(PX-478) alone, and CDDP and Cu(PX-478) in combination.
- FIGURE 1 1 C shows fold-increase in extracellular ATP measured against untreated cells for the following treatments of CT26 cells: 4,4’,4”-tri-ter-butyl- 2,2 :6 ‘ ,2” - terpyridine (TTT) alone, cisplatin (CDDP) alone, TTT and CDDP, Cu(TTT) 2 alone, and cisplatin and Cu(TTT) 2 in combination.
- FIGURE 1 I D shows fold-increase in extracellular ATP measured against untreated cells for the following treatments of CT26 cells: flavopiridol (FLV) alone, mitoxantrone (Mito) alone, FLV and Mi to, Cu(FLV) alone, and Mito and Cu(FLV) in combination.
- FIGURE 1 1 E shows fold-increase in extracellular ATP measured against untreated cells for the following treatments of CT26 cells: clioquinol (CQ) alone, gemcitabine (Gem) alone, CQ and Gem in combination, Cu(CQ)2 alone, and Gem and CU(CQ) 2 in combination.
- FIGURE 1 I F shows fold-increase in extracellular ATP measured against untreated cells for the following treatments of CT26 cells: 4 (4-chlorophenyl) - 2,2':6 ' ,2”-terpyridine (4CPT) alone, cisplatin (CDDP) alone, 4CPT and CDDP, CU(4CPT) 2 alone, and CDDP and Cu(4CPT) 2 in combination.
- FIGURE 12A shows secretion of HMGBi (ng/mL) from CT26 cells for the following: untreated, PX-478 alone, gemcitabine (Gem) alone, PX-478 and Gem, Cu(PX-478) alone, and Gem and Cu(PX-478) in combination.
- FIGURE 12B shows secretion of HMGBi (ng/mL) from CT26 cells for the following: untreated, flavopiridol (FLV) alone, mitoxantrone (Mito) alone, FLV and Mito, Cu(FLV) alone, and Mito and Cu(FLV) in combination.
- FIGURE 13A shows ATP release (nM) of CT26 cells for the following:
- FIGURE 13B shows HMGBi release (ng/mL) of CT26 cells for the following: untreated, copper, PX478, gemcitabine (GEM), PX478 and GEM, and copper, PX478 and GEM in combination.
- GEM gemcitabine
- FIGURE 13C shows CRT exposure (fold-increase) of CT26 cell for the following: untreated, copper, PX478, gemcitabine (GEM), PX478 and GEM, and copper, PX478 and GEM in combination.
- GEM gemcitabine
- compositions and methods described in the present disclosure enhance immunogenic cell death phenotypes, which is also referred to herein as“JCD".
- JCD immunogenic cell death phenotypes
- the inventors have examined the ability of various agents to induce ICD by utilizing in vitro assays to measure molecular determinants indicative of ICD and have found that copper in combination with an agent can exhibit an increased ICD response relative to the agent alone.
- the increase in ICD response is attributable to the copper.
- the copper potentiates the ICD response of the agent.
- the agent is most advantageously loaded into delivery vehicles. This may include loading of the agent into pre-formed liposomes comprising copper, using, for example, methods described herein.
- other delivery vehicles can be utilized as well for delivery of the metal and/or the agents.
- DAMPs damage-associated molecular patterns
- ATP is one such DAMP that is secreted during ICD and, without being limiting, is believed to attract monocytes to the site of tumour cell death, which differentiate to dendritic cells (DC) expressing inflammatory DC markers (e.g., CD1 l c+ CDl l b+ Ly6C hl ).
- DC dendritic cells
- inflammatory DC markers e.g., CD1 l c+ CDl l b+ Ly6C hl
- ATP secretion can be measured in vitro by exposing cells in culture to the agents being tested for a pre-determ ined period of time. ATP is subsequently measured by a reaction in which the ATP secreted is measured by luminescence.
- ATP secretion is measured in vitro as follows. Prior to measuring ATP secretion, cells of interest, such as CT26 murine colon cancer cells, are seeded at an appropriate density in multi-well plates, such as 24-well plates. At 24 hours, cells are treated with the agents and the metal ion. Optionally, the metal ion and agent are incubated under conditions that are optimal for metal complexation formation prior to their addition to the cells. However, as noted, in certain embodiments, 1CD induction is not dependent on formation of a complex between the metal and the agent.
- the cells are exposed to the tested agents (copper and/or agent) for 24 hours (or left untreated), after which the supernatant of each well is transferred to a new plate.
- the Promega CellTiter-GloTM 2.0 Kit is then used, as per the manufacturer ' s instructions, to determine the extracellular ATP level under each treatment condition.
- the assay measures ATP by luminescence. Luciferin is converted to oxyluciferin in the presence of ATP, Mg 2+ and 0 2 . Oxyluciferin in turn emits light that is quantified by a luminometer and is correlated with the amount of ATP present by a standard curve.
- a further DAMP is the high-mobility group box 1 , also referred to herein as "HMGBi”.
- HMGBi high-mobility group box 1
- HMGBi has been reported to be a late apoptotic marker and its release to the extracellular space facilitate dendritic cell maturation and promote tumour antigen presentation to induce CDS T cells. This molecular determinant of 1CD can be measured by the assay described below.
- cells of interest such as CT26 murine colon cancer cells
- cells of interest are seeded at an appropriate density in 24-well plates.
- cells are treated with the agents of interest.
- the metal ion may be added to the agent at a predefined molar ratio.
- the metal complexes are formed at a pre-determined metal-to- ligand ratio prior to addition to the cells for optimal metal complexation.
- Cells are exposed to the copper and/or tested agents for 24 hours, after which the supernatant of each well is collected and the amount of HMGBi release is assessed using a commercial ELISA kit purchased from IBL InternationalTM.
- the test quantifies HMGBi by an anti-HMGB antibody that is immobilized.
- HMGBi binds to the antibody and a second enzyme marked antibody recognizes the immobilized antibody.
- Substrate reaction catalyzed by the enzyme leads to a change in colour intensity, which is quantified by known methodology.
- Calreticulin is another DAMP that is exposed on the surface of tumour cells during cell death. Without being limited by theory, Ecto-CRT is reported to act as a signal that activates phagocytosis by certain antigen presenting cells, contributing to the induction of an immune response. CRT can be measured by flow cytometric analysis of CRT cell surface expression. According to this assay, after 24 hours of treatment of the cells, such as CT26 cells, with copper and/or an agent, the copper and/or agent is removed and replaced with culture media. Cells are harvested at 0, 24, 48, and 96 hours post-treatment and stained with anti-CRT antibody followed by Alexa-488-conjugated secondary antibody.
- Propidium iodide is used as a counterstain to differentiate between viable and dead cells.
- the relative CRT mean fluorescence intensity (MF1) of Pi-negative viable cells is determined by subtracting the MFI of isotype control-stained cells from the MFI of anti-CRT stained cells, and then normalising to HBSS-treated cells.
- An immunofluorescence assay may also be used to visualize cell surface expression of CRT on cells treated with an agent for 4 hours and stained with the anti-CRT antibody (green), as described above.
- the cell membrane may be labelled with wheat germ agglutinin (red) and the nucleus with Hoechst 33342 (blue). Images may be taken using an IN Cell Analyzer 2200TM and the fluorescent signal (green) can be quantitated and analyzed at a per-well and per-cell level to analyze relative cell surface CRT expression.
- ICD induction by copper is evidenced by each of the three assays or any combination of two of any of the three assays or one of any of the three assays.
- the presence of copper increases the levels of ATP secretion over that of the ICD inducer alone.
- the presence of copper enhances the level of FIMGB released with or without an agent that is an ICD inducer.
- ICD induction by copper is evidenced by CRT expression.
- the ICD response of the copper and the agent in combination as measured by extracellular ATP, HMBG 1 and/or CRT exposure is 1.2 to 10,000, 1.5 to 10,000, 2 to 10,000, 3 to 10,000, 4 to 10,000, or 5 to 10,000 times that of the agent alone measured under otherwise identical conditions.
- the ICD response of the copper and the agent in combination as measured by extracellular ATP, HMBG 1 and/or CRT exposure is at least 1.2, 1.3, 1.4, 1.5, 1 .6, 1.7, 1.8, 1.9, 2, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 or more times that of the agent alone measured under otherwise identical conditions.
- the upper limit of the ICD response of the copper and the agent in combination as measured by extracellular ATP, HMBG 1 and/or CRT exposure may be 800, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000 or 10,000 times relative to agent alone. Any combination of the foregoing lower and upper limits is included in select embodiments of the disclosure.
- the ICD of an agent in combination with copper is determined by measuring a phenotype of ICD (ATP secretion, CRT release and/or HMGBi), also referred to herein as a“marker” of ICD.
- a phenotype of ICD ATP secretion, CRT release and/or HMGBi
- the cytotoxicity of a particular pharmaceutical formulation and/or treatment regime can be measured using a cytotoxicity assay.
- an additive or synergistic cytotoxic effect between copper and an agent may be exhibited (non- antagonistic). Such determination may be made by the Chou Talalay method, which is known to those of ordinary skill in the art.
- the cytotoxicity assay can be used to determine whether a cytotoxic effect of the agent of interest in combination with copper is present.
- the composition or treatment w ill be considered to have a cytotoxic effect if any combination selected from at least two of (i) copper, (ii) an agent, (iii) a second agent if two or more are used, and (iii) radiation treatment exhibits a cytotoxic effect in vitro.
- a pharmaceutical composition comprises copper as well as a first and a second agent in combination, and if the second agent exhibits a cytotoxic effect in combination with copper, but the first agent in combination with copper does not, nor the first agent in combination with the second agent, the pharmaceutical composition will still be considered to have a cytotoxic effect as used herein.
- a pharmaceutical composition comprises copper and a first and a second agent in combination, and if the first agent exhibits a cytotoxic effect in combination with the second agent, but the first or second agent in combination with copper does not, the pharmaceutical composition will still be considered to have a cytotoxic effect as used herein.
- a treatment comprises an agent in combination with the metal and additionally radiation treatment, and if the agent and the radiation treatment exhibit a cytotoxic effect in combination, then the treatment will be considered cytotoxic, even if the metal and agent demonstrate no cytotoxic effect in combination. Additional examples will be readily envisioned by those of ordinary skill in the art.
- the cells are seeded in plates and treated with the agent of interest with or without copper for 24 hours, after which the cells are irradiated at different doses (2, 4, 6, 8, 10 Gy).
- the irradiated cells are immediately plated in culture dishes to achieve 100-500 colonies two weeks later.
- the colonies are stained with aqueous malachite green or crystal violet and dried overnight prior to colony counting.
- the dose response curves from each treatment are then compared to assess treatment efficacy in vitro.
- the metal ion of select embodiments is copper.
- the metal ion may also be an ion of a transition metal or a Group lllb metal.
- the transition metal may be from Group IB, 2B, 3B, 4B, 5B, 6B, 7B and 8B (groups 3- 12). Examples of transition metals include copper, zinc, vanadium, manganese, iron, cobalt and nickel.
- the metal has d-orbitals.
- the Group lllb metal is from the boron family, which includes boron, aluminum, gallium, and indium. In one embodiment, the metal is in the 2 + oxidation state.
- agent any compound or molecular species to treat, ameliorate, and/or prevent a disease or condition; a compound or molecular species that serves as a carrier for transporting the metal ion to facilitate loading into a given delivery vehicle and/or to facilitate delivery of the metal after administration; or a compound or molecular species that serves as an adjuvant.
- the disease or condition includes any proliferate disorder or disease and includes, but is not limited to, cancer, infectious diseases or any other condition in which 1CD is desirable.
- the agent of select embodiments used in combination with copper typically comprises at least one complexation moiety or ligand to enable complexation with the copper.
- the agent for use in the pharmaceutical compositions described herein need not complex with the metal.
- the agent may have a chemical group, often referred to as a ligand, which has atoms that allow for the coordination of the agent with the metal.
- the chemical group may be selected from an S-donor, O-donor, N, O-donor, a Schiff base, hydrazones, P- donor phosphine, N-donor or a combination thereof.
- the moiety is a hard electron donor.
- Other moieties or ligands known to those of skill in the art suitable for complexation with a metal ion are included within the scope of certain embodiments as well.
- the complexation via metal coordination facilitates drug loading as described in co-owned WO 2017/100925 (Bally et al.). This could be measured by incubating an agent of interest with a liposome containing copper, or other metal ion of interest, under optimal conditions to facilitate drug uptake and measuring drug uptake after one hour.
- the drug uptake after one hour is at least 30%, 40%, 50%, 60%, 70% or 80% as determined by measuring the drugilipid ratio relative to the theoretical drugdipid ratio that could ideally be obtained.
- the drug uptake after one hour is between 60% and 100% as measured above.
- the agent tnay be selected from antineoplastic agents, for example cytotoxic agents.
- Such agents include, but are not limited to, nucleoside analogues, anthracyclines, anti-folates, topoisomerase I inhibitors, taxanes, vinca alkaloids, alkylating agents, platinum compounds, targeted antineoplastics, including monoclonal antibodies, tyrosine kinase inhibitors, mTOR inhibitors, CDK inhibitors, retinoids, immunomodulatory agents and histone deacetylase inhibitors, terpyridine derivatives and Cu-dependent cytotoxics.
- an agent suitable for use in certain embodiments is selected from PX-478, Emodin, clioquinol (CQ), pyrithione (PYR), flavopiridol (FLV), diethyldithiocarbamate (DDC), epigallocathecin gallate (EPGG), cisplatin (CDDP), gemcitabine (GEM), mitoxantrone (MITO), 4' (4-chlorophenyl) - 2.2 ‘ :6 ' ,2 " -terpyridine (4CPT), 4, 4 ⁇ 4”-tri-ter-butyl-2,2':6 ⁇ 2" - terpyridine (TTT), shikonin, wogonin, cytarabine (ARA-C), oxaliplatin (OXP), salubrinal (SAL), abemaciclib (ABE), palbociclib (PAL) and ribociclib (RIB), 2,2';6’,2"-terpyridine (
- the agent may function as a carrier moiety, such as an ionophore.
- the ionophore may facilitate loading of the metal into a delivery vehicle.
- the agent may function as an ionophore to facilitate the delivery of a metal complexed therewith across a lipid bilayer of a delivery vehicle, such as a liposome.
- the agent and copper may be added to the external solution of a liposome preparation.
- the agent may form a complex with the metal in the solution external to the liposome and the resultant copper-ionophore complex may
- the carrier agent may facilitate the transport of the bound copper in the complex to a disease site.
- a carrier agent may facilitate the transport of the metal across a cellular membrane.
- the agent additionally exerts a therapeutic effect rather than simply functioning as a carrier agent.
- Non-limiting examples of agents that complex with copper and that may function as a carrier for the metal include diacetyl-bis(N4-methylthiosemicarbazone) (ATSM), clioquinol (CQ) and diethyldithiocarbamate (DDC).
- ATSM diacetyl-bis(N4-methylthiosemicarbazone)
- CQ clioquinol
- DDC diethyldithiocarbamate
- Cu-ATSM is permeable across cellular membranes and can be reduced in hypoxic tissue. A reduction in hypoxic conditions (such as at a tumour site) may allow the copper atom to dissociate from the ATSM. In some embodiments, this may allow copper to be continuously deposited in hypoxic tissues and cells.
- the agent for use in select embodiments may be poorly soluble in solution prior to or after complexation with the metal ion.
- the poorly soluble agent in free form has a solubility of less than 1 mg/iriL in either water or a solution of the metal ion which complexes with the agent.
- Solubility of the agent in water or in the presence of the metal ion (10 mM to 500 mM) is measured at conditions of physiological pH and temperature after 60 minutes of incubation under these conditions. Measurement of solubility of the agent is conducted as described in co-owned WO 2017/100925.
- the agent has a solubility that is greater than 1 mg/mL in either water or a metal ion solution, as determined by the assay described above (see WO 2017/100925).
- the agent and metal may be formulated in any known delivery vehicle.
- delivery vehicle means a particle of a size between 10 nm and 2000 nm that contains associated therewith the agent, copper or a combination thereof.
- the delivery vehicle typically controls the rate of release of its cargo.
- the agent and copper are both administered in free form or one of the agent and the metal are formulated in the delivery vehicle, while the other is in free form (such as formulated with a pharmaceutically acceptable carrier, such as a salt).
- a pharmaceutically acceptable carrier such as a salt
- Whether or not the agent and/or the metal are formulated in the delivery vehicle may depend on the mode of administration. For example, if the agent and copper are introduced directly to a disease site, such as by direct injection, then formulation in a delivery vehicle may not be necessary.
- suitable delivery vehicles include vesicles such as liposomes and exosomes, micelles, polymer-based nanoparticles, emulsions, or nanotubes.
- the agent may also be conjugated with a lipid or polymer to improve its ability to be formulated in a given drug delivery vehicle.
- the agent may be formulated in a liposome.
- the liposome is a vesicle comprising a bilayer having amphipathic lipids enclosing an internal solution.
- the liposome may be a large unilamellar vesicle (LUV), which can be prepared as described below using extrusion.
- the average diameter of the liposome may be between 60 nm and 2,000 nm, 70 and 1 ,000 nm, 70 and 500 nm, 70 and 200 nm or 70 and 180 nm.
- the liposome may comprise lipids including phosphoglycerides and
- sphingolipids representative examples of which include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, pahnitoy!oleoyl phosphatidylcholine, lysophosphatidylcholine, lysophos- phatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine or dilinoleoylphosphatidylcholine.
- the phospholipids may comprise two acyl chains from 6 to 24 carbon atoms selected independently of one another and with varying degrees of unsaturation. Additionally, the amphipathic lipids described above may be mixed with other lipids including triacylglycerols and sterols. As would be appreciated by those of skill in the art, lipids that interfere with liposome formation in the presence of a metal should typically be avoided. Whether or not a given lipid is suitable for liposome formation in the presence of a metal ion can be determined by those of skill in the art.
- the liposome comprises the lipids 1 ,2- distearoyl-sn-glycero-3- phosophocholine (DSPC) and cholesterol.
- DSPC distearoyl-sn-glycero-3- phosophocholine
- the liposomes or other drug delivery vehicles may also comprise a hydrophilic polymer-lipid conjugate.
- the hydrophilic polymer may be a
- the hydrophilic polymer-lipid conjugate is generally prepared from a lipid that has a functional group at the polar head moiety that is chemically conjugated to the hydrophilic polymer.
- An example of such a lipid is phosphatidylethanolamine.
- the inclusion of such hydrophilic polymer-lipid conjugates in a liposome can increase its circulation longevity in the bloodstream after administration.
- the hydrophilic polymer is biocompatible and has a solubility in water that permits the polymer to extend away from the outer surface of the liposome.
- the polymer is generally flexible and may provide uniform surface coverage of the liposome outer surface.
- the liposome may include a hydrophilic polymer, such as polyethylene glycol (PEG) at between 1 and 20 mol% or between 2 and 10 mol%.
- PEG polyethylene glycol
- a non-limiting example of a liposomal formulation comprising PEG is DSPC/CHOL/PEG (50:45:5, mole ratio) or DSPC /PEG (95:5, mole ratio).
- the specific ratios of the lipids may vary according to embodiments that would be apparent to those of ordinary skill in the art.
- Liposomes can be prepared by any of a variety of suitable techniques known to those of skill in the art.
- An example of one suitable method involves cycles of freeze-thaw and subsequent extrusion of lipid preparations.
- lipids selected for inclusion in a liposome may be desiccated and dissolved in a solvent, such as an organic solvent, at a desired ratio. After removal of the solvent, the resultant lipids are hydrated in an aqueous solution. The solution in which the lipids are hydrated forms the internal solution of the liposomes. Subsequently the hydrated lipids may be subjected to cycles of freezing and thawing. The hydrated lipids are passed through an extrusion apparatus to obtain liposomes of a defined size. The size of the resulting liposomes may be determined using quasi-electric light scattering (e.g., using a NanoBrook ZetaPALSTM Potential Analyzer).
- the liposomes may be prepared so that they comprise an internal solution comprising the metal ion.
- the lipids when preparing liposomes by freeze-thaw and subsequent extrusion as described above, the lipids are hydrated in a solution comprising a metal ion.
- the liposomes so formed will comprise the metal ion not only in the internal solution of the liposomes, but also in the external solution.
- Unencapsulated metal ion may be removed from the external solution of the liposome prior to loading of the agent.
- the external copper solution may be exchanged with a solution containing substantially no copper ions by passage through a column equilibrated with a buffer.
- the solution that exchanges with the metal-containing solution is a buffer, although other solutions may be used as desired.
- the liposomes may be subsequently concentrated to a desired lipid concentration by any suitable concentration method, such as by using tangential flow dialysis.
- the solution external to the liposome contains substantially no metal ions that complex with the poorly soluble agent. By this it is meant that the concentration of metal ions in the external solution is less than that of the metal ion concentration in the liposome, for example less than one fifth of the concentration of metal ion in the liposome.
- the external solution may comprise a chelating agent that chelates with the metal ions.
- the metal ion may be encapsulated in the liposome as a metal salt. Examples include copper sulfate, copper chloride or copper gluconate.
- the pre-formed liposomes comprising the metal ion may be incubated with the agent to facilitate uptake via metal complexation.
- the agent may be added in any suitable form, including as a powder or as a solution. If the agent is insoluble in water, it can be added as a powder. The amount of free agent in solution can subsequently be increased by increasing the temperature.
- Incubation of the pre formed liposomes with the one or more agents is performed under conditions sufficient to allow the agent to move across the phospholipid bi layer of the liposome into the internal solution thereof. Such a method is referred to by those of skill in the art as "loading". Movement of the agent across the phospholipid bilayer of the pre formed liposome during loading may occur independently of any pH gradient across the bilayer.
- the loading may, however, be dependent on other factors.
- the loading conditions can be readily selected by those of skill in the art to achieve a desired rate of loading.
- the diffusion of the agent across the bi layer may be dependent on the temperature and/or lipid composition of the liposome.
- the formation of the drug-metal complex incorporated in the pre-formed liposomes may be characterized as an inorganic synthesis reaction.
- the uptake of drug during the loading reaction is visualized as a colour change as many metal complexed agents have different spectral characteristics that can be detected by eye. For example, a colour change to purple, brown, green or yellow can be observed during loading with copper.
- the drug-to-lipid ratio may be at least 0.3: 1 0.4: 1 , 0.5: 1 , at least 0.6: 1 , at least 0.7: 1 , or at least 0.8: 1.
- the drug-to-lipid ratio is about 0.1 : 1 to about 0.6: 1 (molanol), about 0.15: 1 to about 0.5: 1 (molanol) or about 0.2: 1 to about 0.4: 1 (molanol).
- Such a high drug-to-lipid ratio may be dependent on the number of metal ions inside the liposome and/or the nature of the complex formed. Formation of a transition metal complex with a given agent may be rapid (e.g., Cu(DDC) 2 ), occurring in minutes, or more gradual. The complexation reaction rate may be temperature dependent. The rate of metal-agent complex formation may also be dependent on the rate at which the externally added agent crosses the lipid bilayer of the liposome. As will be appreciated by those of skill in the art, these variables can be adjusted as desired to achieve a desired rate of uptake of the agent or metal-agent complexation if the latter mechanism facilitates loading.
- loading of an agent may be facilitated by a carrier agent that acts as an ionophore, as described previously.
- the liposome formulations may be either passively or actively loaded with the agent.
- active loading is preferred as it may result in higher trapping efficiencies and drug retention properties relative to passive loading.
- a combination of passive and active entrapment methods can be utilized.
- an agent may be passively entrapped in a liposome with a metal ion and a second agent could be loaded by the metal loading method described above.
- the liposome has a trapping efficiency of at least 80% with respect to at least one agent encapsulated in the liposome.
- the trapping efficiency is at least 90%. Such high trapping efficiencies may result from active loading. By contrast, passive loading may result in comparatively lower trapping efficiencies.
- an exosome may serve as a delivery vehicle for one or more of the agents and/or copper.
- An exosome comprises a lipid bilayer enclosing an internal aqueous space and is derived from a living or dead organism, explanted tissues or organs and/or cultured cells.
- An exosome is typically derived from plasma membrane budding from a producer cell as described in U.S. Patent No. 10, 195,290, which is incorporated herein by reference. Exosomes may be utilized to deliver a variety of agents, such as DNA, RNA, protein and lipid.
- exosome may allow them to be transported to a cellular target site in the body and be taken up efficiently by cells.
- exosome as used herein is meant to include a hybridosome®, which is a hybrid of a lipid nanoparticle and exosome, and vexosomes that transport a virus into cells via an exosome.
- the delivery vehicle may include other particles that are not based on vesicle formation, although such delivery vehicles may include a surface stabilizing lipid bilayer or monolayer.
- An example is a micelle that is any particle or assembly that forms a core that is hydrophobic and comprises more hydrophilic regions that surround the core and that are in contact with the surrounding solvent. Micelles are generally used for the delivery of hydrophobic or water insoluble agents that are contained in the hydrophobic core.
- the micelle may be made up of lipids with hydrophilic heads facing outwardly and the hydrophobic tails facing inwardly to form the hydrophobic core of the particle.
- micelles can vary widely and include molecules such as lipoproteins, including high and low density lipoproteins, chylomicrons, fatty acids, lipids, polymers or any suitable combination.
- micelles can be made up of a single molecular species or may include particles with a hydrophobic core composed of one molecular species and a hydrophilic region contacting the surrounding solvent that is composed of a different molecular species.
- a core may be made of fatty acids surrounded by a mono or bilayer of lipids or other amphipathic molecules.
- the delivery vehicle may also be a polymer-based nanoparticle or microparticle.
- the polymer-based nanoparticles or microparticles are typically produced from synthetic polymers, but can include natural materials as well as lipid monolayers and bi layers.
- Non-limiting examples of nanoparticles and microparticles comprise a core of drug surrounded by a polymeric shell or a drug dispersed throughout a polymeric matrix.
- the drug may be dispersed as a solid or liquid in the nanoparticle or microparticle.
- the agent may also be covalently bonded or associated with the particle by an electrostatic interaction.
- the nanoparticle or microparticle may be a polymer/lipid hybrid, such as a polymer core containing an agent surrounded by a lipid monolayer or bilayer.
- the emulsion may be an oil-in-water emulsion that contains one or more water insoluble agents in the oil phase.
- a nanotube is a carbon-based cylindrical tube in which an agent can be loaded.
- the particles may comprise a graphene sheet that forms into the shape of a cylinder.
- a metal and an agent within a liposome or other drug delivery vehicle
- other embodiments encompass formulations for 1CD induction in which copper is administered to a patient separately from the delivery vehicle comprising the agent.
- the copper is typically administered separately or together with the delivery vehicle.
- a copper chelate is administered by injection.
- copper chelates include Cu(ATSM) and Cu(GTSM).
- the copper is formulated as a salt.
- copper gluconate may be administered orally.
- the agent may be administered as a liposomal or other drug delivery formulation before, during or after administration of the free copper.
- the agent may be loaded into the liposome using active loading methods besides metal loading, such as pH gradient loading, or passive loading methods as known to those of ordinary skill in the art.
- active loading methods such as pH gradient loading, or passive loading methods as known to those of ordinary skill in the art.
- the free copper may complex with the agent or can remain uncomplexed.
- a liposome or other drug delivery vehicle comprising copper is administered separately or together with one or more ICD inducing agents that are in free form.
- the copper may complex with the agent in free form, or remain uncomplexed.
- one or more additional agents may be used to induce ICD as described herein.
- the combinations of two or more drugs may exhibit increased ATP secretion, HMBG ] release and/or CRT expression relative to treatment with one agent alone and/or complexed with metal.
- the one or more additional agent may also complex with the metal or may be present in free form.
- antineoplastic agents such as cytotoxic agents, including nucleoside analogues, anthracyclines, anti-folates, topoisomerase 1 inhibitors, taxanes, vinca alkaloids, alkylating agents, platinum compounds, targeted antineoplastics, including monoclonal antibodies, tyrosine kinase inhibitors, mTOR inhibitors, CDK inhibitors, retinoids, immunomodulatory agents, histone deacetylase inhibitors, terpyridine derivatives and Cu-dependent cytotoxics.
- cytotoxic agents including nucleoside analogues, anthracyclines, anti-folates, topoisomerase 1 inhibitors, taxanes, vinca alkaloids, alkylating agents, platinum compounds, targeted antineoplastics, including monoclonal antibodies, tyrosine kinase inhibitors, mTOR inhibitors, CDK inhibitors, retinoids, immunomodulatory agents, histone deacetylase inhibitors,
- embodiments is selected from PX-478, Emodin, clioquinol (CQ), pyrithione (PYR), flavopiridol (FLV), diethyldithiocarbamate (DDC), epigallocathecin gallate (EPGG), cisplatin (CDDP), gemcitabine (GEM), mitoxantrone (MITO), 4 ' (4-chlorophenyl) - 2,2':6 ' ,2"-terpyridine (4CPT), 4,4’,4’ ' -tri-ter-butyl-2,2’:6’,2 ” - terpyridine (TTT), shikonin, wogonin.
- CQ clioquinol
- PYR pyrithione
- FLV flavopiridol
- DDC diethyldithiocarbamate
- EPGG epigallocathecin gallate
- EPGG epigallocathecin gallate
- CDDP epigalloc
- cytarabine ARA-C
- OXP oxaliplatin
- SAL salubrinal
- ABE abemaciclib
- PAL palbociclib
- RIB ribociclib
- 2,2';6',2"-terpyridine TER
- the agent is not daunorubicin or doxorubicin.
- one or more agents complexed with a metal may be used to treat cancer in combination with an additional therapy such as radiation.
- the methods described in select embodiments herein can be used to load multiple agents, either simultaneously or sequentially into a liposome or other delivery vehicle. If each of the agents is incorporated into a liposome, the agents can be loaded by the complexation method described herein. Moreover, the liposomes into which the agent is loaded may themselves be prepared so that the internal solution comprises not only the metal ion but also an additional agent.
- Loading of an agent in this manner is often referred to as passive loading.
- the subsequent loading of a second agent which complexes with the metal in the pre formed liposome (as described above) will result in incorporation of two agents in the liposome, one of which is loaded passively and the other actively via complexation. Since the passively loaded agent need not complex with a metal ion to effect loading, this approach provides greater flexibility in preparing liposome-encapsulated agent combinations for use to treat or prevent a disease of interest.
- the two or more agents may be loaded at a predetermined ratio that exhibits synergistic or additive effects as elucidated by the Chou-Talalay determination, which is a known methodology for measuring such effects.
- a formulation of liposomes may also comprise two or more liposome populations, which incorporate the same or different agents, comprise different lipid formulations, or comprise liposomes of different vesicle sizes.
- one or more agents may be in free form, while one or more agents may be incorporated in a liposome.
- the copper may be in free form and the agent or agents incorporated in a liposome.
- the copper may be incorporated in a liposome and the agent or agents may be in free form in the pharmaceutical composition.
- the combinations of agents selected for the pharmaceutical formulation may achieve greater therapeutic efficacy, safety, prolonged drug release or targeting after administration.
- a second agent in free form is included in a treatment regime such that the drug becomes active in the presence of the metal ion.
- drug combinations include co-encapsulation of metal-CQ and free DSF, the precursor of DDC.
- the DSF is metabolized to form DDC and DDC and is subsequently activated in the presence of a metal ion, such as copper, at the tumour site.
- liposomal formulations incorporating copper and two or more agents are described, it is also possible to incorporate the copper and/or two or more agents into other delivery vehicles, such as those described previously.
- the pharmaceutical composition is generally administered to treat and/or prevent cancer, although other diseases, infections and conditions are contemplated in which 1CD induction by a metal provides a prophylactic (preventive), ameliorative or a therapeutic benefit.
- the pharmaceutical composition will be administered at any suitable dosage.
- the pharmaceutical compositions is administered parentally, i.e., intra-arterially, intravenously, subcutaneously or intramuscularly.
- the pharmaceutical compositions described herein may be administered topically.
- the pharmaceutical compositions described herein may be administered orally.
- the pharmaceutical compositions are for pulmonary administration by aerosol or powder dispersion.
- the pharmaceutical compositions are for intra- tumoural administration. If intra-tumoural injection is employed, incorporation of the metal and agent in a drug delivery vehicle is optional.
- compositions described herein may be administered to a patient.
- patient as used herein includes a human or a non-human subject.
- Example 1 Copper enhances ATP release
- CT26 murine colon cancer cells were seeded at 200,000 cells/well in 24-well plates. After 24 hours, the cells were treated with copper or zinc alone and in combination with the indicated agents, which included:
- DNR daunorubicin
- GEM gemcitabine
- CDK inhibitors including abemaciclib (ABE), palbociclib (PAL) and ribociclib (RIB) ( Figure 1 A);
- terpyridine derivatives including 2,2';6',2"-terpyridine (TER), 4’-(4- chlorophenyl)-2, 2':6, 2"-terpyridine (CPT), 4,4 ⁇ 4"-tri-tert-butyl- 2,2 ' :6 ? ,2"-terpyridine (TTBT) ( Figure I B);
- Cu-dependent cytotoxics including 8-hydroxyquinoline (8HQ), clioquinol (CQ), diethyldithiocarbamate (DDC) and pyrithione (PYR) ( Figure 1 B); and
- the metal ion was added to the ligand (agent) at the indicated molar ratio such that metal complexes between the copper or zinc and the anti-cancer agent were formed prior to addition to the cells. (However, it should be understood that the invention is not limited to the formation of any metal-agent complexes, as discussed below).
- the cells were exposed to the tested agents for 24 hours, after which the supernatant of each well was transferred to a new plate.
- the Promega CellTiter-Glo® 2.0 Kit was then used immediately, as per the manufacturer ' s instructions, to determine the extracellular ATP level under each treatment condition.
- the assay for measuring extracellular ATP involves measuring luminescence as described previously.
- Example 2 Conner enhances at least two ICD markers including ATP and HMGBi release
- HMGBi release was tested following the procedure set forth in Example 1.
- CT26 murine colon cancer cells were seeded at an appropriate density in 24-well plates. At 24 hours, cells are treated with the agents of interest. Cells were exposed to the copper and/or tested agents for 24 hours, after which the supernatant of each well was collected and the amount of HMGBi release was assessed using a commercial ELISA kit purchased from IBL InternationalTM.
- the test quantifies HMGBi by an anti- HMGBi antibody that is immobilized. HMGBi binds to the antibody and a second enzyme marked antibody recognizes the immobilized antibody. A substrate reaction catalyzed by the enzyme leads to a change in colour intensity, which is quantified by known methodology as described previously.
- Example 3 Conner enhances each of the three markers of ICD The ability of copper to enhance all three markers of ICD, as measured by ATP secretion, HMGB, release and CRT exposure was also examined.
- ATP secretion and HMGB ] release were determined in vitro as described previously.
- a 96-well black-walled, clear bottom plate was coated with 50 pL/well of 25 pg/mL poly-D-lysine in water for 1 h at room temperature to facilitate cell adherence.
- the coating solution was aspirated and 20,000 CT26 cells/well were seeded at 100 pL/well. Cells were grown for 24 h and thereafter 100 pL of anti-cancer agents or medium was added to each well.
- the cells were exposed to the drugs for 24 h, at which time the cells were washed 3x with 100 pL/well of HBSS with calcium and magnesium without phenol red and fixed with 50 pL/well of 4% methanol-free formaldehyde diluted in phosphate-buffered saline (PBS) for 20 min at room temperature. Methanol-free formaldehyde was used to minimize permeabilization of cell membranes during fixation. Cells were washed 3x with HBSS and then 50 pL/well of primary antibody against CRT (diluted 1 :200 in staining buffer) was added for 1 h on ice.
- PBS phosphate-buffered saline
- Cells were washed 3x with HBSS and stained with 50 pL/well of secondary antibody conjugated to DyLighf 1* 488 (diluted 1 :500 in staining buffer) for 30 min on ice and in the dark.
- the plate was washed 3x with HBSS and 50 pL/well of Hoechst 33342 (diluted 1 :2000 in PBS) and CellMaskTM Deep Red (diluted 1 : 1000) was added for 20 min at room temperature to stain the cell nuclei.
- Cells were finally washed 3x with HBSS and imaged in 100 pL/well of PBS with the IN Cell Analyzer 2200. Twenty images were acquired for each well. The flat field correction was applied during the image acquisition.
- Mean fluorescence intensity of CRT on the cell membranes was quantified for each individual cell by conducting a multi-target analysis using the IN Cell 2200 Workstation 3.7 software (GE Healthcare). The average of mean fluorescence intensities for all cells under the same condition was calculated and normalized to untreated cells in order to express the results as fold-increase in CRT exposure.
- anti-cancer agents to increase HMGET release or CRT exposure with and without complex formation with copper was tested.
- the agents examined that complex with copper include daunorubicin (DNR).
- PX478 and salubrinal (SAL) and those that are not capable of forming complexes include CDDP, gemcitabine (GEM) and cytarabine (ARA-C).
- FlMGBi release and CRT exposure was determined according to the above-described HMGB and CRT assays in CT26 cells.
- Example 5 Enhancement of ICD as measured by at least ATP secretion is copper specific
- Figure 7 shows similar results with various copper salts (copper acetate, copper chloride, copper gluconate and copper sulfate), as well as cobalt, iron, manganese, nickel and zinc.
- ROS reactive oxygen species
- Combinations of drugs and metals were prepared in the same way (pre-mixing in water and further dilution in medium) for the cytotoxicity, ATP, HMGB 1 and CRT assays described above.
- To detect ROS cells were incubated with the compounds for 4 h. The amount of ROS generated was measured by the ROS-GloTM FEO2 assay (Promega, Madison, WI, USA).
- 10 pL/well of H2O2 substrate solution was added directly to each well.
- 50 mT/well of ROS-GloTM detection solution was applied to all wells, and the plate was incubated for 20 min at room temperature. The luminescence signal was measured in arbitrary units using the FLUOstar OPTIMA microplate reader (BMG Labtech, Ortenberg, Germany).
- Figure 8 shows the results of the ROS studies using various metals. The results are shown as a fold-increase relative to an untreated control. As shown, the copper salts examined exhibited increases in ROS, while most of the other metals tested, including zinc, iron, manganese, and vanadium, did not exhibit such effects. Menadione (MEN) was utilized as a positive control.
- MEN Menadione
- Figure 9A shows fold-increase of ROS as a function of concentration (0.25- 250 mM) of copper or zinc addition to the cells.
- concentration (0.25- 250 mM) of copper or zinc addition to the cells.
- the magnitude of the fold increase of ROS increased with increasing concentrations of copper from 0 to 125 mM (left graph). However, these effects were not observed with zinc (right graph). ROS were reported as fold-increase relative to an untreated control.
- Figures 9B and 9C show the results of ROS studies using copper or zinc in combination with daunorubicin (DNR), PX478, salubrinal (SAL), cisplatin (CDDP), gemcitabine (GEM) and cytarabine (ARA-C).
- DNR daunorubicin
- PX478, SAL, CDDP and ARA-C displayed increased ROS relative to the agent alone or the agent in combination with zinc (as measured by a fold increase in ROS relative to an untreated control).
- Example 6 In vivo vaccination studies with combinations of copper and anti cancer agents
- Immune competent Balb/c mice (6-8 weeks old) were “vaccinated” by inoculating the left flank subcutaneously (s.c.) with 3 x 10 6 CT26 cells pretreated in vitro. Briefly 2 x 10 7 CT26 cells were seeded in each 175 cm 2 culture flask. At 24 hours later, the cells were treated with 1 mM MITO, 25 mM CDDP, 25 mM copper sulfate (C11SO 4 ) or combinations of the cytotoxic agent with CuS0 4 at the same doses. The copper concentration was selected based on studies demonstrating that a significant increase in intracellular copper levels could be achieved when cells were exposed to 25 mM CuS0 4 (not shown).
- Tumour volumes were calculated using the equation L X W 2 /2 with the length (mm) being the longer axis of the tumour. Mice were considered tumour-free until the first day that tumours were defined as measurable (64 mm ) using calipers. Tumours were allowed to grow to a maximum of 1000 innT before termination. Control studies were completed to ensure that the tumour take rate of untreated CT26 cells was 100% in order to establish the validity of the vaccination assay.
- mice that were not vaccinated developed tumours by day 12 following inoculation of untreated cells show' that mice that were not vaccinated developed tumours by day 12 following inoculation of untreated cells (Figure 10A).
- Vaccination w'ith cells pretreated w ith MITO resulted in 58% of the mice being tumour-free on Day 39 post cancer challenge ( Figure 10B).
- the proportion of tumour-free mice increased to 70% when mice were vaccinated with cells pretreated with MITO + copper.
- Vaccination with CDDP or CDDP + copper pretreated cells resulted in 10% and 0% tumour-free mice, respectively.
- Example 7 Combinations of anti-cancer agents with copper exhibit increased ATP secretion
- CT26 cells were treated with an anti-cancer agent of interest alone, in combination with copper, or further in combination with copper and certain clinically approved anti-cancer agents.
- the level of ATP secretion was determined by analyzing the supernatant at 24 hours following treatment.
- the first anti-cancer agents tested have copper-binding ligands and included PX-478 (25 mM), flavopiridol (FLV; I mM), clioquinol (CQ; 25mM), and analogues of terpyridine 4,4 ' 4”-Tri-ter- Butyl-2,2':6 ‘ ,2"-terpyridine (TTT; 25 mM) and 4 ' -(4-chlorophenyl)-2,2':6',2”- terpyridine (CPT; 25 mM).
- the clinically approved anti-cancer agents include the following drugs: mitoxantrone (Mito; 1 mM), gemcitabine (Gem; 0.25 m
- Example 8 HMGB release of anti-cancer agent combinations with copper
- CT26 murine colon cancer cells were seeded at 200,000 cells/well in 24-well plates. After 24 hours, cells were treated with the indicated anti-cancer agents.
- the release of FlMGBi was assessed for the combination of PX-478 or Cu(PX-478) and gemcitabine (Gem) and the combination of flavopiridol (FLV) or Cu(FLV) with mitoxantrone (Mito). Where copper or zinc complexes were tested, the metal ion was added to the ligand at the indicated molar ratio such that the metal complexes were formed prior to addition to the cells.
- FIG. 12B shows that the use of flavopidirol (FLV), either in free form or as a copper complex, leads to greater HMGBi secretion compared to mitoxantrone (Mito) alone.
- FLV flavopidirol
- FIG. 12A combining PX-478 or Cu(PX-478) with gemcitabine (Gem) led to a modest to comparable increase in HMGB, release relative to gemcitabine alone.
- Example 9 Combinations of copper and two anti-cancer agents can induce at least two markers of ICD
- the ability of a combination of anti-cancer agents with copper to induce all three markers of ICD was also examined.
- the anti-cancer agents selected for the study included PX478 and GEM.
- the ICD markers, including release of ATP and HMGBi and CRT exposure were examined.
- the ICD marker assays were carried out as described previously and the anti-cancer agents and copper were added at the concentrations indicated in Figure 13.
- Combinations of copper, PX478 and GEM showed enhanced levels of ATP and HMGB 1 release relative to the agents or copper measured alone and a combination of the two agents ( Figure 13A and 13B).
- the results show that the response for a combination of copper.
- PX478 and GEM was comparable to that attained with the two anti-cancer agents measured in combination or GEM alone ( Figure 13C).
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- General Chemical & Material Sciences (AREA)
- Inorganic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Provided herein are compositions and methods for the use of copper and at least one agent to induce immunogenic cell death (ICD), in combination or sequentially, to treat or prevent a disease such as cancer, an infection or a condition in a patient. A combination of the copper and the agent provide an increase in ICD, compared to the agent alone, as determined by measuring at least one of ATP secretion, HMGB| release, and CRT exposure of cells. The copper, the agent, or both, may be formulated in a liposome or other delivery vehicles.
Description
METAL CONTAINING PRODUCTS AND COMPOSITIONS TO 1NDUCE IMMUNOGENIC CELL DEATH AND USES THEREOF
TECHNICAL FIELD
This invention generally relates to cell biology and cancer treatment.
Disclosed herein are pharmaceutical compositions and uses thereof to induce immunogenic cell death for the treatment or prevention of cancer or other disease conditions. Further disclosed are methods for identifying compositions with enhanced ability to induce immunogenic cell death. Additional embodiments are directed to treatment methods to induce immunogenic cell death.
BACKGROUND
Immunogenic Cell Death (ICD) is a phenomenon whereby cancer cells die in a manner that activates the immune system. Activation of the immune system can in turn elicit an immune response against the cancer. Cells infected as part of an infectious disease condition can also initiate an adaptive immune response. Without being limited by theory, the phenomenon of ICD is distinct from regular apoptosis in which cell death does not generally lead to the activation of the immune system, although mechanisms involved in apoptosis may contribute to induction of ICD- related molecular markers.
In particular, during immunogenic cell death, dying tumour cells or infected cells emit signals known as damage-associated molecular patterns (DAMPs). ICD is often characterized by three distinct DAMPs, which are evidenced by the presence of one or more of the following molecular determinants (referred to in the literature as "markers") of ICD: (1 ) the pre-apoptotic expression of endoplasmic reticulin (ER) calreticulin (CRT). (2) the secretion of adenosine triphosphate (ATP), and (3) the secretion of nuclear high mobility group box 1 (HMGBi). It has been reported that the presence of these DAMPs during tumour cell death leads to the release of pro- inflammatory cytokines, activation of innate immune cells such as dendritic cells (DCs) and macrophages, and ultimately stimulation of the adaptive immune response.
A number of agents used in cancer treatment are capable of inducing immunogenic cell death as determined by one or more assays to detect one or more of the above markers. Generally, ICD inducers can be categorized as Type I or Type II inducers. It has been reported that Type I inducers induce ICD as an off-target effect,
while Type II inducers typically cause primarily ER stress, which is directly related to ICD induction.
However, there is an ongoing need in the art for additional or improved compositions for inducing ICD for the treatment of cancer and other disease conditions. The present disclosure seeks to address that need or to provide useful alternatives to known approaches.
SUMMARY
According to one aspect of the disclosure, there is provided an
immunostimulatory pharmaceutical composition to treat, ameliorate and/or prevent a cancer or other condition by inducing Immunogenic Cell Death (ICD) comprising: copper and at least one agent, which is optionally an anti-cancer agent. The agent is optionally capable of complexing with copper to form a metal complex. The combination of the copper and the agent provide an increase in ICD, compared to the agent alone. The increase in the ICD response of the combination as measured by at least one of extracellular adenosine triphosphate (ATP), HMGBi release, and CRT exposure of cells in vitro may be 1 .2 to 10,000 times that of the agent alone measured under otherwise identical conditions. In alternative embodiments, the copper, the agent, or both, in certain embodiments are formulated in a liposome or other delivery vehicle known to those of skill in the art.
According to a further aspect of the disclosure, there is provided the use of copper and at least one agent, such as an anti-cancer agent, that induces ICD, in combination or sequentially, to a patient in need thereof. The combination of the copper and the agent provide an increase in ICD, compared to the agent alone, as determined by an assay that measures at least one of ATP secretion, HMGBi release, and CRT exposure of cells. The copper, the agent, or both, in certain embodiments, are formulated in a liposome or other delivery vehicles known to those of skill in the art.
According to another aspect of the disclosure, there is provided a method of inducing ICD comprising administering copper and at least one agent, such as an anti cancer agent, in combination or sequentially, to a patient in need thereof. The combination of the copper and the agent provide an increase in ICD, compared to the agent alone, as determined by measuring at least one of adenosine triphosphate (ATP)
secretion, HMGB release, and CRT exposure. The copper, the agent, or both, may be formulated in a liposome or other delivery vehicle.
In yet a further aspect of the disclosure there is provided a method of producing an immunostimulatory pharmaceutical composition for inducing ICD, comprising the steps of:
(i) exposing an agent, such as an anti-cancer agent, and copper, alone and in combination, to an assay that measures at least one of ATP secretion, HMGB release, and CRT exposure in cell culture;
(ii) determining whether the agent and the copper in combination exhibit an increase in ICD response compared to results in which the ICD of the agent is measured alone; and
(iii) wherein, if the combination of the copper and the agent provide an increase in ICD, compared to the agent alone, formulating the agent, the copper, or both in one or more pharmaceutical formulations, such as a liposome or any other drug delivery vehicle, to treat, ameliorate or to prevent cancer, either sequentially or in combination, to a patient in need thereof.
In one embodiment of any of the foregoing aspects, the copper and the agent are capable of inducing the ICD response with a further, second agent.
In yet a further aspect of the disclosure, there is provided a kit comprising copper and an agent, such as an anti-cancer agent, to induce ICD, wherein a combination of the copper and the agent provide an increase in ICD, compared to the agent alone, as determined by measuring ATP secretion, HMGB| release, CRT exposure or a combination thereof, and wherein the copper, the agent, or both, in non limiting embodiments, are formulated in a liposome or other drug delivery vehicle. The kit may comprise instructions to induce immunogenic cell death, e.g., dosaging or administration instructions, to a patient in need thereof. Alternatively, or in addition, the kit may comprise instructions for loading the copper and/or the agent into the delivery vehicle.
According to any of the foregoing aspects of the disclosure, in alternative embodiments, the copper and the at least one agent are capable of inducing immunogenic cell death with radiation.
According to any of the foregoing aspects of the disclosure, in alternative embodiments, the increase in the ICD response in the presence of copper is measured by ATP secretion.
In a further embodiment of the any of the foregoing aspects, in alternative embodiments, the copper is incorporated in the same or a different liposome or other delivery vehicle as the agent.
According to a further embodiment of any of the above aspects, in alternative embodiments, a further, second agent is utilized.
According to any of the foregoing aspects of the disclosure, in alternative embodiments, a cytotoxic effect is realized, as measured by a cytotoxic assay.
According to any of the foregoing aspects of the disclosure, in alternative embodiments, the liposome is a pre-formed liposome. That is, the liposome is prepared in solution and subsequently the agent, such as an anti-cancer agent, is loaded into the liposome by complexation with the metal.
In alternative embodiments of any of the foregoing aspects, the agent is a therapeutic agent that can treat or prevent a disease, infection or condition. The agent may or may not complex with a metal. The agent may be an anti-cancer agent, an anti-viral agent or other therapeutic agent to treat or prevent disease, for example by inducing ICD in the presence of a metal. Moreover, in a further embodiment of any of the foregoing aspects, a nanoparticle or other suitable delivery vehicle is utilized instead of a liposome. Such alternate drug delivery vehicles are known to those of ordinary skill in the art, and may include, without limitation, a vesicle such as an exosome, a micelle, a lipid-based nanoparticle, a polymer-based nanoparticle, an emulsion or a nanotube.
BRIEF DESCRIPTION OF FIGURES FIGURE 1 A shows ATP release of CT26 murine colon cancer cells (referred to herein as“CT26 cells") treated with copper, zinc, approved chemotherapeutics, CDK inhibitors and combinations of the metal and anti-cancer agents. The approved chemotherapeutics tested included cytarabine (ARA-C), daunorubicin (DNR) and gemcitabine (GEM), and the approved CDK inhibitors included abemaciclib (ABE), palbociclib (PAL) and ribociclib (RIB). The concentration of the metal and anti-
cancer agent is as indicated in the figure. The ATP release is indicated as a fold- increase in ATP release relative to an untreated control.
FIGURE 1 B shows ATP release of CT26 cells treated with copper, zinc, terpyridine derivatives or Cu-dependent cytotoxics and combinations of the metal and anti-cancer agents. The terpyridine derivatives include 2,2';6',2"-terpyridine (TER), 4‘-(4-chlorophenyl)-2, 2":6, 2"-terpyridine (CPT), 4,4\4"-tri-tert-butyl-2,2':6,,2"- terpyridine (TTBT). The Cu-dependent cytotoxics include 8-hydroxyquinoline (8HQ), clioquinol (CQ), diethyldithiocarbamate (DDC) and pyrithione (PYR). The concentration of the metal and anti-cancer agent is as indicated in the figure. The ATP release is indicated as a fold-increase in ATP release relative to an untreated control.
FIGURE 1 C shows ATP release of CT26 cells treated with copper, zinc and other chemical compounds, as well as combinations of the metals and chemical compounds. The chemical compounds tested include emodin (EMD),
epigallocathecin gallate (EPGG), physcion (P14Y) and PX478. The concentration of the metal and anti-cancer agent is as indicated in the figure. The ATP release is indicated as a fold-increase in ATP release relative to an untreated control.
FIGURE 2 shows the ATP and HMGBi release of CT26 cells treated with copper, zinc and an anti-cancer agent as well as combinations of the metals and the anti-cancer agent. The anti-cancer agents tested include 8-Hydroxyquinoline (8HQ), clioquinol (CQ), diethyldithiocarbamate (DDC) and 4"-Tri-tert-Butyl-2,2’:6\2"- terpyridine (TTBT). The concentration of the metal and anti-cancer agent is as indicated in the figure. The ATP release is indicated as a fold-increase in ATP release relative to an untreated control and HMGBi release is reported as ng/mL.
FIGURE 3 shows ATP and HMGBi release, as well as CRT exposure, of CT26 cells treated with copper and 4,4',4"-Tri-tert-Butyl-2,2':6',2"-terpyridine (TTBT) alone or in combination. The concentration of the metal and anti-cancer agent is as indicated in the figure. The ATP release is indicated as a concentration in nM. HMGBi release is reported as ng/mL and CRT exposure is measured as a fold- increase relative to an untreated control.
FIGURE 4A shows the HMGBi release from CT26 cells treated with various agents, with and without copper, which can complex with the metal. The agents tested include daunorubicin (DNR), PX478 and salubrinal (SAL)). HMGBi release was measured in ng/mL. The concentration of copper and the anti-cancer agents is shown in the figure.
FIGURE 4B shows the HMGB i release from CT26 cells treated with various agents, with and without copper, which cannot complex with the metal. The agents tested include cisplatin (CDDP), gemcitabine (GEM) and cytarabine (ARA-C).
HMGBi release was measured in ng/mL. The concentration of copper and the anti cancer agents is shown in the figure.
FIGURE 5 A shows the CRT exposure from CT26 cells of various agents, with and without copper, which can complex with the metal. The agents tested include daunorubicin (DNR), PX478 and salubrinal (SAL). CRT exposure was measured as a fold-increase relative to an untreated control. The concentration of copper and the anti-cancer agents is shown in the figure.
FIGURE 5B shows the CRT exposure from CT26 cells of various agents with and without copper that cannot complex with the metal. The agents tested include cisplatin (CDDP), gemcitabine (GEM) and cytarabine (ARA-C). CRT exposure was measured as a fold-increase relative to an untreated control. The concentration of copper and the anti-cancer agents is shown in the figure.
FIGURE 6A shows the ATP release from CT26 cells after treatment with daunorubicin (DNR) and the metals copper, zinc or iron, alone or in combination with the anti-cancer agent. The concentration of the anti-cancer agent and the metal is as indicated in the graph. Results are shown as a fold-increase in ATP release relative to an untreated control.
FIGURE 6B shows the ATP release from CT26 cells after treatment with daunorubicin (DNR) and the metals manganese, cobalt and vanadium, alone or in combination with the anti-cancer agent. The concentration of the anti-cancer agent and the metal is as indicated in the graph. Results are shown as a fold-increase in ATP release relative to an untreated control.
FIGURE 6C shows the ATP release from CT26 cells after treatment with gemcitabine (GEM) and the metals copper, zinc or iron, alone or in combination with the anti-cancer agent. The concentration of gemcitabine and the metal is as indicated
in the graph. Results are shown as a fold-increase in ATP release relative to an untreated control.
FIGURE 6D shows the ATP release from CT26 cells after treatment with gemcitabine (GEM) and metals, including manganese, cobalt and vanadium, alone or in combination with the anti-cancer agent. The concentration of the anti-cancer agent and the metal is as indicated in the graph. Results are shown as a fold-increase in ATP release relative to an untreated control.
FIGURE 6E shows the ATP release from CT26 cells after treatment with 4’- (4-chlorophenyl)-2,2’:6',2'"-terpyridine (CPT) and the metals copper, zinc or iron, alone or in combination with the anti-cancer agent. The concentration of the anti cancer agent and the metal is as indicated in the graph. Results are shown as a fold- increase in ATP release relative to an untreated control.
FIGURE 6F shows the ATP release from CT26 cells after treatment with CPT and the metals manganese, cobalt and vanadium, alone or in combination with the anti-cancer agent. The concentration of the anti-cancer agent and the metal is as indicated in the graph. Results are shown as a fold-increase in ATP release relative to an untreated control.
FIGURE 6G shows the ATP release from CT26 cells after treatment with 4,4',4”-tri-tert-butyl-2,2’:6',2''-terpyridine (TTBT) and the metals copper, zinc or iron, alone or in combination with the anti-cancer agent. The concentration of the anti-cancer agent and the metal is as indicated in the graph. Results are shown as a fold-increase in ATP release relative to an untreated control.
FIGURE 6H shows the ATP release from CT26 cells after treatment with 4,4’,4”-tri-tert-butyl-2.2":6‘,2”-terpyridine (TTBT) and the metals manganese, cobalt and vanadium, alone or in combination with the anti-cancer agent. The concentration of the anti-cancer agent and the metal is as indicated in the graph. Results are shown as a fold-increase in ATP release relative to an untreated control.
FIGURE 61 shows the ATP release from CT26 cells after treatment with abemaciclib (ABE) and the metals copper, zinc or iron, alone or in combination with the anti-cancer agent. The concentration of the anti-cancer agent and the metal is as indicated in the graph. Results are shown as a fold-increase in ATP release relative to an untreated control.
FIGURE 6J shows the ATP release from CT26 cells after treatment with with abemaciclib (ABE) and the metals manganese, cobalt and vanadium, alone or in combination with the anti-cancer agent. The concentration of the anti-cancer agent and the metal is as indicated in the graph. Results are shown as a fold-increase in ATP release relative to an untreated control.
FIGURE 7A shows the ATP release from CT26 cells after treatment with various copper salts (copper acetate, copper chloride, copper gluconate and copper sulfate), as well as cobalt, iron, manganese, nickel and zinc. The concentration of the anti-cancer agent and the metal is as indicated in the graph. Results are shown as a fold-increase in ATP release relative to an untreated control.
FIGURE 7B shows the ATP release from CT26 cells after treatment with 4,4'.4"-tri-tert-butyl-2,2’:6\2”-terpyridine (TTBT) alone and in combination with various copper salts (copper acetate, copper chloride, copper gluconate and copper sulfate), as well as cobalt, iron, manganese, nickel and zinc. The concentration of the TTBT and the metal is as indicated in the graph. Results are shown as a fold-increase in ATP release relative to an untreated control.
FIGURE 7C shows the ATP release from CT26 cells after treatment with diethyldithiocarbamate (DDC) alone and in combination with various copper salts (copper acetate, copper chloride, copper gluconate and copper sulfate), as well as cobalt, iron, manganese, nickel and zinc. The concentration of the anti-cancer agent and the metal is as indicated in the graph. Results are shown as a fold-increase in ATP release relative to an untreated control.
FIGURE 8 shows the fold-increase in reactive oxygen species (ROS) from CT26 cells after treatment with different copper salts and various metals. The copper salts examined include: copper sulfate, copper acetate, copper chloride and copper gluconate. The other metals besides copper tested for ROS were zinc, iron, manganese, cobalt and vanadium. Results are shown as a fold-increase in ROS relative to an untreated control.
FIGURE 9A shows ROS concentrations as a function of concentration (0.25- 250 mM) of copper or zinc addition to CT26 cells. Results are shown as a fold- increase in ROS relative to an untreated control.
FIGURE 9B shows the results of ROS studies with CT26 cells using copper or zinc in combination with daunorubicin (DNR), PX478 and salubrinal (SAL). Results
are shown as a fold-increase in ROS relative to an untreated control. The metals were added at equimolar concentrations.
FIGURE 9C shows the results of ROS studies with CT26 cells using copper or zinc in combination with cisplatin (CDDP), gemcitabine (GEM) and cytarabine (ARA-C). Results are shown as a fold-increase in ROS relative to an untreated control. The metals were added at equimolar concentrations.
FIGURE 10A shows in vivo vaccination results with immune competent Balb/c mice that were inoculated with CT26 cells which were pre-treated with mitoxantrone (Mito), Mito and copper, cisplatin (CDDP), and CDDP and copper (Day -7). The results are reported as the number of tumour-free mice as a function of days post-challenge by inoculating the flank opposite to the “vaccination site"’ with untreated CT26 cells (Day 0). The untreated control did not receive any pre-treatment or inoculation, serving as an“unvaccinated” control.
FIGURE 10B shows the percentage of tumour free Balb/c mice on Day 39 post-challenge. The mice were inoculated on Day -7 with CT26 cells which were previously treated with mitoxantrone (Mito), Mito and copper, cisplatin (CDDP), and CDDP and copper. Day 0 refers to the day when the mice were challenged with untreated CT26 cells a week after“vaccination” with pre-treated cells. The untreated control did not receive any pre-treatment or inoculation, serving as an“unvaccinated” control.
FIGURE 1 1 A shows fold-increase in extracellular ATP measured against untreated cells for the following treatments of CT26 cells: PX-478 alone, gemcitabine (Gem) alone, PX-478 and Gem, Cu(PX-478) alone, and Gem and Cu(PX-478) in combination.
FIGURE 1 I B shows fold-increase in extracellular ATP measured against untreated cells for the following treatments of CT26 cells: PX-478 alone, cisplatin (CDDP) alone, PX-478 and CDDP, Cu(PX-478) alone, and CDDP and Cu(PX-478) in combination.
FIGURE 1 1 C shows fold-increase in extracellular ATP measured against untreated cells for the following treatments of CT26 cells: 4,4’,4”-tri-ter-butyl- 2,2 :6‘,2” - terpyridine (TTT) alone, cisplatin (CDDP) alone, TTT and CDDP, Cu(TTT)2 alone, and cisplatin and Cu(TTT)2 in combination.
FIGURE 1 I D shows fold-increase in extracellular ATP measured against untreated cells for the following treatments of CT26 cells: flavopiridol (FLV) alone, mitoxantrone (Mito) alone, FLV and Mi to, Cu(FLV) alone, and Mito and Cu(FLV) in combination.
FIGURE 1 1 E shows fold-increase in extracellular ATP measured against untreated cells for the following treatments of CT26 cells: clioquinol (CQ) alone, gemcitabine (Gem) alone, CQ and Gem in combination, Cu(CQ)2 alone, and Gem and CU(CQ)2 in combination.
FIGURE 1 I F shows fold-increase in extracellular ATP measured against untreated cells for the following treatments of CT26 cells: 4 (4-chlorophenyl) - 2,2':6',2”-terpyridine (4CPT) alone, cisplatin (CDDP) alone, 4CPT and CDDP, CU(4CPT)2 alone, and CDDP and Cu(4CPT)2 in combination.
FIGURE 12A shows secretion of HMGBi (ng/mL) from CT26 cells for the following: untreated, PX-478 alone, gemcitabine (Gem) alone, PX-478 and Gem, Cu(PX-478) alone, and Gem and Cu(PX-478) in combination.
FIGURE 12B shows secretion of HMGBi (ng/mL) from CT26 cells for the following: untreated, flavopiridol (FLV) alone, mitoxantrone (Mito) alone, FLV and Mito, Cu(FLV) alone, and Mito and Cu(FLV) in combination.
FIGURE 13A shows ATP release (nM) of CT26 cells for the following:
untreated, copper, PX478, gemcitabine (GEM), PX478 and GEM, and copper, PX478 and GEM in combination.
FIGURE 13B shows HMGBi release (ng/mL) of CT26 cells for the following: untreated, copper, PX478, gemcitabine (GEM), PX478 and GEM, and copper, PX478 and GEM in combination.
FIGURE 13C shows CRT exposure (fold-increase) of CT26 cell for the following: untreated, copper, PX478, gemcitabine (GEM), PX478 and GEM, and copper, PX478 and GEM in combination.
The details of one or more exemplary embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
All publications, patents, patent applications cited herein are hereby expressly incorporated by reference for all purposes.
DETAILED DESCRIPTION
Induction and measurement of Immunogenic Cell Death (ICD)
The compositions and methods described in the present disclosure enhance immunogenic cell death phenotypes, which is also referred to herein as“JCD". In particular, the inventors have examined the ability of various agents to induce ICD by utilizing in vitro assays to measure molecular determinants indicative of ICD and have found that copper in combination with an agent can exhibit an increased ICD response relative to the agent alone. According to one embodiment, the increase in ICD response is attributable to the copper. In another embodiment, the copper potentiates the ICD response of the agent. In certain non-limiting embodiments, after selection of suitable combinations by the foregoing assay, the agent is most advantageously loaded into delivery vehicles. This may include loading of the agent into pre-formed liposomes comprising copper, using, for example, methods described herein. However, as discussed below, other delivery vehicles can be utilized as well for delivery of the metal and/or the agents.
As noted, during ICD, cells emit signals known as damage-associated molecular patterns (DAMPs). ATP is one such DAMP that is secreted during ICD and, without being limiting, is believed to attract monocytes to the site of tumour cell death, which differentiate to dendritic cells (DC) expressing inflammatory DC markers (e.g., CD1 lc+CDl l b+Ly6Chl). Without being limited by any particular theory, it has been reported that tumours treated with mitoxantrone are infiltrated by
CD1 E+CD 1 l b+ Ly6Chl cells within 12 h of treatment and subsequently by
macrophages. ATP secretion can be measured in vitro by exposing cells in culture to the agents being tested for a pre-determ ined period of time. ATP is subsequently measured by a reaction in which the ATP secreted is measured by luminescence.
ATP secretion is measured in vitro as follows. Prior to measuring ATP secretion, cells of interest, such as CT26 murine colon cancer cells, are seeded at an appropriate density in multi-well plates, such as 24-well plates. At 24 hours, cells are treated with the agents and the metal ion. Optionally, the metal ion and agent are
incubated under conditions that are optimal for metal complexation formation prior to their addition to the cells. However, as noted, in certain embodiments, 1CD induction is not dependent on formation of a complex between the metal and the agent.
Subsequently, the cells are exposed to the tested agents (copper and/or agent) for 24 hours (or left untreated), after which the supernatant of each well is transferred to a new plate. The Promega CellTiter-Glo™ 2.0 Kit is then used, as per the manufacturer's instructions, to determine the extracellular ATP level under each treatment condition. The assay measures ATP by luminescence. Luciferin is converted to oxyluciferin in the presence of ATP, Mg2+ and 02. Oxyluciferin in turn emits light that is quantified by a luminometer and is correlated with the amount of ATP present by a standard curve. The assay for use in the present disclosure is described in Promega Technical Manual, CellTiter-Glo® 2.0 Assay, Instructions for Use of Products, G9241 , G9242 and G9243 available on-line at www.promega.com and which is incorporated herein by reference.
A further DAMP is the high-mobility group box 1 , also referred to herein as "HMGBi”. Without being limited to any particular mechanism, HMGBi has been reported to be a late apoptotic marker and its release to the extracellular space facilitate dendritic cell maturation and promote tumour antigen presentation to induce CDS T cells. This molecular determinant of 1CD can be measured by the assay described below.
Similar to the assay for ATP release, cells of interest, such as CT26 murine colon cancer cells, are seeded at an appropriate density in 24-well plates. At 24 hours, cells are treated with the agents of interest. Where copper in combination with an agent or copper complexes are tested, the metal ion may be added to the agent at a predefined molar ratio. For example, in those embodiments in which complex formation is desirable, the metal complexes are formed at a pre-determined metal-to- ligand ratio prior to addition to the cells for optimal metal complexation. Cells are exposed to the copper and/or tested agents for 24 hours, after which the supernatant of each well is collected and the amount of HMGBi release is assessed using a commercial ELISA kit purchased from IBL International™. The test quantifies HMGBi by an anti-HMGB antibody that is immobilized. HMGBi binds to the antibody and a second enzyme marked antibody recognizes the immobilized antibody.
Substrate reaction catalyzed by the enzyme leads to a change in colour intensity, which is quantified by known methodology.
Calreticulin (CRT) is another DAMP that is exposed on the surface of tumour cells during cell death. Without being limited by theory, Ecto-CRT is reported to act as a signal that activates phagocytosis by certain antigen presenting cells, contributing to the induction of an immune response. CRT can be measured by flow cytometric analysis of CRT cell surface expression. According to this assay, after 24 hours of treatment of the cells, such as CT26 cells, with copper and/or an agent, the copper and/or agent is removed and replaced with culture media. Cells are harvested at 0, 24, 48, and 96 hours post-treatment and stained with anti-CRT antibody followed by Alexa-488-conjugated secondary antibody. Propidium iodide (PI) is used as a counterstain to differentiate between viable and dead cells. The relative CRT mean fluorescence intensity (MF1) of Pi-negative viable cells is determined by subtracting the MFI of isotype control-stained cells from the MFI of anti-CRT stained cells, and then normalising to HBSS-treated cells. An immunofluorescence assay may also be used to visualize cell surface expression of CRT on cells treated with an agent for 4 hours and stained with the anti-CRT antibody (green), as described above. To enhance visualization, the cell membrane may be labelled with wheat germ agglutinin (red) and the nucleus with Hoechst 33342 (blue). Images may be taken using an IN Cell Analyzer 2200™ and the fluorescent signal (green) can be quantitated and analyzed at a per-well and per-cell level to analyze relative cell surface CRT expression.
As would be appreciated by those of skill in the art, the procedures and/or reaction conditions for the ATP, HMGB 1 and/or CRT assays described above can be modified to achieve optimal results relative to an untreated control.
In a further embodiment, ICD induction by copper is evidenced by each of the three assays or any combination of two of any of the three assays or one of any of the three assays. In a further embodiment, the presence of copper increases the levels of ATP secretion over that of the ICD inducer alone. In yet a further embodiment, the presence of copper enhances the level of FIMGB released with or without an agent that is an ICD inducer. According to yet further embodiments described herein, ICD induction by copper is evidenced by CRT expression. In one embodiment, the ICD response of the copper and the agent in combination as measured by extracellular
ATP, HMBG 1 and/or CRT exposure is 1.2 to 10,000, 1.5 to 10,000, 2 to 10,000, 3 to 10,000, 4 to 10,000, or 5 to 10,000 times that of the agent alone measured under otherwise identical conditions. In another embodiment, the ICD response of the copper and the agent in combination as measured by extracellular ATP, HMBG 1 and/or CRT exposure is at least 1.2, 1.3, 1.4, 1.5, 1 .6, 1.7, 1.8, 1.9, 2, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 or more times that of the agent alone measured under otherwise identical conditions. In alternative embodiments, the upper limit of the ICD response of the copper and the agent in combination as measured by extracellular ATP, HMBG 1 and/or CRT exposure may be 800, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000 or 10,000 times relative to agent alone. Any combination of the foregoing lower and upper limits is included in select embodiments of the disclosure.
As discussed, the ICD of an agent in combination with copper is determined by measuring a phenotype of ICD (ATP secretion, CRT release and/or HMGBi), also referred to herein as a“marker” of ICD. In alternative embodiments, the cytotoxicity of a particular pharmaceutical formulation and/or treatment regime can be measured using a cytotoxicity assay. In certain non-limiting embodiments, an additive or synergistic cytotoxic effect between copper and an agent may be exhibited (non- antagonistic). Such determination may be made by the Chou Talalay method, which is known to those of ordinary skill in the art.
Without being limiting, in one embodiment, the cytotoxicity assay can be used to determine whether a cytotoxic effect of the agent of interest in combination with copper is present. Moreover, in those embodiments that additionally include radiation and/or a second agent, the composition or treatment w ill be considered to have a cytotoxic effect if any combination selected from at least two of (i) copper, (ii) an agent, (iii) a second agent if two or more are used, and (iii) radiation treatment exhibits a cytotoxic effect in vitro. By way of example, if a pharmaceutical composition comprises copper as well as a first and a second agent in combination, and if the second agent exhibits a cytotoxic effect in combination with copper, but the first agent in combination with copper does not, nor the first agent in combination with the second agent, the pharmaceutical composition will still be considered to have a cytotoxic effect as used herein. Yet in a further example, if a pharmaceutical composition comprises copper and a first and a second agent in combination, and if
the first agent exhibits a cytotoxic effect in combination with the second agent, but the first or second agent in combination with copper does not, the pharmaceutical composition will still be considered to have a cytotoxic effect as used herein. In another non-limiting, illustrative example, if a treatment comprises an agent in combination with the metal and additionally radiation treatment, and if the agent and the radiation treatment exhibit a cytotoxic effect in combination, then the treatment will be considered cytotoxic, even if the metal and agent demonstrate no cytotoxic effect in combination. Additional examples will be readily envisioned by those of ordinary skill in the art.
As noted, whether a cytotoxic effect is observed can be measured using an in vitro assay. Cells are seeded in 384-well plates and treated with various
concentrations of copper and agent (0.001 nM to 10 mM) for 72 hours. Cells are then stained with Hoechst 33342 and ethidium homodimer-1 for total and non-viable (cells that have lost membrane integrity), respectively. The cells are subsequently imaged using an automated fluorescent microscopic platform such as the IN Cell Analyzer 2200. All images are processed through software such as the IN Cell Developer Toolbox which provides total and dead cell counts. The viable cell counts are used to generate dose responsive curves using curve-fitting programs such as PRISM™ (GraphPad) to determine the potency of each treatment as determined by IC5o values.
For combinations involving radiation, the cells are seeded in plates and treated with the agent of interest with or without copper for 24 hours, after which the cells are irradiated at different doses (2, 4, 6, 8, 10 Gy). The irradiated cells are immediately plated in culture dishes to achieve 100-500 colonies two weeks later. The colonies are stained with aqueous malachite green or crystal violet and dried overnight prior to colony counting. The surviving fraction (SF) is calculated using the equation SF =
[no. of colonies formed after treatment / (no. of cells seeded x plating efficiency)].
The dose response curves from each treatment are then compared to assess treatment efficacy in vitro.
As noted, the metal ion of select embodiments is copper. The metal ion may also be an ion of a transition metal or a Group lllb metal. The transition metal may be from Group IB, 2B, 3B, 4B, 5B, 6B, 7B and 8B (groups 3- 12). Examples of transition metals include copper, zinc, vanadium, manganese, iron, cobalt and nickel. In another embodiment, the metal has d-orbitals. The Group lllb metal is from the
boron family, which includes boron, aluminum, gallium, and indium. In one embodiment, the metal is in the 2+ oxidation state.
Agents
By the term "agent", as used herein it is meant any compound or molecular species to treat, ameliorate, and/or prevent a disease or condition; a compound or molecular species that serves as a carrier for transporting the metal ion to facilitate loading into a given delivery vehicle and/or to facilitate delivery of the metal after administration; or a compound or molecular species that serves as an adjuvant. The disease or condition includes any proliferate disorder or disease and includes, but is not limited to, cancer, infectious diseases or any other condition in which 1CD is desirable.
In alternative embodiments, the agent of select embodiments used in combination with copper typically comprises at least one complexation moiety or ligand to enable complexation with the copper. However, it will be appreciated that, in certain embodiments, the agent for use in the pharmaceutical compositions described herein need not complex with the metal.
In those non-limiting embodiments in which the agent complexes with a metal, the agent may have a chemical group, often referred to as a ligand, which has atoms that allow for the coordination of the agent with the metal. The chemical group may be selected from an S-donor, O-donor, N, O-donor, a Schiff base, hydrazones, P- donor phosphine, N-donor or a combination thereof. In another embodiment, the moiety is a hard electron donor. Other moieties or ligands known to those of skill in the art suitable for complexation with a metal ion are included within the scope of certain embodiments as well. In a further embodiment, the complexation via metal coordination facilitates drug loading as described in co-owned WO 2017/100925 (Bally et al.). This could be measured by incubating an agent of interest with a liposome containing copper, or other metal ion of interest, under optimal conditions to facilitate drug uptake and measuring drug uptake after one hour. In one embodiment, the drug uptake after one hour is at least 30%, 40%, 50%, 60%, 70% or 80% as determined by measuring the drugilipid ratio relative to the theoretical drugdipid ratio that could ideally be obtained. In another embodiment, the drug uptake after one hour is between 60% and 100% as measured above.
The agent tnay be selected from antineoplastic agents, for example cytotoxic agents. Such agents include, but are not limited to, nucleoside analogues, anthracyclines, anti-folates, topoisomerase I inhibitors, taxanes, vinca alkaloids, alkylating agents, platinum compounds, targeted antineoplastics, including monoclonal antibodies, tyrosine kinase inhibitors, mTOR inhibitors, CDK inhibitors, retinoids, immunomodulatory agents and histone deacetylase inhibitors, terpyridine derivatives and Cu-dependent cytotoxics.
In one embodiment, an agent suitable for use in certain embodiments is selected from PX-478, Emodin, clioquinol (CQ), pyrithione (PYR), flavopiridol (FLV), diethyldithiocarbamate (DDC), epigallocathecin gallate (EPGG), cisplatin (CDDP), gemcitabine (GEM), mitoxantrone (MITO), 4' (4-chlorophenyl) - 2.2‘:6',2"-terpyridine (4CPT), 4, 4\4”-tri-ter-butyl-2,2':6\ 2" - terpyridine (TTT), shikonin, wogonin, cytarabine (ARA-C), oxaliplatin (OXP), salubrinal (SAL), abemaciclib (ABE), palbociclib (PAL) and ribociclib (RIB), 2,2';6’,2"-terpyridine (TER), 4'-(4-chlorophenyl)-2, 2’:6, 2”-terpyridine (CPT), 4,4',4"-tri-tert-butyl- 2,2':6\2”-terpyridine (referred to herein as“TTBT" or“TTT"), 8-hydroxyquinoline (8HQ), physcion (PHY), diacetyl-bis(N4-methylthiosemicarbazone) (ATSM) and glyoxal-bis (N4-methylthiosemicarbazone) (GTSM). In one embodiment, the agent is not daunorubicin or doxorubicin.
In those embodiments in which the agent complexes with copper, or another metal, the agent may function as a carrier moiety, such as an ionophore. The ionophore may facilitate loading of the metal into a delivery vehicle. For example, the agent may function as an ionophore to facilitate the delivery of a metal complexed therewith across a lipid bilayer of a delivery vehicle, such as a liposome. According to such embodiments, the agent and copper may be added to the external solution of a liposome preparation. The agent may form a complex with the metal in the solution external to the liposome and the resultant copper-ionophore complex may
subsequently cross the lipid bilayer and enter the aqueous interior of the delivery vehicle.
Moreover, without being limiting, after administration of a delivery vehicle comprising the metal-carrier complex, the carrier agent may facilitate the transport of the bound copper in the complex to a disease site. Without being bound by theory, such a carrier agent may facilitate the transport of the metal across a cellular
membrane. In one embodiment, the agent additionally exerts a therapeutic effect rather than simply functioning as a carrier agent.
Non-limiting examples of agents that complex with copper and that may function as a carrier for the metal include diacetyl-bis(N4-methylthiosemicarbazone) (ATSM), clioquinol (CQ) and diethyldithiocarbamate (DDC). Without being limiting, in certain embodiments, Cu-ATSM is permeable across cellular membranes and can be reduced in hypoxic tissue. A reduction in hypoxic conditions (such as at a tumour site) may allow the copper atom to dissociate from the ATSM. In some embodiments, this may allow copper to be continuously deposited in hypoxic tissues and cells.
Without being limiting, the agent for use in select embodiments may be poorly soluble in solution prior to or after complexation with the metal ion. By this it is meant that the poorly soluble agent in free form has a solubility of less than 1 mg/iriL in either water or a solution of the metal ion which complexes with the agent.
Solubility of the agent in water or in the presence of the metal ion (10 mM to 500 mM) is measured at conditions of physiological pH and temperature after 60 minutes of incubation under these conditions. Measurement of solubility of the agent is conducted as described in co-owned WO 2017/100925.
In yet further embodiments, the agent has a solubility that is greater than 1 mg/mL in either water or a metal ion solution, as determined by the assay described above (see WO 2017/100925).
Preparation of pharmaceutical formulations
The agent and metal may be formulated in any known delivery vehicle. As used herein, the term“delivery vehicle” means a particle of a size between 10 nm and 2000 nm that contains associated therewith the agent, copper or a combination thereof. The delivery vehicle typically controls the rate of release of its cargo.
In some embodiments, the agent and copper are both administered in free form or one of the agent and the metal are formulated in the delivery vehicle, while the other is in free form (such as formulated with a pharmaceutically acceptable carrier, such as a salt). Whether or not the agent and/or the metal are formulated in the delivery vehicle may depend on the mode of administration. For example, if the agent and copper are introduced directly to a disease site, such as by direct injection, then formulation in a delivery vehicle may not be necessary. Examples of suitable delivery
vehicles include vesicles such as liposomes and exosomes, micelles, polymer-based nanoparticles, emulsions, or nanotubes. The agent may also be conjugated with a lipid or polymer to improve its ability to be formulated in a given drug delivery vehicle.
As discussed, in alternative embodiments, the agent may be formulated in a liposome. In alternative embodiments, the liposome is a vesicle comprising a bilayer having amphipathic lipids enclosing an internal solution. The liposome may be a large unilamellar vesicle (LUV), which can be prepared as described below using extrusion. In one embodiment, the average diameter of the liposome may be between 60 nm and 2,000 nm, 70 and 1 ,000 nm, 70 and 500 nm, 70 and 200 nm or 70 and 180 nm. The liposome may comprise lipids including phosphoglycerides and
sphingolipids, representative examples of which include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, pahnitoy!oleoyl phosphatidylcholine, lysophosphatidylcholine, lysophos- phatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine or dilinoleoylphosphatidylcholine. Other compounds lacking in phosphorus, such as sphingolipid and glycosphingolipid families are also encompassed by certain embodiments. The phospholipids may comprise two acyl chains from 6 to 24 carbon atoms selected independently of one another and with varying degrees of unsaturation. Additionally, the amphipathic lipids described above may be mixed with other lipids including triacylglycerols and sterols. As would be appreciated by those of skill in the art, lipids that interfere with liposome formation in the presence of a metal should typically be avoided. Whether or not a given lipid is suitable for liposome formation in the presence of a metal ion can be determined by those of skill in the art. In one embodiment, the liposome comprises the lipids 1 ,2- distearoyl-sn-glycero-3- phosophocholine (DSPC) and cholesterol. The precise ratios of the lipids may vary as required. A non-limiting example of a suitable ratio of DSPC/Cholesterol is 55:45 mokmol.
The liposomes or other drug delivery vehicles may also comprise a hydrophilic polymer-lipid conjugate. The hydrophilic polymer may be a
polyalkylether, such as polyethylene glycol. The hydrophilic polymer-lipid conjugate is generally prepared from a lipid that has a functional group at the polar head moiety that is chemically conjugated to the hydrophilic polymer. An example of such a lipid
is phosphatidylethanolamine. The inclusion of such hydrophilic polymer-lipid conjugates in a liposome can increase its circulation longevity in the bloodstream after administration. The hydrophilic polymer is biocompatible and has a solubility in water that permits the polymer to extend away from the outer surface of the liposome. The polymer is generally flexible and may provide uniform surface coverage of the liposome outer surface. In addition, it has been found herein that the inclusion of such a hydrophilic polymer-lipid conjugate can increase the amount of the transition metal encapsulated in the liposome. This can be used as a methodology to increase the amount of the agent encapsulated in the liposome. In one embodiment, the liposome may include a hydrophilic polymer, such as polyethylene glycol (PEG) at between 1 and 20 mol% or between 2 and 10 mol%. A non-limiting example of a liposomal formulation comprising PEG is DSPC/CHOL/PEG (50:45:5, mole ratio) or DSPC /PEG (95:5, mole ratio). The specific ratios of the lipids, however, may vary according to embodiments that would be apparent to those of ordinary skill in the art.
Liposomes can be prepared by any of a variety of suitable techniques known to those of skill in the art. An example of one suitable method involves cycles of freeze-thaw and subsequent extrusion of lipid preparations. According to one such method, lipids selected for inclusion in a liposome may be desiccated and dissolved in a solvent, such as an organic solvent, at a desired ratio. After removal of the solvent, the resultant lipids are hydrated in an aqueous solution. The solution in which the lipids are hydrated forms the internal solution of the liposomes. Subsequently the hydrated lipids may be subjected to cycles of freezing and thawing. The hydrated lipids are passed through an extrusion apparatus to obtain liposomes of a defined size. The size of the resulting liposomes may be determined using quasi-electric light scattering (e.g., using a NanoBrook ZetaPALS™ Potential Analyzer).
Without being limiting, the liposomes may be prepared so that they comprise an internal solution comprising the metal ion. For example, when preparing liposomes by freeze-thaw and subsequent extrusion as described above, the lipids are hydrated in a solution comprising a metal ion. Generally, the liposomes so formed will comprise the metal ion not only in the internal solution of the liposomes, but also in the external solution. Unencapsulated metal ion may be removed from the external solution of the liposome prior to loading of the agent. For example, the external copper solution may be exchanged with a solution containing substantially no copper
ions by passage through a column equilibrated with a buffer. Other techniques may be employed such as centrifugation, dialysis, the addition of a chelating agent, such as EDTA (to chelate the metal) or related technologies. Typically the solution that exchanges with the metal-containing solution is a buffer, although other solutions may be used as desired. The liposomes may be subsequently concentrated to a desired lipid concentration by any suitable concentration method, such as by using tangential flow dialysis. In one embodiment, the solution external to the liposome contains substantially no metal ions that complex with the poorly soluble agent. By this it is meant that the concentration of metal ions in the external solution is less than that of the metal ion concentration in the liposome, for example less than one fifth of the concentration of metal ion in the liposome. Alternatively, or in addition, the external solution may comprise a chelating agent that chelates with the metal ions. As noted, the metal ion may be encapsulated in the liposome as a metal salt. Examples include copper sulfate, copper chloride or copper gluconate.
The pre-formed liposomes comprising the metal ion may be incubated with the agent to facilitate uptake via metal complexation. The agent may be added in any suitable form, including as a powder or as a solution. If the agent is insoluble in water, it can be added as a powder. The amount of free agent in solution can subsequently be increased by increasing the temperature. Incubation of the pre formed liposomes with the one or more agents is performed under conditions sufficient to allow the agent to move across the phospholipid bi layer of the liposome into the internal solution thereof. Such a method is referred to by those of skill in the art as "loading". Movement of the agent across the phospholipid bilayer of the pre formed liposome during loading may occur independently of any pH gradient across the bilayer. The loading may, however, be dependent on other factors. As will be appreciated, the loading conditions can be readily selected by those of skill in the art to achieve a desired rate of loading. For example, the diffusion of the agent across the bi layer may be dependent on the temperature and/or lipid composition of the liposome.
Without being bound by theory, the formation of the drug-metal complex incorporated in the pre-formed liposomes may be characterized as an inorganic synthesis reaction. In certain embodiments, the uptake of drug during the loading reaction is visualized as a colour change as many metal complexed agents have
different spectral characteristics that can be detected by eye. For example, a colour change to purple, brown, green or yellow can be observed during loading with copper.
By formulating complexes through such an inorganic synthesis reaction occurring within the pre-formed liposome, a high drug-to-lipid ratio may be attained according to certain embodiments. For example, the drug-to-lipid ratio may be at least 0.3: 1 0.4: 1 , 0.5: 1 , at least 0.6: 1 , at least 0.7: 1 , or at least 0.8: 1. In further embodiments, the drug-to-lipid ratio is about 0.1 : 1 to about 0.6: 1 (molanol), about 0.15: 1 to about 0.5: 1 (molanol) or about 0.2: 1 to about 0.4: 1 (molanol). Such a high drug-to-lipid ratio may be dependent on the number of metal ions inside the liposome and/or the nature of the complex formed. Formation of a transition metal complex with a given agent may be rapid (e.g., Cu(DDC)2), occurring in minutes, or more gradual. The complexation reaction rate may be temperature dependent. The rate of metal-agent complex formation may also be dependent on the rate at which the externally added agent crosses the lipid bilayer of the liposome. As will be appreciated by those of skill in the art, these variables can be adjusted as desired to achieve a desired rate of uptake of the agent or metal-agent complexation if the latter mechanism facilitates loading.
In addition, loading of an agent may be facilitated by a carrier agent that acts as an ionophore, as described previously.
The liposome formulations may be either passively or actively loaded with the agent. However, active loading is preferred as it may result in higher trapping efficiencies and drug retention properties relative to passive loading. Nonetheless, in order to encapsulate more than one agent, a combination of passive and active entrapment methods can be utilized. For example, an agent may be passively entrapped in a liposome with a metal ion and a second agent could be loaded by the metal loading method described above.
In one embodiment, the liposome has a trapping efficiency of at least 80% with respect to at least one agent encapsulated in the liposome. In another
embodiment, the trapping efficiency is at least 90%. Such high trapping efficiencies may result from active loading. By contrast, passive loading may result in comparatively lower trapping efficiencies.
While liposomes are discussed above, other vesicles besides liposomes can be used to formulate the copper, the agent or both. For example, an exosome may serve
as a delivery vehicle for one or more of the agents and/or copper. An exosome comprises a lipid bilayer enclosing an internal aqueous space and is derived from a living or dead organism, explanted tissues or organs and/or cultured cells. An exosome is typically derived from plasma membrane budding from a producer cell as described in U.S. Patent No. 10, 195,290, which is incorporated herein by reference. Exosomes may be utilized to deliver a variety of agents, such as DNA, RNA, protein and lipid. Unique protein and carbohydrate molecules on the surface of exosomes may allow them to be transported to a cellular target site in the body and be taken up efficiently by cells. The term exosome as used herein is meant to include a hybridosome®, which is a hybrid of a lipid nanoparticle and exosome, and vexosomes that transport a virus into cells via an exosome.
The delivery vehicle may include other particles that are not based on vesicle formation, although such delivery vehicles may include a surface stabilizing lipid bilayer or monolayer. An example is a micelle that is any particle or assembly that forms a core that is hydrophobic and comprises more hydrophilic regions that surround the core and that are in contact with the surrounding solvent. Micelles are generally used for the delivery of hydrophobic or water insoluble agents that are contained in the hydrophobic core. The micelle may be made up of lipids with hydrophilic heads facing outwardly and the hydrophobic tails facing inwardly to form the hydrophobic core of the particle. However, the components that make up a micelle can vary widely and include molecules such as lipoproteins, including high and low density lipoproteins, chylomicrons, fatty acids, lipids, polymers or any suitable combination. Moreover, micelles can be made up of a single molecular species or may include particles with a hydrophobic core composed of one molecular species and a hydrophilic region contacting the surrounding solvent that is composed of a different molecular species. For example, a core may be made of fatty acids surrounded by a mono or bilayer of lipids or other amphipathic molecules.
The delivery vehicle may also be a polymer-based nanoparticle or microparticle. The polymer-based nanoparticles or microparticles are typically produced from synthetic polymers, but can include natural materials as well as lipid monolayers and bi layers. Non-limiting examples of nanoparticles and microparticles comprise a core of drug surrounded by a polymeric shell or a drug dispersed throughout a polymeric matrix. The drug may be dispersed as a solid or liquid in the
nanoparticle or microparticle. The agent may also be covalently bonded or associated with the particle by an electrostatic interaction. The nanoparticle or microparticle may be a polymer/lipid hybrid, such as a polymer core containing an agent surrounded by a lipid monolayer or bilayer.
Other delivery vehicles include emulsions or nanotubes. The emulsion may be an oil-in-water emulsion that contains one or more water insoluble agents in the oil phase. A nanotube is a carbon-based cylindrical tube in which an agent can be loaded. The particles may comprise a graphene sheet that forms into the shape of a cylinder.
Alternative delivery vehicles may be employed besides those specifically exemplified above.
Although the incorporation of a metal and an agent, optionally as a metal- agent complex, within a liposome or other drug delivery vehicle is described, other embodiments encompass formulations for 1CD induction in which copper is administered to a patient separately from the delivery vehicle comprising the agent. According to such embodiments, the copper is typically administered separately or together with the delivery vehicle. Typically, a copper chelate is administered by injection. Examples of copper chelates include Cu(ATSM) and Cu(GTSM). In another embodiment, the copper is formulated as a salt. For example, copper gluconate may be administered orally. The agent may be administered as a liposomal or other drug delivery formulation before, during or after administration of the free copper. In those embodiments utilizing liposomal delivery of the agent wdthout co encapsulation with copper, the agent may be loaded into the liposome using active loading methods besides metal loading, such as pH gradient loading, or passive loading methods as known to those of ordinary skill in the art. After administration, the free copper may complex with the agent or can remain uncomplexed. In a further embodiment, a liposome or other drug delivery vehicle comprising copper is administered separately or together with one or more ICD inducing agents that are in free form. Likewise, after administration, the copper may complex with the agent in free form, or remain uncomplexed.
Additional combinations of anti-cancer agents and metal ions for inducing ICD
According to certain features of select embodiments, one or more additional agents may be used to induce ICD as described herein. The combinations of two or
more drugs may exhibit increased ATP secretion, HMBG] release and/or CRT expression relative to treatment with one agent alone and/or complexed with metal. The one or more additional agent may also complex with the metal or may be present in free form. These additional agents may be selected from antineoplastic agents such as cytotoxic agents, including nucleoside analogues, anthracyclines, anti-folates, topoisomerase 1 inhibitors, taxanes, vinca alkaloids, alkylating agents, platinum compounds, targeted antineoplastics, including monoclonal antibodies, tyrosine kinase inhibitors, mTOR inhibitors, CDK inhibitors, retinoids, immunomodulatory agents, histone deacetylase inhibitors, terpyridine derivatives and Cu-dependent cytotoxics. In one embodiment, a second agent suitable for use in certain
embodiments is selected from PX-478, Emodin, clioquinol (CQ), pyrithione (PYR), flavopiridol (FLV), diethyldithiocarbamate (DDC), epigallocathecin gallate (EPGG), cisplatin (CDDP), gemcitabine (GEM), mitoxantrone (MITO), 4' (4-chlorophenyl) - 2,2':6',2"-terpyridine (4CPT), 4,4’,4’'-tri-ter-butyl-2,2’:6’,2” - terpyridine (TTT), shikonin, wogonin. cytarabine (ARA-C), oxaliplatin (OXP), salubrinal (SAL), abemaciclib (ABE), palbociclib (PAL) and ribociclib (RIB), 2,2';6',2"-terpyridine (TER), 4’-(4-chlorophenyl)-2, 2':6, 2"-terpyridine (CPT), 4,4’,4!‘-tri-tert-butyl- 2,2’:6',2”-terpyridine (TTBT), 8-hydroxyquinoline (8HQ), physcion (PHY), diacetyl- bis(N4-methylthiosemicarbazone) (ATSM) and glyoxal-bis (N4- methylthiosemicarbazone) (GTSM). In one embodiment, the agent is not daunorubicin or doxorubicin.
In a further embodiment, one or more agents complexed with a metal may be used to treat cancer in combination with an additional therapy such as radiation.
Advantageously, the methods described in select embodiments herein can be used to load multiple agents, either simultaneously or sequentially into a liposome or other delivery vehicle. If each of the agents is incorporated into a liposome, the agents can be loaded by the complexation method described herein. Moreover, the liposomes into which the agent is loaded may themselves be prepared so that the internal solution comprises not only the metal ion but also an additional agent.
Loading of an agent in this manner is often referred to as passive loading. The subsequent loading of a second agent which complexes with the metal in the pre formed liposome (as described above) will result in incorporation of two agents in the liposome, one of which is loaded passively and the other actively via complexation.
Since the passively loaded agent need not complex with a metal ion to effect loading, this approach provides greater flexibility in preparing liposome-encapsulated agent combinations for use to treat or prevent a disease of interest. Moreover, in certain non-limiting embodiments, the two or more agents may be loaded at a predetermined ratio that exhibits synergistic or additive effects as elucidated by the Chou-Talalay determination, which is a known methodology for measuring such effects.
A formulation of liposomes may also comprise two or more liposome populations, which incorporate the same or different agents, comprise different lipid formulations, or comprise liposomes of different vesicle sizes. Moreover, one or more agents may be in free form, while one or more agents may be incorporated in a liposome. For example, the copper may be in free form and the agent or agents incorporated in a liposome. In another embodiment, the copper may be incorporated in a liposome and the agent or agents may be in free form in the pharmaceutical composition. The combinations of agents selected for the pharmaceutical formulation may achieve greater therapeutic efficacy, safety, prolonged drug release or targeting after administration.
According to one embodiment, a second agent in free form is included in a treatment regime such that the drug becomes active in the presence of the metal ion. Examples of such drug combinations include co-encapsulation of metal-CQ and free DSF, the precursor of DDC. The DSF is metabolized to form DDC and DDC and is subsequently activated in the presence of a metal ion, such as copper, at the tumour site.
Although liposomal formulations incorporating copper and two or more agents are described, it is also possible to incorporate the copper and/or two or more agents into other delivery vehicles, such as those described previously.
Administration
In alternative embodiments, the pharmaceutical composition is generally administered to treat and/or prevent cancer, although other diseases, infections and conditions are contemplated in which 1CD induction by a metal provides a prophylactic (preventive), ameliorative or a therapeutic benefit. The pharmaceutical composition will be administered at any suitable dosage. In one embodiment, the pharmaceutical compositions is administered parentally, i.e., intra-arterially, intravenously, subcutaneously or intramuscularly. In other embodiments, the
pharmaceutical compositions described herein may be administered topically. In still further alternative embodiments, the pharmaceutical compositions described herein may be administered orally. In yet a further embodiment, the pharmaceutical compositions are for pulmonary administration by aerosol or powder dispersion.
In yet a further embodiment, the pharmaceutical compositions are for intra- tumoural administration. If intra-tumoural injection is employed, incorporation of the metal and agent in a drug delivery vehicle is optional.
The compositions described herein may be administered to a patient. The term patient as used herein includes a human or a non-human subject.
The following examples are given for the purpose of illustration only and not by way of limitation on the scope of the invention.
EXAMPLES
Example 1 : Copper enhances ATP release
This example shows that ATP secretion, which is a marker of immunogenic cell death, is enhanced in vitro when cells are treated with copper, but not zinc. The cells were treated with various combinations of copper, zinc and metal-binding anti cancer agents of interest. The results are shown in Figure 1 A, I B and 1 C.
CT26 murine colon cancer cells were seeded at 200,000 cells/well in 24-well plates. After 24 hours, the cells were treated with copper or zinc alone and in combination with the indicated agents, which included:
(i) approved chemotherapeutics including cytarabine (ARA-C),
daunorubicin (DNR), gemcitabine (GEM) (Figure 1 A);
(ii) CDK inhibitors including abemaciclib (ABE), palbociclib (PAL) and ribociclib (RIB) (Figure 1 A);
(iii) terpyridine derivatives, including 2,2';6',2"-terpyridine (TER), 4’-(4- chlorophenyl)-2, 2':6, 2"-terpyridine (CPT), 4,4\4"-tri-tert-butyl- 2,2':6?,2"-terpyridine (TTBT) (Figure I B);
(iv) Cu-dependent cytotoxics, including 8-hydroxyquinoline (8HQ), clioquinol (CQ), diethyldithiocarbamate (DDC) and pyrithione (PYR) (Figure 1 B); and
(v) other chemical compounds including emodin (EMD), epigallocathecin gal late (EPGG), physcion (PHY) and PX478 (Figure 1C).
Where copper or zinc complexes were tested, the metal ion was added to the ligand (agent) at the indicated molar ratio such that metal complexes between the copper or zinc and the anti-cancer agent were formed prior to addition to the cells. (However, it should be understood that the invention is not limited to the formation of any metal-agent complexes, as discussed below). The cells were exposed to the tested agents for 24 hours, after which the supernatant of each well was transferred to a new plate. The Promega CellTiter-Glo® 2.0 Kit was then used immediately, as per the manufacturer's instructions, to determine the extracellular ATP level under each treatment condition. The assay for measuring extracellular ATP involves measuring luminescence as described previously.
The results of ATP secretion from CT26 cells for each anti-cancer agent in the presence and absence of copper or zinc are shown in Figures 1 A- 1 C. Copper and zinc were mixed with the anti-cancer agent at the indicated molar ratio (ligandunetal ion). The data in Figures 1 A to 1 C demonstrate that treatment of the cells with the anti cancer agents yields increased ATP release when the agents were used in combination with Cu2+, but not with Zn2+. In all cases, the increase in extracellular ATP of the combination of the agent and Cu2+ was increased by a statistically significant amount relative to treatment of cells with agent alone. It is also notable that enhancements in ATP release in the presence of copper were still observed for agents that do not bind copper (GEM and ARA-C), suggesting that copper complexation may not be required for induction of ICD markers in vitro.
Example 2: Conner enhances at least two ICD markers including ATP and HMGBi release
The ability of copper to enhance two markers of ICD, namely ATP secretion and HMGBi release, was also examined.
Copper or zinc were added to cells, either alone or together with 8- hydroxyquinoline (8HQ), clioquinol (CQ), diethyldithiocarbamate (DDC) and 4,4',4”- tri-tert-butyl-2,2':6\2”-terpyridine (TTBT) at the molar concentrations indicated, followed by measurement of extracellular ATP levels and HMGBi release.
ATP secretion was tested following the procedure set forth in Example 1. For the measurement of HMGBi release, similar to the assay for ATP release, CT26 murine colon cancer cells were seeded at an appropriate density in 24-well plates. At 24 hours, cells are treated with the agents of interest. Cells were exposed to the copper and/or tested agents for 24 hours, after which the supernatant of each well was collected and the amount of HMGBi release was assessed using a commercial ELISA kit purchased from IBL International™. The test quantifies HMGBi by an anti- HMGBi antibody that is immobilized. HMGBi binds to the antibody and a second enzyme marked antibody recognizes the immobilized antibody. A substrate reaction catalyzed by the enzyme leads to a change in colour intensity, which is quantified by known methodology as described previously.
Copper and zinc were added to cells, together with 8-hydroxyquinoline (8HQ), clioquinol (CQ), diethyldithiocarbamate (DDC) or 4,4’,4”-tri-tert-butyl-2,2’:6’,2”- terpyridine (TTBT) at the molar concentrations indicated, followed by measurement of the HMGBi .
As shown in Figure 2, combinations with copper and the anti-cancer agents examined showed enhancements in ATP release relative to untreated cells and the agent alone, while zinc did not display such enhancements (top panel). As further shown in Figure 2. copper in combination with the anti-cancer agents examined showed enhancements in HMGBi release relative to untreated cells and the agent alone (bottom panel). Similar to the ATP studies, the addition of zinc did not result in any enhancements in HMGBi release.
Thus, the data shown in Figure 2 reveals that copper can enhance both ATP and HMGB i release in combination with an anti-cancer agent and that the effects observed are greater than those observed with the anti-cancer agent alone.
Example 3: Conner enhances each of the three markers of ICD
The ability of copper to enhance all three markers of ICD, as measured by ATP secretion, HMGB, release and CRT exposure was also examined.
Copper and 4, 4',4"-Tri-tert-Butyl-2,2':6\2”-terpyridine (TTBT) alone or in combination were added to cells at concentrations of 25 mM.
ATP secretion and HMGB] release were determined in vitro as described previously.
To quantify CRT exposed on the cell membranes, a 96-well black-walled, clear bottom plate was coated with 50 pL/well of 25 pg/mL poly-D-lysine in water for 1 h at room temperature to facilitate cell adherence. The coating solution was aspirated and 20,000 CT26 cells/well were seeded at 100 pL/well. Cells were grown for 24 h and thereafter 100 pL of anti-cancer agents or medium was added to each well. The cells were exposed to the drugs for 24 h, at which time the cells were washed 3x with 100 pL/well of HBSS with calcium and magnesium without phenol red and fixed with 50 pL/well of 4% methanol-free formaldehyde diluted in phosphate-buffered saline (PBS) for 20 min at room temperature. Methanol-free formaldehyde was used to minimize permeabilization of cell membranes during fixation. Cells were washed 3x with HBSS and then 50 pL/well of primary antibody against CRT (diluted 1 :200 in staining buffer) was added for 1 h on ice. Cells were washed 3x with HBSS and stained with 50 pL/well of secondary antibody conjugated to DyLighf1* 488 (diluted 1 :500 in staining buffer) for 30 min on ice and in the dark. The plate was washed 3x with HBSS and 50 pL/well of Hoechst 33342 (diluted 1 :2000 in PBS) and CellMask™ Deep Red (diluted 1 : 1000) was added for 20 min at room temperature to stain the cell nuclei. Cells were finally washed 3x with HBSS and imaged in 100 pL/well of PBS with the IN Cell Analyzer 2200. Twenty images were acquired for each well. The flat field correction was applied during the image acquisition. Mean fluorescence intensity of CRT on the cell membranes was quantified for each individual cell by conducting a multi-target analysis using the IN Cell 2200 Workstation 3.7 software (GE Healthcare). The average of mean fluorescence intensities for all cells under the same condition was calculated and normalized to untreated cells in order to express the results as fold-increase in CRT exposure.
Primary antibody against CRT (product number PA3-900), secondary antibody conjugated to DyLight® 488 (product number 35552), Hoechst 33342 and
CellMask™ Deep Red were obtained from Thermo Fisher Scientific, Waltham, MA, USA.
The results in Figure 3 show that copper in combination with the anti-cancer agent, TTBT, increased each of the three molecular markers associated with 1CD.
Example 4: Determination of HMGB release and CRT exposure with and without copper-agent complexation
The ability of anti-cancer agents to increase HMGET release or CRT exposure with and without complex formation with copper was tested. The agents examined that complex with copper include daunorubicin (DNR). PX478 and salubrinal (SAL) and those that are not capable of forming complexes include CDDP, gemcitabine (GEM) and cytarabine (ARA-C). FlMGBi release and CRT exposure was determined according to the above-described HMGB and CRT assays in CT26 cells.
FlMGBi release in the presence of copper, as shown in Figures 4A and 4B, was comparable for the agents that complex with copper (Figure 4A; DNR, PX478 and SAL) and those that do not (Figure 4B; CDDP, GEM and ARA-C).
Likewise, as shown in Figures 5A and 5B, the addition of copper to the anti cancer agents tested had no significant impact on CRT exposure levels observed, regardless of whether a copper binding agent (Figure 5A; DNR, PX478 and SAL) or a non-binding agent (Figure 5B; CDDP, GEM and ARA-C) was used.
These results suggest that complexation of an agent with copper may not be required for 1CD induction as evidenced by assays that respectively measure HMGB; release and CRT exposure.
Example 5: Enhancement of ICD as measured by at least ATP secretion is copper specific
As suggested by the data above, copper enhances ICD in vitro , as measured by the release of ATP and HMBGi while zinc does not. To determine whether the ICD effect is copper-specific, other metals besides zinc were tested to determine whether or not they could induce ICD as measured by ATP secretion. As shown below, the ICD effect as determined by ATP release suggests that ICD is induced by only copper and not by other metals.
Extracellular ATP release was measured as set forth above with various anti cancer agents in combination with copper, zinc, iron, manganese, cobalt and vanadium at the indicated molar concentrations. The anti-cancer agents examined included daunorubicin (DNR; Fig. 6A and 6B), gemcitabine (GEM; Fig. 6C and 6D), 4’-(4-chIorophenyl)-2, 2':6, 2"-terpyridine (CPT; Fig. 6E and 6F), 4,4’ ,4”-tr i-tert- butyl-2,2’:6’,2”-terpyridine (TTBT; Fig. 6G and 6H) and abemaciclib (ABE; Fig. 61 and 6J). The results in Figures 6A-J show that the induction of ATP in vitro occurs only with copper and an anti-cancer agent, and not with the agent combinations including other metals, namely zinc, iron, manganese, cobalt and vanadium.
Figure 7 shows similar results with various copper salts (copper acetate, copper chloride, copper gluconate and copper sulfate), as well as cobalt, iron, manganese, nickel and zinc. The anti-cancer agents examined alone and in combination with each metal and metal salt included TTBT and DDC. Each of the combinations with the copper salts tested exhibited enhanced extracellular ATP release, while the other metals tested did not exhibit such effects (Fig. 7A and 7B).
The ability of copper and various other metals to induce reactive oxygen species (ROS) was next investigated since it is known that ROS are generated at high levels by ICD-inducing chemotherapeutic agents.
To measure ROS generation by CT26 cells, 10,000 cells/well were seeded on a 96-well white-walled, clear bottom plate (Greiner Bio-One catalog number 655098) in 80 pL/well of medium. Cells were allowed to attach and grow for 24 h. Thereafter, 20 pL/well were replaced with 20 mT/well of medium supplemented with drugs or medium (untreated controls). Combinations of drugs and metals were first pre-mixed to allow complexation of the compounds for those that are able to bind copper or zinc as determined by their chemical structure. Subsequently, the combinations of drugs and metals were diluted to the appropriate concentrations in medium. Combinations of drugs and metals were prepared in the same way (pre-mixing in water and further dilution in medium) for the cytotoxicity, ATP, HMGB 1 and CRT assays described above. To detect ROS, cells were incubated with the compounds for 4 h. The amount of ROS generated was measured by the ROS-Glo™ FEO2 assay (Promega, Madison, WI, USA). Two hours before the end of the experiment, 10 pL/well of H2O2 substrate solution was added directly to each well. At the end of the experiment, 50 mT/well of ROS-Glo™ detection solution was applied to all wells, and the plate was incubated
for 20 min at room temperature. The luminescence signal was measured in arbitrary units using the FLUOstar OPTIMA microplate reader (BMG Labtech, Ortenberg, Germany).
Figure 8 shows the results of the ROS studies using various metals. The results are shown as a fold-increase relative to an untreated control. As shown, the copper salts examined exhibited increases in ROS, while most of the other metals tested, including zinc, iron, manganese, and vanadium, did not exhibit such effects. Menadione (MEN) was utilized as a positive control.
Figure 9A shows fold-increase of ROS as a function of concentration (0.25- 250 mM) of copper or zinc addition to the cells. The magnitude of the fold increase of ROS increased with increasing concentrations of copper from 0 to 125 mM (left graph). However, these effects were not observed with zinc (right graph). ROS were reported as fold-increase relative to an untreated control.
Figures 9B and 9C show the results of ROS studies using copper or zinc in combination with daunorubicin (DNR), PX478, salubrinal (SAL), cisplatin (CDDP), gemcitabine (GEM) and cytarabine (ARA-C). Copper in combination with DNR, PX478, SAL, CDDP and ARA-C displayed increased ROS relative to the agent alone or the agent in combination with zinc (as measured by a fold increase in ROS relative to an untreated control).
Example 6: In vivo vaccination studies with combinations of copper and anti cancer agents
An in vivo study was conducted to investigate if the vaccination of immune competent animals with cells pretreated with copper and selected anti-cancer agents would elicit an anti-tumour immunity by preventing the development of tumours when the same animals were challenged with untreated cancer cells seven days later. Mitoxantrone (MITO), an inducer of 1CD, was selected as a positive control and cisplatin (CDDP) was used as a negative control. The cytotoxicity pattern of MITO and the presence of ICD markers (ATP, FIMGBi and CRT) on treated CT26 cells were confirmed in vitro (not shown).
Immune competent Balb/c mice (6-8 weeks old) were “vaccinated" by inoculating the left flank subcutaneously (s.c.) with 3 x 106 CT26 cells pretreated in vitro. Briefly 2 x 107 CT26 cells were seeded in each 175 cm2 culture flask. At 24
hours later, the cells were treated with 1 mM MITO, 25 mM CDDP, 25 mM copper sulfate (C11SO4) or combinations of the cytotoxic agent with CuS04 at the same doses. The copper concentration was selected based on studies demonstrating that a significant increase in intracellular copper levels could be achieved when cells were exposed to 25 mM CuS04 (not shown). The cells were washed with HBSS and harvested at 24 h post-treatment on Day 0 using enzyme-free cell dissociation buffer (Thermo Fisher Scientific). The cells were counted using a Cellometer Auto T4 (Nexcelom Bioscience, Lawrence, MA, USA) and re-suspended in PBS for inoculation (50 pL per mouse; n = 10 per treatment group). One week later (day 7), all mice were challenged with untreated CT26 cells by inoculating 5 x 105 cells/50 pL s.c. into the right flank. Both flanks were monitored for palpable tumours. Tumour growth was monitored and measured with a calliper. Tumour volumes were calculated using the equation L X W2 /2 with the length (mm) being the longer axis of the tumour. Mice were considered tumour-free until the first day that tumours were defined as measurable (64 mm ) using calipers. Tumours were allowed to grow to a maximum of 1000 innT before termination. Control studies were completed to ensure that the tumour take rate of untreated CT26 cells was 100% in order to establish the validity of the vaccination assay.
The results show' that mice that were not vaccinated developed tumours by day 12 following inoculation of untreated cells (Figure 10A). Vaccination w'ith cells pretreated w ith MITO resulted in 58% of the mice being tumour-free on Day 39 post cancer challenge (Figure 10B). However, the proportion of tumour-free mice increased to 70% when mice were vaccinated with cells pretreated with MITO + copper. Vaccination with CDDP or CDDP + copper pretreated cells resulted in 10% and 0% tumour-free mice, respectively.
These data provide support that the addition of copper can enhance the anti cancer immune response of certain agents in vivo and that this augmentation effect can be sustained over time.
Example 7: Combinations of anti-cancer agents with copper exhibit increased ATP secretion
This example examines the effect of anti-cancer agents alone or in
combination with copper, further in combination with existing anti-cancer agents
(clinically proven) to enhance ATP secretion, a marker of immunogenic cell death. The results are shown in Figure 1 I .
CT26 cells were treated with an anti-cancer agent of interest alone, in combination with copper, or further in combination with copper and certain clinically approved anti-cancer agents. The level of ATP secretion was determined by analyzing the supernatant at 24 hours following treatment. The first anti-cancer agents tested have copper-binding ligands and included PX-478 (25 mM), flavopiridol (FLV; I mM), clioquinol (CQ; 25mM), and analogues of terpyridine 4,4'4”-Tri-ter- Butyl-2,2':6‘,2"-terpyridine (TTT; 25 mM) and 4'-(4-chlorophenyl)-2,2':6',2”- terpyridine (CPT; 25 mM). The clinically approved anti-cancer agents include the following drugs: mitoxantrone (Mito; 1 mM), gemcitabine (Gem; 0.25 mM), and cisplatin (CDDP; 25 mM).
As shown in Figures 1 1 A-F, the combination of the first anti-cancer agent (PX-478, TTT, FLV, CQ and CPT), copper and the clinically approved agent (Mito, Gem and CDDP) yielded greater ATP release than the approved anti-cancer alone.
The use of the copper plus the first anti-cancer agent, instead of the first anti-cancer agent alone, in some cases, leads to additional enhancement in ATP release.
Example 8: HMGB release of anti-cancer agent combinations with copper
The effect of combining copper-binding agents with existing anti-cancer agents, together with copper, to increase HMGB secretion from cells was next examined and the results are presented in Figure 12.
CT26 murine colon cancer cells were seeded at 200,000 cells/well in 24-well plates. After 24 hours, cells were treated with the indicated anti-cancer agents. The release of FlMGBi was assessed for the combination of PX-478 or Cu(PX-478) and gemcitabine (Gem) and the combination of flavopiridol (FLV) or Cu(FLV) with mitoxantrone (Mito). Where copper or zinc complexes were tested, the metal ion was added to the ligand at the indicated molar ratio such that the metal complexes were formed prior to addition to the cells. Cells were exposed to the tested agents for 24 hours, after which the supernatant of each well was collected and the amount of HMGBi release was assessed using a commercial ELISA kit purchased from IBL International™.
Figure 12B shows that the use of flavopidirol (FLV), either in free form or as a copper complex, leads to greater HMGBi secretion compared to mitoxantrone (Mito) alone. As seen in Figure I 2A, combining PX-478 or Cu(PX-478) with gemcitabine (Gem) led to a modest to comparable increase in HMGB, release relative to gemcitabine alone.
Example 9: Combinations of copper and two anti-cancer agents can induce at least two markers of ICD
The ability of a combination of anti-cancer agents with copper to induce all three markers of ICD was also examined. The anti-cancer agents selected for the study included PX478 and GEM. The ICD markers, including release of ATP and HMGBi and CRT exposure were examined. The ICD marker assays were carried out as described previously and the anti-cancer agents and copper were added at the concentrations indicated in Figure 13.
Combinations of copper, PX478 and GEM showed enhanced levels of ATP and HMGB 1 release relative to the agents or copper measured alone and a combination of the two agents (Figure 13A and 13B). For CRT exposure, measured as a fold increase in CRT exposure, the results show that the response for a combination of copper. PX478 and GEM was comparable to that attained with the two anti-cancer agents measured in combination or GEM alone (Figure 13C).
The foregoing description should not be construed as limiting and includes embodiments and equivalents thereof that would be known to those of ordinary skill in the art. A number of embodiments of the invention have been described.
Nevertheless, it can be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.
Claims
1 . An iinmunostimulatory pharmaceutical composition to treat, ameliorate or prevent a disease by inducing immunogenic cell death (ICD) comprising: copper and at least one agent; and
wherein a combination of the copper and the agent provide an increase in the ICD observed, compared to the agent alone, as measured by at least one of extracellular adenosine triphosphate (ATP), HMGB release, and CRT exposure of cells in vitro and wherein the increase in the ICD response of the combination is between about 1.2 to 10,000 times that of the agent alone measured under otherwise identical conditions.
2. The pharmaceutical composition of claim 1 , wherein the agent is capable of complexing with the copper to form a metal complex.
3. The pharmaceutical composition of claim 2, wherein the increase in ICD of the copper and the agent in combination, compared to the agent alone, is measured by extracellular adenosine triphosphate (ATP).
4. The pharmaceutical composition of claim 1 , wherein the copper, the agent, or both, are formulated in a vesicle, micelle, polymer-based nanoparticle, emulsion or nanotube.
5. The pharmaceutical composition of claim 1 , wherein the metal, the agent, or both, are formulated in the vesicle, and wherein vesicle is a liposome or exosome.
6. The pharmaceutical composition of claim 5, wherein vesicle is a liposome.
7. The pharmaceutical composition of claim 6, wherein the copper is
incorporated in the same or a different liposome in the pharmaceutical composition as the agent and wherein the liposome is pre-formed.
8. The pharmaceutical composition of any one of claims 1 to 7, further
comprising at least a second agent.
9. The pharmaceutical composition of any one of claims 1 to 4, wherein the pharmaceutical composition provides a cytotoxic effect, or wherein at least one agent and/or copper is cytotoxic in combination with radiation as measured by a cytotoxic assay.
10. The pharmaceutical composition of any one of claims 1 to 9, wherein the composition is a formulation effective for intra-tumoural injection.
1 1 . The pharmaceutical composition of any one of claims 1 to 10, wherein the composition is a vaccine.
12. The pharmaceutical composition of claim 2, wherein the metal ion complexes with the agent.
13. The pharmaceutical composition of any one of claims 1 to 12, wherein the increase in the ICD is due to the copper.
14. Use of copper and at least one agent to induce immunogenic cell death (ICD), or for use in treating, ameliorating or preventing a disease, a cancer, an infection, or a condition, in combination or sequentially, to an individual or patient in need thereof, wherein a combination of the copper and the agent provide an increase in ICD, compared to the agent alone, as determined by at least one assay that determines in vitro ATP secretion, H GBi release, and CRT exposure.
15. The use of claim 14, wherein the copper, the agent, or both, are formulated in a vesicle, micelle, polymer-based nanoparticle, emulsion, or nanotube.
16. The use of claim 14, wherein the metal, the agent, or both, are formulated in a vesicle.
1 7. The use of claim 16, wherein the vesicle is a liposome or an exosome.
18. The use of any one of claims 14 to 17, wherein the ICD is induced as
measured in vitro with a further, second agent and optionally a third agent.
19. The use of any one of claims 14 to 18, wherein the copper and the at least one agent are capable of inducing ICD with radiation.
20. The use of any one of claims 14 to 19, wherein the increase in the ICD in the presence of copper is measured by ATP secretion.
21 . The use of any one of claims 14 to 20, wherein the metal and/or the agent is part of a formulation effective to treat a subject via intra-tumoural injection.
22. The use of any one of claims 14 to 21 , wherein the metal and/or the agent is part of a vaccine.
23. The use of any one of claims 14 to 21 , wherein the copper in an uncomplexed form is capable of inducing all or a portion of the ICD observed in vitro in the presence or absence of the agent.
24. The use of any one of claims 14 to 23, wherein the metal complexes with the agent.
25. A method of inducing immunogenic cell death (ICD), or for treating, ameliorating or preventing a disease, a cancer, an infection, or a condition in an individual in need thereof, comprising administering copper and at least one agent, in combination or sequentially, to a patient or individual in need thereof, wherein a combination of the copper and the agent provide an increase in ICD, compared to the agent alone, as determined by an in vitro assay that measures at least one of ATP secretion, HMGBi release, and CRT exposure.
26. The method of claim 25, wherein the metal, the agent, or both, are formulated in a liposome, micelle, polymer-based nanoparticle, emulsion, endosome or nanotube.
27. The method of claim 26, wherein the copper, the agent, or both, are formulated in a liposome.
28. The method of claim 25, 26 or 27, further comprising a step of radiation
treatment.
29. The method of any one of claims 25 to 28, wherein the ICD is measured by ATP secretion.
30. A method of producing an immunostimulatory pharmaceutical composition for inducing immunogenic cell death (ICD), or for use in treating, ameliorating or preventing a disease, a cancer, an infection, or a condition, comprising the steps of:
(i) exposing an agent and copper, alone and in combination, to an assay that measures at least one of ATP secretion, HMGBi release, and CRT exposure in cell culture;
(ii) determining whether the combination exhibits an increase in an ICD response relative to the agent measured alone; and
(iii) wherein, if the combination of the copper and the agent provide an increase in ICD, compared to the agent alone, formulating the agent, the copper, or both in one or more pharmaceutical formulations to treat or to prevent cancer, either sequentially or in combination, to a patient in need thereof.
3 I . The method of claim 30, wherein the pharmaceutical formulation comprises copper.
32. A kit comprising copper and an agent, to induce immunogenic cell death (ICD), or for use in treating, ameliorating or preventing a disease, a cancer, an infection, or a condition, wherein a combination of the copper and the agent provide an increase in ICD, compared to the agent alone, as determined by measuring: ATP secretion, HMGBi release, CRT exposure or a combination thereof.
33. The kit of claim 32, further comprising instructions for formulation of the copper, the agent, or both, in a delivery vehicle.
34. The kit of claim 32, further comprising instructions for use thereof to induce immunogenic cell death to a patient in need thereof.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201862688705P | 2018-06-22 | 2018-06-22 | |
US62/688,705 | 2018-06-22 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2019241873A1 true WO2019241873A1 (en) | 2019-12-26 |
Family
ID=68982593
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CA2019/000094 WO2019241873A1 (en) | 2018-06-22 | 2019-06-24 | Metal containing products and compositions to induce immunogenic cell death and uses thereof |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2019241873A1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111658611A (en) * | 2020-04-13 | 2020-09-15 | 四川大学 | Vaccine delivery system based on microemulsion, and preparation method and application thereof |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5632982A (en) * | 1994-06-07 | 1997-05-27 | The Board Of Trustees Of The Leland Stanford Junior University | Cytotoxic enhancement of TNF with copper |
US6548540B2 (en) * | 1998-09-08 | 2003-04-15 | Charlotte-Mecklenburg Hospital Authority | Method of treating cancer using dithiocarbamate derivatives |
-
2019
- 2019-06-24 WO PCT/CA2019/000094 patent/WO2019241873A1/en active Application Filing
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5632982A (en) * | 1994-06-07 | 1997-05-27 | The Board Of Trustees Of The Leland Stanford Junior University | Cytotoxic enhancement of TNF with copper |
US6548540B2 (en) * | 1998-09-08 | 2003-04-15 | Charlotte-Mecklenburg Hospital Authority | Method of treating cancer using dithiocarbamate derivatives |
Non-Patent Citations (5)
Title |
---|
CHAKRABORTY ET AL.: "A copper chelate selectively triggers apoptosis in myeloid-derived suppressor cells in a drug-resistant tumor model and enhances antitumor immune response", IMMUNOPHARMACOL. IMMUNOTOXICOL., vol. 36, no. 2, 10 March 2014 (2014-03-10), pages 165 - 175 * |
MOOKERJEE ET AL.: "A novel copper complex induces ROS generation in doxorubicin resistant Ehrlich ascitis carcinoma cells and increases activity of antioxidant enzymes in vital organs in vivo", BMC CANCER, vol. 6, 15 November 2006 (2006-11-15), pages 267 - 277, XP021023053 * |
SHOWWALTER ET AL., CYTOKINES IN IMMUNOGENIC CELL DEATH: APPLICATIONS FOR CANCER IMMUNOTHERAPY, vol. 97, 22 June 2017 (2017-06-22), pages 123 - 132, XP085123395 * |
TERENZI ET AL.: "Anticancer metal drugs and immunogenic cell death", J INORG BIOCHEM, vol. 165, 16 June 2016 (2016-06-16), pages 71 - 79, XP029834029, DOI: 10.1016/j.jinorgbio.2016.06.021 * |
YOU ET AL.: "Process of immunogenic cell death caused by disulfiram as the anti-colorectal cancer candidate", BIOCHEM BIOPHYS RES COMMUN., vol. 513, no. 4, 16 April 2019 (2019-04-16), pages 891 - 897, XP085686146, DOI: 10.1016/j.bbrc.2019.03.192 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111658611A (en) * | 2020-04-13 | 2020-09-15 | 四川大学 | Vaccine delivery system based on microemulsion, and preparation method and application thereof |
WO2021208860A1 (en) * | 2020-04-13 | 2021-10-21 | 四川大学 | Microemulsion-based vaccine delivery system, preparation method therefor and use thereof |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Xu et al. | Rationally designed rapamycin-encapsulated ZIF-8 nanosystem for overcoming chemotherapy resistance | |
Fan et al. | Intranuclear biophotonics by smart design of nuclear-targeting photo-/radio-sensitizers co-loaded upconversion nanoparticles | |
DK2670394T3 (en) | NANOPARTICLE DELIVERY SYSTEMS, PREPARATION AND APPLICATIONS THEREOF | |
Martina et al. | The effect of magnetic targeting on the uptake of magnetic-fluid-loaded liposomes by human prostatic adenocarcinoma cells | |
US20200009050A1 (en) | Pharmaceutical composition, preparation and uses thereof | |
Xiao et al. | Cancer cell membrane-camouflaged MOF nanoparticles for a potent dihydroartemisinin-based hepatocellular carcinoma therapy | |
AU2016369977A1 (en) | Metal complexed therapeutic agents and lipid-based nanoparticulate formulations thereof | |
WO2012040513A1 (en) | Compositions and methods for the delivery of beta lapachone | |
Kepinska et al. | Fullerene as a doxorubicin nanotransporter for targeted breast cancer therapy: Capillary electrophoresis analysis | |
Mondon et al. | MPEG‐hexPLA micelles as novel carriers for hypericin, a fluorescent marker for use in cancer diagnostics | |
Li et al. | Stepwise targeting and responsive lipid-coated nanoparticles for enhanced tumor cell sensitivity and hepatocellular carcinoma therapy | |
Karpuz et al. | Design and in vitro evaluation of folate-targeted, co-drug encapsulated theranostic liposomes for non-small cell lung cancer | |
Pizzuti et al. | Bilirubin-coated radioluminescent particles for radiation-induced photodynamic therapy | |
Nguyen et al. | Amplified fenton-based oxidative stress utilizing ultraviolet upconversion luminescence-fueled nanoreactors for apoptosis-strengthened ferroptosis anticancer therapy | |
Gao et al. | Multifunctional nanoparticle for cancer therapy | |
Palanikumar et al. | pH-responsive upconversion mesoporous silica nanospheres for combined multimodal diagnostic imaging and targeted photodynamic and photothermal cancer therapy | |
Anwar et al. | Importance of theranostics in rare brain-eating amoebae infections | |
US20140105829A1 (en) | Therapeutic nanoemulsion formulation for the targeted delivery of docetaxel and methods of making and using the same | |
WO2019241873A1 (en) | Metal containing products and compositions to induce immunogenic cell death and uses thereof | |
Li et al. | Multi-Enzyme Cascade-Triggered Nitric Oxide Release Nanoplatform Combined with Chemo Starvation-like Therapy for Multidrug-Resistant Cancers | |
Faghihi et al. | Prospects and challenges of synergistic effect of fluorescent carbon dots, liposomes and nanoliposomes for theragnostic applications | |
Xu et al. | Novel fabrication of marizomib-loaded chitosan-coated hydroxyapatite nanocarriers as a promising system for effective treatment of ovarian cancer | |
US20220008451A1 (en) | Flavin adenine dinucleotide (fad) for use in the prevention and/or treatment of cancer | |
JP2022545786A (en) | Nanocomposite particles and uses thereof | |
Nagdiya et al. | Drug delivery systems of gefitinib for improved cancer therapy: A review |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 19823706 Country of ref document: EP Kind code of ref document: A1 |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 19823706 Country of ref document: EP Kind code of ref document: A1 |