WO2023002509A1 - Lipid formulation for delivery of therapeutic agents - Google Patents
Lipid formulation for delivery of therapeutic agents Download PDFInfo
- Publication number
- WO2023002509A1 WO2023002509A1 PCT/IN2022/050660 IN2022050660W WO2023002509A1 WO 2023002509 A1 WO2023002509 A1 WO 2023002509A1 IN 2022050660 W IN2022050660 W IN 2022050660W WO 2023002509 A1 WO2023002509 A1 WO 2023002509A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- compound
- lipid
- formula
- formulation
- alkyl
- Prior art date
Links
- 239000000203 mixture Substances 0.000 title claims abstract description 141
- 238000009472 formulation Methods 0.000 title claims abstract description 128
- 150000002632 lipids Chemical class 0.000 title claims abstract description 126
- 239000003814 drug Substances 0.000 title claims description 67
- 229940124597 therapeutic agent Drugs 0.000 title claims description 47
- -1 lipid compound Chemical class 0.000 claims abstract description 163
- 238000000034 method Methods 0.000 claims abstract description 57
- LMBFAGIMSUYTBN-MPZNNTNKSA-N teixobactin Chemical compound C([C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(=O)N[C@H](CCC(N)=O)C(=O)N[C@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(=O)N[C@H]1C(N[C@@H](C)C(=O)N[C@@H](C[C@@H]2NC(=N)NC2)C(=O)N[C@H](C(=O)O[C@H]1C)[C@@H](C)CC)=O)NC)C1=CC=CC=C1 LMBFAGIMSUYTBN-MPZNNTNKSA-N 0.000 claims abstract description 38
- 230000008569 process Effects 0.000 claims abstract description 16
- 150000001875 compounds Chemical class 0.000 claims description 138
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 99
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 52
- 125000000217 alkyl group Chemical group 0.000 claims description 48
- 125000003118 aryl group Chemical group 0.000 claims description 43
- 125000000954 2-hydroxyethyl group Chemical group [H]C([*])([H])C([H])([H])O[H] 0.000 claims description 42
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 41
- 125000000008 (C1-C10) alkyl group Chemical group 0.000 claims description 33
- 229910052739 hydrogen Inorganic materials 0.000 claims description 33
- 239000001257 hydrogen Substances 0.000 claims description 33
- 125000004435 hydrogen atom Chemical class [H]* 0.000 claims description 33
- 108020004999 messenger RNA Proteins 0.000 claims description 32
- 239000008194 pharmaceutical composition Substances 0.000 claims description 32
- 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 claims description 31
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 30
- 108020004707 nucleic acids Proteins 0.000 claims description 30
- 102000039446 nucleic acids Human genes 0.000 claims description 30
- 150000007523 nucleic acids Chemical group 0.000 claims description 30
- 125000001424 substituent group Chemical group 0.000 claims description 30
- 125000000623 heterocyclic group Chemical group 0.000 claims description 28
- 125000004452 carbocyclyl group Chemical group 0.000 claims description 25
- 210000004027 cell Anatomy 0.000 claims description 23
- 229940127089 cytotoxic agent Drugs 0.000 claims description 21
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 20
- 125000004356 hydroxy functional group Chemical group O* 0.000 claims description 20
- 150000003839 salts Chemical class 0.000 claims description 20
- 125000003342 alkenyl group Chemical group 0.000 claims description 18
- 239000003795 chemical substances by application Substances 0.000 claims description 18
- 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 claims description 18
- 208000035475 disorder Diseases 0.000 claims description 18
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 17
- 125000005189 alkyl hydroxy group Chemical group 0.000 claims description 17
- 125000000304 alkynyl group Chemical group 0.000 claims description 17
- 239000003153 chemical reaction reagent Substances 0.000 claims description 17
- 229940079593 drug Drugs 0.000 claims description 17
- 239000008103 glucose Substances 0.000 claims description 17
- 238000004519 manufacturing process Methods 0.000 claims description 17
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 16
- 108090000623 proteins and genes Proteins 0.000 claims description 16
- 125000000027 (C1-C10) alkoxy group Chemical group 0.000 claims description 15
- 125000006376 (C3-C10) cycloalkyl group Chemical group 0.000 claims description 15
- 125000006374 C2-C10 alkenyl group Chemical group 0.000 claims description 15
- 231100000599 cytotoxic agent Toxicity 0.000 claims description 15
- 239000002904 solvent Substances 0.000 claims description 15
- 230000008685 targeting Effects 0.000 claims description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- MWRBNPKJOOWZPW-CLFAGFIQSA-N dioleoyl phosphatidylethanolamine Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC(COP(O)(=O)OCCN)OC(=O)CCCCCCC\C=C/CCCCCCCC MWRBNPKJOOWZPW-CLFAGFIQSA-N 0.000 claims description 13
- 102000004169 proteins and genes Human genes 0.000 claims description 13
- 150000003431 steroids Chemical class 0.000 claims description 13
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 12
- 239000002254 cytotoxic agent Substances 0.000 claims description 12
- 201000010099 disease Diseases 0.000 claims description 12
- 210000004185 liver Anatomy 0.000 claims description 12
- 108020004459 Small interfering RNA Proteins 0.000 claims description 11
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 10
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 claims description 10
- 108020004414 DNA Proteins 0.000 claims description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- 239000011780 sodium chloride Substances 0.000 claims description 10
- 239000012453 solvate Substances 0.000 claims description 10
- 102000005427 Asialoglycoprotein Receptor Human genes 0.000 claims description 9
- 101710112752 Cytotoxin Proteins 0.000 claims description 9
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 9
- 229930006000 Sucrose Natural products 0.000 claims description 9
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- 108010006523 asialoglycoprotein receptor Proteins 0.000 claims description 9
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 9
- LGJMUZUPVCAVPU-UHFFFAOYSA-N beta-Sitostanol Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(C)CCC(CC)C(C)C)C1(C)CC2 LGJMUZUPVCAVPU-UHFFFAOYSA-N 0.000 claims description 9
- 239000002619 cytotoxin Substances 0.000 claims description 9
- ZZUFCTLCJUWOSV-UHFFFAOYSA-N furosemide Chemical compound C1=C(Cl)C(S(=O)(=O)N)=CC(C(O)=O)=C1NCC1=CC=CO1 ZZUFCTLCJUWOSV-UHFFFAOYSA-N 0.000 claims description 9
- 239000002955 immunomodulating agent Substances 0.000 claims description 9
- JYJIGFIDKWBXDU-MNNPPOADSA-N inulin Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)OC[C@]1(OC[C@]2(OC[C@]3(OC[C@]4(OC[C@]5(OC[C@]6(OC[C@]7(OC[C@]8(OC[C@]9(OC[C@]%10(OC[C@]%11(OC[C@]%12(OC[C@]%13(OC[C@]%14(OC[C@]%15(OC[C@]%16(OC[C@]%17(OC[C@]%18(OC[C@]%19(OC[C@]%20(OC[C@]%21(OC[C@]%22(OC[C@]%23(OC[C@]%24(OC[C@]%25(OC[C@]%26(OC[C@]%27(OC[C@]%28(OC[C@]%29(OC[C@]%30(OC[C@]%31(OC[C@]%32(OC[C@]%33(OC[C@]%34(OC[C@]%35(OC[C@]%36(O[C@@H]%37[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O%37)O)[C@H]([C@H](O)[C@@H](CO)O%36)O)[C@H]([C@H](O)[C@@H](CO)O%35)O)[C@H]([C@H](O)[C@@H](CO)O%34)O)[C@H]([C@H](O)[C@@H](CO)O%33)O)[C@H]([C@H](O)[C@@H](CO)O%32)O)[C@H]([C@H](O)[C@@H](CO)O%31)O)[C@H]([C@H](O)[C@@H](CO)O%30)O)[C@H]([C@H](O)[C@@H](CO)O%29)O)[C@H]([C@H](O)[C@@H](CO)O%28)O)[C@H]([C@H](O)[C@@H](CO)O%27)O)[C@H]([C@H](O)[C@@H](CO)O%26)O)[C@H]([C@H](O)[C@@H](CO)O%25)O)[C@H]([C@H](O)[C@@H](CO)O%24)O)[C@H]([C@H](O)[C@@H](CO)O%23)O)[C@H]([C@H](O)[C@@H](CO)O%22)O)[C@H]([C@H](O)[C@@H](CO)O%21)O)[C@H]([C@H](O)[C@@H](CO)O%20)O)[C@H]([C@H](O)[C@@H](CO)O%19)O)[C@H]([C@H](O)[C@@H](CO)O%18)O)[C@H]([C@H](O)[C@@H](CO)O%17)O)[C@H]([C@H](O)[C@@H](CO)O%16)O)[C@H]([C@H](O)[C@@H](CO)O%15)O)[C@H]([C@H](O)[C@@H](CO)O%14)O)[C@H]([C@H](O)[C@@H](CO)O%13)O)[C@H]([C@H](O)[C@@H](CO)O%12)O)[C@H]([C@H](O)[C@@H](CO)O%11)O)[C@H]([C@H](O)[C@@H](CO)O%10)O)[C@H]([C@H](O)[C@@H](CO)O9)O)[C@H]([C@H](O)[C@@H](CO)O8)O)[C@H]([C@H](O)[C@@H](CO)O7)O)[C@H]([C@H](O)[C@@H](CO)O6)O)[C@H]([C@H](O)[C@@H](CO)O5)O)[C@H]([C@H](O)[C@@H](CO)O4)O)[C@H]([C@H](O)[C@@H](CO)O3)O)[C@H]([C@H](O)[C@@H](CO)O2)O)[C@@H](O)[C@H](O)[C@@H](CO)O1 JYJIGFIDKWBXDU-MNNPPOADSA-N 0.000 claims description 9
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- 150000003384 small molecules Chemical class 0.000 claims description 9
- 239000005720 sucrose Substances 0.000 claims description 9
- 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 claims description 8
- IJFVSSZAOYLHEE-SSEXGKCCSA-N 1,2-dilauroyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCC IJFVSSZAOYLHEE-SSEXGKCCSA-N 0.000 claims description 8
- 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 claims description 8
- 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 claims description 8
- 229920001202 Inulin Polymers 0.000 claims description 8
- 235000012000 cholesterol Nutrition 0.000 claims description 8
- 125000004093 cyano group Chemical group *C#N 0.000 claims description 8
- RTZKZFJDLAIYFH-UHFFFAOYSA-N ether Substances CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 8
- 229940029339 inulin Drugs 0.000 claims description 8
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 8
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- 239000000243 solution Substances 0.000 claims description 7
- SLKDGVPOSSLUAI-PGUFJCEWSA-N 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine zwitterion Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(=O)OCCN)OC(=O)CCCCCCCCCCCCCCC SLKDGVPOSSLUAI-PGUFJCEWSA-N 0.000 claims description 6
- LVNGJLRDBYCPGB-UHFFFAOYSA-N 1,2-distearoylphosphatidylethanolamine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(COP([O-])(=O)OCC[NH3+])OC(=O)CCCCCCCCCCCCCCCCC LVNGJLRDBYCPGB-UHFFFAOYSA-N 0.000 claims description 6
- 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 claims description 6
- ZLGYVWRJIZPQMM-HHHXNRCGSA-N 2-azaniumylethyl [(2r)-2,3-di(dodecanoyloxy)propyl] phosphate Chemical compound CCCCCCCCCCCC(=O)OC[C@H](COP(O)(=O)OCCN)OC(=O)CCCCCCCCCCC ZLGYVWRJIZPQMM-HHHXNRCGSA-N 0.000 claims description 6
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- CZWCKYRVOZZJNM-USOAJAOKSA-N dehydroepiandrosterone sulfate Chemical compound C1[C@@H](OS(O)(=O)=O)CC[C@]2(C)[C@H]3CC[C@](C)(C(CC4)=O)[C@@H]4[C@@H]3CC=C21 CZWCKYRVOZZJNM-USOAJAOKSA-N 0.000 claims description 6
- 150000004677 hydrates Chemical class 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 231100000065 noncytotoxic Toxicity 0.000 claims description 6
- 230000002020 noncytotoxic effect Effects 0.000 claims description 6
- 239000003638 chemical reducing agent Substances 0.000 claims description 5
- 208000008439 Biliary Liver Cirrhosis Diseases 0.000 claims description 4
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- 102000000844 Cell Surface Receptors Human genes 0.000 claims description 4
- 108010001857 Cell Surface Receptors Proteins 0.000 claims description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 4
- 208000012654 Primary biliary cholangitis Diseases 0.000 claims description 4
- INQOMBQAUSQDDS-UHFFFAOYSA-N iodomethane Chemical group IC INQOMBQAUSQDDS-UHFFFAOYSA-N 0.000 claims description 4
- 235000010378 sodium ascorbate Nutrition 0.000 claims description 4
- PPASLZSBLFJQEF-RKJRWTFHSA-M sodium ascorbate Substances [Na+].OC[C@@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RKJRWTFHSA-M 0.000 claims description 4
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- JVBXVOWTABLYPX-UHFFFAOYSA-L sodium dithionite Chemical compound [Na+].[Na+].[O-]S(=O)S([O-])=O JVBXVOWTABLYPX-UHFFFAOYSA-L 0.000 claims description 4
- PPASLZSBLFJQEF-RXSVEWSESA-M sodium-L-ascorbate Chemical compound [Na+].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RXSVEWSESA-M 0.000 claims description 4
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 4
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- BQPPJGMMIYJVBR-UHFFFAOYSA-N (10S)-3c-Acetoxy-4.4.10r.13c.14t-pentamethyl-17c-((R)-1.5-dimethyl-hexen-(4)-yl)-(5tH)-Delta8-tetradecahydro-1H-cyclopenta[a]phenanthren Natural products CC12CCC(OC(C)=O)C(C)(C)C1CCC1=C2CCC2(C)C(C(CCC=C(C)C)C)CCC21C BQPPJGMMIYJVBR-UHFFFAOYSA-N 0.000 claims description 3
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- 125000004169 (C1-C6) alkyl group Chemical group 0.000 claims description 3
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- NRJAVPSFFCBXDT-UHFFFAOYSA-N 2,3-di(octadecanoyloxy)propyl 2-(trimethylazaniumyl)ethyl phosphate Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCCCCCC NRJAVPSFFCBXDT-UHFFFAOYSA-N 0.000 claims description 3
- MBZYKEVPFYHDOH-BQNIITSRSA-N 24,25-dihydrolanosterol Chemical compound C([C@@]12C)C[C@H](O)C(C)(C)[C@@H]1CCC1=C2CC[C@]2(C)[C@@H]([C@H](C)CCCC(C)C)CC[C@]21C MBZYKEVPFYHDOH-BQNIITSRSA-N 0.000 claims description 3
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- FPTJELQXIUUCEY-UHFFFAOYSA-N 3beta-Hydroxy-lanostan Natural products C1CC2C(C)(C)C(O)CCC2(C)C2C1C1(C)CCC(C(C)CCCC(C)C)C1(C)CC2 FPTJELQXIUUCEY-UHFFFAOYSA-N 0.000 claims description 3
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- JZCCFEFSEZPSOG-UHFFFAOYSA-L copper(II) sulfate pentahydrate Chemical compound O.O.O.O.O.[Cu+2].[O-]S([O-])(=O)=O JZCCFEFSEZPSOG-UHFFFAOYSA-L 0.000 description 1
- GLNDAGDHSLMOKX-UHFFFAOYSA-N coumarin 120 Chemical compound C1=C(N)C=CC2=C1OC(=O)C=C2C GLNDAGDHSLMOKX-UHFFFAOYSA-N 0.000 description 1
- 229940109239 creatinine Drugs 0.000 description 1
- 125000001995 cyclobutyl group Chemical group [H]C1([H])C([H])([H])C([H])(*)C1([H])[H] 0.000 description 1
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- 125000001511 cyclopentyl group Chemical group [H]C1([H])C([H])([H])C([H])([H])C([H])(*)C1([H])[H] 0.000 description 1
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- ZBCBWPMODOFKDW-UHFFFAOYSA-N diethanolamine Chemical compound OCCNCCO ZBCBWPMODOFKDW-UHFFFAOYSA-N 0.000 description 1
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- 229910001873 dinitrogen Inorganic materials 0.000 description 1
- QLBHNVFOQLIYTH-UHFFFAOYSA-L dipotassium;2-[2-[bis(carboxymethyl)amino]ethyl-(carboxylatomethyl)amino]acetate Chemical compound [K+].[K+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O QLBHNVFOQLIYTH-UHFFFAOYSA-L 0.000 description 1
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- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- MWDZOUNAPSSOEL-UHFFFAOYSA-N kaempferol Natural products OC1=C(C(=O)c2cc(O)cc(O)c2O1)c3ccc(O)cc3 MWDZOUNAPSSOEL-UHFFFAOYSA-N 0.000 description 1
- 235000019136 lipoic acid Nutrition 0.000 description 1
- 210000005229 liver cell Anatomy 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 150000002688 maleic acid derivatives Chemical class 0.000 description 1
- 229910052748 manganese Inorganic materials 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
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- 125000001421 myristyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- WNYIBZHOMJZDKN-UHFFFAOYSA-N n-(2-acetamidoethyl)acetamide Chemical compound CC(=O)NCCNC(C)=O WNYIBZHOMJZDKN-UHFFFAOYSA-N 0.000 description 1
- 125000004123 n-propyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 125000001624 naphthyl group Chemical group 0.000 description 1
- 239000002858 neurotransmitter agent Substances 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 150000002823 nitrates Chemical class 0.000 description 1
- 125000004433 nitrogen atom Chemical group N* 0.000 description 1
- VIKNJXKGJWUCNN-XGXHKTLJSA-N norethisterone Chemical compound O=C1CC[C@@H]2[C@H]3CC[C@](C)([C@](CC4)(O)C#C)[C@@H]4[C@@H]3CCC2=C1 VIKNJXKGJWUCNN-XGXHKTLJSA-N 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 125000002347 octyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
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- 150000007530 organic bases Chemical class 0.000 description 1
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- 239000003960 organic solvent Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 229960001592 paclitaxel Drugs 0.000 description 1
- 125000000913 palmityl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 125000001147 pentyl group Chemical group C(CCCC)* 0.000 description 1
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical class OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
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- 125000003367 polycyclic group Chemical group 0.000 description 1
- 238000002600 positron emission tomography Methods 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 235000011181 potassium carbonates Nutrition 0.000 description 1
- JKANAVGODYYCQF-UHFFFAOYSA-N prop-2-yn-1-amine Chemical compound NCC#C JKANAVGODYYCQF-UHFFFAOYSA-N 0.000 description 1
- 125000004368 propenyl group Chemical group C(=CC)* 0.000 description 1
- 210000001938 protoplast Anatomy 0.000 description 1
- 229960001285 quercetin Drugs 0.000 description 1
- 235000005875 quercetin Nutrition 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 230000001177 retroviral effect Effects 0.000 description 1
- 108091092562 ribozyme Proteins 0.000 description 1
- 150000003873 salicylate salts Chemical class 0.000 description 1
- 150000003335 secondary amines Chemical class 0.000 description 1
- 239000004017 serum-free culture medium Substances 0.000 description 1
- 238000010898 silica gel chromatography Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 125000003003 spiro group Chemical group 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 125000004079 stearyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 150000003890 succinate salts Chemical class 0.000 description 1
- 125000004434 sulfur atom Chemical group 0.000 description 1
- 150000003467 sulfuric acid derivatives Chemical class 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 150000003892 tartrate salts Chemical class 0.000 description 1
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 description 1
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 150000003512 tertiary amines Chemical class 0.000 description 1
- KYMBYSLLVAOCFI-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SCN1CC1=CN=C(C)N=C1N KYMBYSLLVAOCFI-UHFFFAOYSA-N 0.000 description 1
- 235000019157 thiamine Nutrition 0.000 description 1
- 229960003495 thiamine Drugs 0.000 description 1
- 239000011721 thiamine Substances 0.000 description 1
- 229960002663 thioctic acid Drugs 0.000 description 1
- 125000005490 tosylate group Chemical group 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 150000003626 triacylglycerols Chemical class 0.000 description 1
- 125000005270 trialkylamine group Chemical group 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 239000002691 unilamellar liposome Substances 0.000 description 1
- 238000001845 vibrational spectrum Methods 0.000 description 1
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
- 239000011592 zinc chloride Substances 0.000 description 1
- 235000005074 zinc chloride Nutrition 0.000 description 1
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C231/00—Preparation of carboxylic acid amides
- C07C231/02—Preparation of carboxylic acid amides from carboxylic acids or from esters, anhydrides, or halides thereof by reaction with ammonia or amines
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H7/00—Compounds containing non-saccharide radicals linked to saccharide radicals by a carbon-to-carbon bond
- C07H7/06—Heterocyclic radicals
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
Definitions
- the present disclosure relates to a formulation.
- the present disclosure relates to a lipid formulation comprising first lipid compound, second lipid compound and co- lipids. It further relates to compounds of Formula I and Formula II, their structures and preparation process.
- the present disclosure relates to the pharmaceutical composition comprising the formulation for treatment or prevention of disease or a condition or a disorder mediated by asialoglycoprotein receptors.
- liposomes are used to overcome various challenges in drug delivery such as limited solubility, serum stability, circulation half-life, biodistribution, and target tissue selectivity.
- the drugs which benefit the most from liposomal delivery are those that are chemically labile, subject to enzymatic degradation and have an intracellular site of action.
- nucleic acids NAs
- liposomes as carriers of nucleic acids (NAs), either as plasmid vectors for gene therapy applications or to deliver smaller nucleic acid species such as antisense oligonucleotides, ribozymes and, more recently, mRNA, siRNA for the purposes of downregulating target genes.
- liposomal drug delivery is that the pharmacokinetics, biodistribution, and intracellular delivery of the liposome payload are largely determined by the physicochemical properties of the carrier i.e., the lipid formulation. Additionally, liposomes are used to formulate hydrophobic drugs that would otherwise be difficult to administer in aqueous dosage form. Hydrophobic drugs rapidly exchange into lipoproteins or other lipid-rich environments soon after injection, resulting in comparably uncontrolled pharmacology. Therefore design and formulation of liposome are the key factors which provide all the advantages in use of liposomes as a carrier in a therapeutic procedure.
- JP6463810B2 provides compositions and methods for intracellular delivery of mRNA in a liposome transfer vehicle to one or more target cells for the production of therapeutic levels of functional secreted proteins, particularly those resulting from protein and / or enzyme deficiencies.
- US20050287202A1 discloses a lipid formula for gene therapy for the introduction of nucleic acids into cells for a desired therapeutic agent.
- the lipid formula is a polyamine which comprises a carbohydrate moiety.
- there are various liposome formulation are available for gene delivery, there exists a need for specific targeted delivery of therapeutic agents or nucleic acids. And it is also essential that the targeted liposomes are stable and active till the targeted delivery.
- a formulation comprising: i. a first lipid compound of Formula I wherein is selected from C5-22 alkyl, C5-22 carbocyclyl, or C5-22 heterocyclyl, wherein C5-22 alkyl, C5-22 carbocyclyl, or C5-22 heterocyclyl optionally further substituted with one or more substituents selected from hydroxy, C1-10 alkyl, C1-10 alkylhydroxy, -NH-C(O)-C1-10 alkyl, or C1-10 alkoxy; and C1-10 alkoxy is optionally further substituted with one or more substituents selected from hydroxy, C 1-10 alkyl, C 1-10 alkylhydroxy, or -OC(O)R 1 ;
- X is a bond or selected from -OR 1 -, -O-C(O)-N(R
- a second lipid compound of Formula II Formula II wherein R 3 , and R 4 are independently selected from hydrogen, hydroxy, amino, halo, cyano, or -C 6-22 aryl; Y is a bond or selected from -OR7-, -O-C(O)-N(R7)-, -N(R7)-C(O)-O-, - C(O)N(R 7 )-, -N(R 7 )-C(O)-, or -O-C(O)R 7 -; R 5 , and R 6 are independently selected from C 5-22 alkyl, C 5-22 alkenyl, C 5-22 alkynyl, or C6-22 aryl; R7 is independently selected from hydrogen, C1-10 alkyl, C2-10 alkenyl, C2- 1 0 alkynyl, C 3-10 cycloalkyl, or -C 6-22 aryl; and p, q, r, and s are independently selected from 1 to 15
- a reconstituted formulation comprising the formulation comprising (i) a first lipid compound of Formula I; (ii) a second lipid compound of Formula II; and (iii) a co-lipid, with 2-25% (w/w) of a reconstituting agent, wherein the reconstituting agent is selected from saline, glucose, sucrose, or inulin, wherein the first lipid to the second lipid to the co-lipid is in the mole ratio range of 1:0.1:0.1 to 1:4:4.
- a pharmaceutical composition comprising the formulation comprising (i) a first lipid compound of Formula I; (ii) a second lipid compound of Formula II; and (iii) a co-lipid with pharmaceutically acceptable salts thereof.
- a pharmaceutical composition comprising the formulation comprising (i) a first lipid compound of Formula I; (ii) a second lipid compound of Formula II; and (iii) a co-lipid with one or more pharmaceutically active compound.
- a formulation comprising (i) a first lipid compound of Formula I; (ii) a second lipid compound of Formula II; and (iii) a co-lipid or a pharmaceutical composition comprising the formulation with pharmaceutically acceptable salts thereof or with one or more pharmaceutically active compound, for use in the treatment and/or prevention of a condition mediated by asialoglycoprotein receptor.
- X is a bond or selected from -OR 1 -, -O-C(O)-N
- a second lipid compound of Formula II or its pharmaceutically acceptable salts, complexes, hydrates, solvates, tautomers, polymorphs, stereoisomers, pharmaceutically active derivatives thereof Formula II wherein R3, and R4 are independently selected from hydrogen, hydroxy, amino, halo, cyano, or -C 6-22 aryl; Y is a bond or selected from -OR 7 -, -O-C(O)-N(R 7 )-, -N(R 7 )- C(O)-O-, -C(O)N(R 7 )-, -N(R 7 )-C(O)-, or -O-C(O)R 7 -; R 5 , and R 6 are independently selected from C5-22 alkyl, C5-22 alkenyl, C5-22 alkynyl, or C6-22 aryl; R7 is independently selected from hydrogen, C 1-10 alkyl, C 2
- a process for preparing the compound of Formula I comprising, reacting compound of Formula A with compound of Formula B in the presence of a first reagent and a first solvent to result in a compound of Formula C; and treating the compound of Formula C in the presence of a second reagent at a temperature in the range of -5 to 30° C to obtain the compound of Formula I, wherein is selected from C5-22 alkyl, C5-22 carbocyclyl, or C5-22 heterocyclyl, wherein C5-22 alkyl, C5-22 carbocyclyl, or C5-22 heterocyclyl optionally further substituted with one or more substituents selected from hydroxy, halogen, azide, C1-10 alkyl, C1-10 alkylhydroxy, -NH-C(O)-C1-10 alkyl, or C1-10 alkoxy.
- a process for preparing the compound of Formula II comprising: reacting compound of Formula D with a compound of Formula E in the presence of a reducing agent at a temperature in the range of 120 to 140° C to obtain a compound of Formula F; and converting the compound of Formula F in the presence of a third reagent to obtain the compound of Formula II, wherein X is hydrogen or C 1-6 alkyl.
- a method of treatment or prevention of a condition, or a disease, or a disorder the method comprising administering the formulation, or the pharmaceutical composition, to a subject in need thereof.
- a method of introducing one or more therapeutic agent into a cell comprising contacting a eukaryotic cell with the formulation, or the pharmaceutical composition.
- a method for the treatment or prevention of disease or disorder comprising administering to a subject suffering from disease or disorder a therapeutically effective amount of the formulation or the pharmaceutical composition, with other clinically relevant cytotoxic agents or non-cytotoxic agents to a subject in need thereof.
- a system for delivering one or more therapeutic agent to a subject in need comprising (a) the formulation, or the pharmaceutical composition; and one or more therapeutic agent; wherein the weight ratio of the formulation to the therapeutic agent is in the range of 1:1 to 50: 1.
- the compounds, or the pharmaceutical composition for the treatment or prevention of disease or disorder, together with other clinically relevant cytotoxic agents or non- cytotoxic agents.
- Figure 1 illustrate the gene delivery efficacies of the second lipid compounds in HepG2 cells, in an accordance with an implementation of the present disclosure.
- Figure 2 illustrate the gene delivery efficacies of the first lipid compounds in HepG2 cells, in an accordance with an implementation of the present disclosure.
- Figure 3 illustrate (a) mRNA transfections; and (b) mRNA transfections in 3D, in an accordance with an implementation of the present disclosure.
- Figure 4 illustrates the in vivo distribution of the first lipid compounds in the mice, in an accordance with an implementation of the present disclosure.
- Figure 5a depicts the process of obtaining of lyophilized and reconstituted formulation
- Figure 5b illustrates particle size distribution of lyophilized and reconstituted lipoplexes
- Figure 5c depicts the zeta potential of the targeting lipoplexes to the non-targeting lipoplexes, in an accordance with an implementation of the present disclosure.
- Figure 6 illustrate the (a) lipid formulation of the present disclosure and (b) the non-targeting lipid formulation, complexed with luciferase expressing pDNA and injected intraperitoneal (i.p) into mice, in an accordance with implementation of the present disclosure.
- Figure 7 illustrate luminescence expression seen in various organs after injecting pDNA loaded lipid formulations into mice, in accordance with an implementation of the present disclosure.
- Figure 8 illustrate the luminescence expression with respect to body weight of the mice organs after injecting pDNA loaded lipid formulations, in an accordance with implementation of the present disclosure.
- Figure 9 illustrate the luminescence expression with respect to (a) body weight of the mice organs and (b) number of hours after injecting mRNA loaded lipid formulations, in an accordance with implementation of the present disclosure.
- Figure 10 illustrates the luminescence expression with respect to number of weeks after injecting mRNA loaded lipid formulations into mice, in an accordance with implementation of the present disclosure.
- Figure 11 illustrates a graph comparing the body weights of mice with respect to number of days after injecting lipid formulation, in an accordance with implementation of the present disclosure.
- Figure 12 illustrates a graph showing spleen weights of mice with respect to number of days after injecting lipid formulation, in an accordance with implementation of the present disclosure.
- Figure 13 illustrates the representative images of H&E (hematoxylin and eosin) analysis in mice, in an accordance with implementation of the present disclosure.
- Figure 14 illustrates the representative images showing fibrosis in mice, in an accordance with implementation of the present disclosure.
- DETAILED DESCRIPTION OF THE INVENTION [0036] Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all such steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features.
- the compounds of Formula I and Formula II can be its derivatives, analogs, complexes, tautomeric forms, stereoisomer’s, diastereomers, geometrical isomers, polymorphs, solvates, or pharmaceutically acceptable salts and compositions.
- substituted is contemplated to include all permissible substituents of organic compounds.
- the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds.
- Illustrative substituents, for example, include those described hereinabove.
- the permissible substituents can be one or more and the same or different for appropriate organic compounds.
- the heteroatoms such as nitrogen may have hydrogen substituents, and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms.
- alkyl refers to straight or branched aliphatic hydrocarbon groups having 1 to 22 carbon atoms, which are attached to the rest of the molecule by a single atom, which may be optionally substituted by one or more substituents.
- alkyl groups include, without limitation, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, hexadecyl, tetradecyl, octadecyl, and the like.
- alkenyl refers to straight or branched aliphatic hydrocarbon groups having 2 to 22 carbon atoms with at least one carbon-carbon double bond, which are attached to the rest of the molecule by a single atom, which may be optionally substituted by one or more substituents.
- alkenyl groups include, without limitation, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and the like.
- alkynyl refers to straight or branched aliphatic hydrocarbon groups having 2 to 22 carbon atoms with at least one carbon-carbon triple bond, which are attached to the rest of the molecule by a single atom, which may be optionally substituted by one or more substituents.
- Preferred alkyl groups include, without limitation, ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like.
- aryl refers to aromatic radicals having 6 to 22 carbon atoms, which may be optionally substituted by one or more substituents.
- Preferred aryl groups include, without limitation, phenyl, naphthyl, indanyl, biphenyl, and the like.
- alkoxy refers to an alkyl group, as defined above, having an oxygen radical attached thereto. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy and the like.
- An "ether” is two hydrocarbons covalently linked by an oxygen.
- alkylhydroxy refers to an alkyl group, as defined above, having a hydroxy radical attached thereto. Representative alkylhydroxy groups include - CH2OH, -C2H4OH, - C3H7OH, and the like.
- carbocyclyl refers to a saturated, unsaturated or partially saturated ring having 5 to 22 carbon atoms forming cyclic systems.
- Carbocyclic groups may be spiral or bridged systems, may be substituted. Carbocyclyl groups may be optionally substituted with one or more heteroatoms. Carbocyclyl may include cycloalkyl, aryl, heteroaryl having 5 to 22 carbon atoms and may be optionally substituted. [0052]
- the term “heterocyclyl” refers to a heterocyclic ring radical having 5 to 22 carbon atoms which may be optionally substituted by one or more substituents.
- the heterocyclyl ring radical may be attached to the main structure at any heteroatom or carbon atom that results in the creation of a stable structure.
- heterocyclyl refers to a stable 5 to 22 membered rings radical, which consists of carbon atoms and from one to five heteroatoms selected from nitrogen, phosphorus, oxygen, and sulfur.
- the heterocyclic ring radical may be monocyclic, bicyclic or tricyclic ring systems, and the nitrogen, phosphorus, carbon, or sulfur atoms in the heterocyclic ring radical may be optionally oxidized to various oxidation states.
- the nitrogen atom may be optionally quaternized; and the ring radical may be partially or fully saturated.
- cycloalkyl refers to non-aromatic mono or polycyclic ring system of about 3 to 10 carbon atoms, which may be optionally substituted by one or more substituents.
- the polycyclic ring denotes hydrocarbon systems containing two or more ring systems with one or more ring carbon atoms in common, i.e., a spiro, fused or bridged structures.
- Preferred cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, groups.
- Compounds disclosed herein include isotopes of hydrogen, carbon, oxygen, fluorine, chlorine, iodine and sulfur which can be incorporated into the compounds, such as not limited to 2 H (D), 3 H (T), 11 C, 13 C, 14 C, 15 N, 18 F, 35 S, 36 Cl and 125 I.
- Compounds of this invention where in atoms were isotopically labeled for example radioisotopes such as 3 H, 13 C, 14 C, and the like can be used in metabolic studies, kinetic studies and imaging techniques such as positron emission tomography used in understanding the tissue distribution of the drugs.
- Compounds of the invention where hydrogen is replaced with deuterium may improve the metabolic stability and pharmacokinetics properties of the drug such as in vivo half-life.
- Compounds of the invention where isotopically labeled 18 F can be useful as PET imaging studies.
- pharmaceutically acceptable refers to compounds or compositions that are physiologically tolerable and do not typically produce allergic or similar untoward reaction, including but not limited to gastric upset or dizziness when administered to subjects.
- salts forming part of this invention include salts derived from inorganic bases such as like Li, Na, K, Ca, Mg, Fe, Cu, Zn and Mn and ammonium, substituted ammonium salts, aluminum salts and the like.; salts of organic bases such as N, N’-diacetylethylenediamine, glucamine, triethylamine, choline, dicyclohexylamine, benzylamine, trialkylamine, thiamine, guanidine, diethanolamine, ⁇ -phenylethylamine, piperidine, morpholine, pyridine, hydroxyethylpyrrolidine, hydroxyethylpiperidine and the like, salts also include amino acid salts such as glycine, alanine, cystine, cysteine, lysine, arginine, phenylalanine, guanidine etc.
- inorganic bases such as like Li, Na, K, Ca, Mg, Fe, Cu
- Salts may include acid addition salts where appropriate which are sulphates, nitrates, phosphates, perchlorates, borates, hydrohalides, acetates, tartrates, maleates, fumarates, citrates, succinates, palmoates, methanesulphonates, tosylates, benzoates, salicylates, hydroxynaphthoates, benzenesulfonates, ascorbates, glycerophosphates, ketoglutarates and the like.
- polymorphs refers to crystal forms of the same molecule, and different polymorphs may have different physical properties such as, for example, melting temperatures, heats of fusion, solubilities, dissolution rates and/or vibrational spectra as a result of the arrangement or conformation of the molecules in the crystal lattice.
- the compounds described herein may also exhibit polymorphism. This invention further includes different polymorphs of the compounds of the present invention.
- polymorph refers to a particular crystalline state of a substance, having particular physical properties such as X-ray diffraction, IR spectra, melting point and the like.
- the compounds described herein can also be prepared in any solid or liquid physical form, for example, the compound can be in a crystalline form, in amorphous form and have any particle size. Furthermore, the compound particles may be micronized or nanosized, or may be agglomerated, particulate granules, powders, oils, oily suspensions or any other form of solid or liquid physical forms. [0063]
- complexes as used herein, can be interchangeably used as "coordination complex,” or "metal coordination complex,” and the like.
- complexes refers to a complex of an organic compound with a metal that can be empirically differentiated from a simple metal salt of the organic compound based on physiochemical and/or spectroscopic properties, with a coordination complex typically having enhanced covalency as compared to a salt.
- complexes also involves a combination of coordinate covalent bonds and/or ionic bonds.
- the term “complexes” also includes molecules that lack an ionic component (e.g., such as a neutral coordination complex prior to deprotonation, where pKa of the coordination complex falls within a physiologically acceptable range).
- solvate refers to a compound formed by the interaction of a solvent and a solute.
- solvate refers to a compound formed by the interaction of solvent and the compounds of Formula I of the present disclosure.
- hydrate refers to a solvate wherein the solvent is water.
- receptor refers to a region of tissue, or a molecule in a cell membrane, which responds specifically to a particular neurotransmitter, hormone, antigen, or other substance.
- effective amount means an amount of a compound or composition which is sufficient enough to significantly and positively modify the symptoms and/or conditions to be treated (e.g., provide a positive clinical response).
- first lipid compound refers to the compounds of Formula I as disclosed herein and are the targeting lipids of the present disclosure. These lipids are sensitive towards selective receptors and binds to the selective receptors. In the present disclosure, the first lipid compounds or the targeting lipids specifically binds to asialoglycoreceptors.
- second lipid compound refers to the compounds of Formula II as disclosed herein and are the cationic lipids of the present disclosure. These lipids possess positive charge and may exists in salt form. These are used to interact with negatively charged species in a cell and hence are capable of delivering specific therapeutic agent into the cells.
- co-lipid refers to lipid compounds which are binding sites for intra and intercellular proteins. These lipids serves as a primary cellular component.
- asialoglycoreceptors refers to a specific receptor present in the hepatic cells or liver cells
- liposomes refers to the formulation comprising the targeting lipid, cationic lipid with co-lipids.
- Liposomes may also act as pharmaceutically effective compound by itself. Liposomes may encapsulate therapeutic agents such as nucleic acids (RNA, mRNA, siRNA, DNA), locked nucleic acids, small molecules, drugs, proteins, chemotherapeutic agent, a cytotoxin, a steroid, an immunotherapeutic agent, a targeted therapeutic agent, or combinations thereof.
- RNA nucleic acids
- mRNA miRNA
- siRNA DNA
- locked nucleic acids small molecules
- drugs proteins
- chemotherapeutic agent a cytotoxin
- a steroid an immunotherapeutic agent
- a targeted therapeutic agent or combinations thereof.
- lipoplex refers to a complex of nucleic acids mixed with lipids that spontaneously forms a vesicle containing therapeutic agent.
- the lipoplexes are formed by mixing pDNA or mRNA with the lipid formulation or liposome of the present disclosure.
- the term “reconstituted formulation” refers to a stabilized liposomal formulation which are obtained by mixing dried liposome with a reconstituting agent.
- the term “reconstituting agent” refers to a buffer which has physiological pH and isotonicity.
- the reconstituted formulation comprises the formulation of the present disclosure and the reconstituting agent selected from saline, glucose, sucrose, or inulin
- one or more pharmaceutically active compound refers to other pharmaceutically active compound which can be used in combinatorial with the pharmaceutical composition of the present disclosure.
- the other pharmaceutically active compound may include but not limited to hydrophobic drugs including quercetin, 7-amino-4-methylcoumarin, paclitaxel, atorvastatin, and so on.
- hydrophobic drugs including quercetin, 7-amino-4-methylcoumarin, paclitaxel, atorvastatin, and so on.
- a well-designed liposomal delivery system are capable of reducing the toxicity and increasing the potency of gene/nucleic acid/ drugs by optimizing gene/nucleic acid /drug delivery to target tissues.
- There are various properties such as stability, size, charge, hydrophobicity, interaction with serum proteins, and interaction with target/nontarget cell surfaces which needs to considered while designing the liposome.
- the compatibility and binding property towards the targeted cell is crucial property to be considered.
- the present disclosure provides novel targeting lipids and cationic lipids in specific to asialoglycoreceptors.
- the present disclosure provides a lipid formulation or liposome comprising the targeting lipids, cationic lipids and co- lipids.
- the present disclosure also provides process for preparing the lipids and the liposomes.
- the present disclosure further provides liposome for delivery of nucleic acids such as RNA, mRNA, SiRNA, DNA and locked nucleic acids, small molecules, drugs, proteins, chemotherapeutic agent, a cytotoxin, a steroid, an immunotherapeutic agent, or a targeted therapeutic agent to the hepatocytes for treating liver related diseases, condition or disorder.
- nucleic acids such as RNA, mRNA, SiRNA, DNA and locked nucleic acids, small molecules, drugs, proteins, chemotherapeutic agent, a cytotoxin, a steroid, an immunotherapeutic agent, or a targeted therapeutic agent to the hepatocytes for treating liver related diseases, condition or disorder.
- a formulation comprising: . i. a first lipid compound of Formula I; Formula I wherein is selected from C5-22 alkyl, C5-22 carbocyclyl, or C5-22 heterocyclyl, wherein C 5-22 alkyl, C 5-22 carbocyclyl, or C 5-22 heterocyclyl optionally further substituted with one or more substituents selected from hydroxy, C1-10 alkyl, C1-10 alkylhydroxy, -NH-C(O)-C1-10 alkyl, or C1-10 alkoxy; and C1-10 alkoxy is optionally further substituted with one or more substituents selected from hydroxy, C 1-10 alkyl, C 1-10 alkylhydroxy, or -OC(O)R 1 ; X is a bond or selected from -OR1-, -O-C(O)-N(R1)-, -N(R1)-C(O)-O-, - C(O)N(R
- a second lipid compound of Formula II Formula II wherein R 3 , and R 4 are independently selected from hydrogen, hydroxy, amino, halo, cyano, or -C6-22 aryl; Y is a bond or selected from -OR7-, -O-C(O)-N(R7)-, -N(R7)-C(O)-O-, - C(O)N(R 7 )-, -N(R 7 )-C(O)-, or -O-C(O)R 7 -; R 5 , and R 6 are independently selected from C 5-22 alkyl, C 5-22 alkenyl, C 5-22 alkynyl, or C6-22 aryl; R7 is independently selected from hydrogen, C1-10 alkyl, C2-10 alkenyl, C2- 1 0 alkynyl, C 3-10 cycloalkyl, or -C 6-22 aryl; and p, q, r, and s are independently selected from 1 to 15
- a formulation comprising: i.
- a first lipid compound of Formula I as disclosed herein wherein is selected from C5-12 carbocyclyl, or C5-12 heterocyclyl, wherein C5-12 carbocyclyl, or C5-12 heterocyclyl optionally further substituted with one or more substituents selected from hydroxy, or C 1-10 alkyl;
- X is a bond;
- R and R’ are independently selected from C7-18 alkyl;
- m and m’ are independently selected from 1 to 12;
- n and n’ are independently selected from 0 to 12; ii.
- R3, and R4 are independently selected from hydrogen, hydroxy, amino, halo, cyano, or -C6-22 aryl
- Y is a bond or selected from -OR 7 -, -O-C(O)-N(R 7 )-, -N(R 7 )-C(O)-O-, - C(O)N(R7)-, -N(R7)-C(O)-, or -O-C(O)R7-
- R5, and R6 are independently selected from C5-22 alkyl, C5-22 alkenyl, C5-22 alkynyl, or C 6-22 aryl
- R7 is independently selected from hydrogen, C1-10 alkyl, C2-10 alkenyl, C2- 10alkynyl, C3-10 cycloalkyl, or -C6-22 aryl
- p, q, r, and s are independently selected from 1 to 15; and iii)
- a formulation comprising:. i. a first lipid compound of Formula I as disclosed herein; wherein is selected from C 5-12 carbocyclyl, or C 5-12 heterocyclyl, wherein C 5-12 carbocyclyl, or C5-12 heterocyclyl optionally further substituted with one or more substituents selected from hydroxy, or C1-10 alkyl;
- X is a bond;
- R and R’ are independently selected from C7-18 alkyl;
- m and m’ are independently selected from 1 to 12; and
- n and n’ are independently selected from 0 to 12; ii.
- a second lipid compound of Formula II wherein R 3 , and R 4 are independently selected from hydrogen, or hydroxy; Y is a bond or selected from -C(O)N(R7), or -N(R7)-C(O)-; R5, and R6 are independently selected from C5-22 alkyl; R7 is independently selected from hydrogen, C 1-10 alkyl; and p, q, r, and s are independently selected from 1 to 5; and; and iii) a co-lipid wherein the first lipid to the second lipid to the co-lipid is in the mole ratio range of 1:0.1:0.1 to 1:4:4. [0082] In an embodiment of the present disclosure, there is provided a formulation comprising: i.
- a first lipid compound of Formula I as disclosed herein wherein is selected from C 5-10 heterocyclyl, wherein C 5-10 heterocyclyl optionally further substituted with one or more of hydroxy;
- X is a bond;
- R and R’ are independently selected from C 10-15 alkyl;
- m and m’ are independently selected from 1 to 2;
- n and n’ are independently selected from 0 to 2; iii.
- a second lipid compound of Formula II wherein R 3 , and R 4 are independently selected from hydrogen, or hydroxy; Y is a bond or -N(R 7 )-C(O)-; R 5 , and R 6 are independently selected from C10-18 alkyl; R7 is hydrogen; and p, q, r, and s are independently selected from 1 to 5; and; and iii) a co-lipid wherein the first lipid to the second lipid to the co-lipid is in the mole ratio range of 1:0.1:0.1 to 1:4:4.
- a formulation as disclosed herein wherein the formulation comprises (i) a first lipid compound of Formula I; (ii) a second lipid compound of Formula II; (iii) a co-lipid; and (iv) one or more therapeutic agent, wherein the first lipid to the second lipid to the co-lipid is in the mole ratio range of 1:0.5:0.5 to 1:4:4.
- the first lipid to the second lipid to the co-lipid is in the mole ratio range of 1:1:1 to 1:4:4.
- a formulation as disclosed herein wherein the first lipid to the second lipid to the co-lipid is in the mole ratio of 1:4:4.
- the formulation comprises one or more therapeutic agent.
- a formulation as disclosed herein wherein the formulation comprises (i) a first lipid compound of Formula I as disclosed herein; (ii) a second lipid compound of Formula II as disclosed herein; (iii) a co-lipid; and (iv) one or more therapeutic agent, wherein the first lipid to the second lipid to the co-lipid is in the mole ratio range of 1:0.1:0.1 to 1:4:4.
- a formulation as disclosed herein wherein the co-lipid is selected from steroids, cholesterol, sitostanol, sitosterol, ß-sitosterol-d6, campesterol, campesterol-d6, desmosterol, desmosterol-d6, 7-dehydrodesmosterol, zymosterol, zymosterol-d5, zymostenol, zymostenol-d7, diosgenin, stigmasterol, lathosterol, lathosterol-d7, lanosterol, lanosterol-d6, lanostenol, dihydrolanosterol-d7, 14-demethyl-lanosterol, 14-demethyl-lanosterol-d6, 14-demethyl-14-dehydrolanosterol (FF-MAS), cholesterol sulfate, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulphate (DHEA), DHEA
- the co-lipid is selected from steroids, cholesterol, dioleoylphosphatidylethanolamine (DOPE), phosphatidylcholine, dilauroylphosphatidylcholine (DLPC), dimirystoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC) dioleoylphosphatidylcholine (DOPC), or combinations thereof.
- the co-lipid is selected from cholesterol (Chol), dioleoylphosphatidylethanolamine (DOPE), or combinations thereof.
- RNA, mRNA, SiRNA, DNA nucleic acids
- locked nucleic acids small molecules, drugs, proteins, chemotherapeutic agent, a cytotoxin, a steroid, an immunotherapeutic agent, a targeted therapeutic agent, or combinations thereof.
- the therapeutic agent is selected from nucleic acids such as RNA, mRNA, SiRNA, DNA, or locked nucleic acids.
- a formulation as disclosed herein wherein the formulation comprises (i) a first lipid compound of Formula I as disclosed herein; (ii) a second lipid compound of Formula II as disclosed herein; (iii) a co-lipid; and (iv) one or more therapeutic agent, wherein the first lipid to the second lipid to the co-lipid is in the mole ratio range of 1:0.1:0.1 to 1:4:4; and the therapeutic agent is selected from nucleic acids (RNA, mRNA, SiRNA, DNA), locked nucleic acids, small molecules, drugs, proteins, chemotherapeutic agent, a cytotoxin, a steroid, an immunotherapeutic agent, a targeted therapeutic agent, or combinations thereof.
- nucleic acids RNA, mRNA, SiRNA, DNA
- a formulation as disclosed herein wherein the formulation further comprises a reconstituting agent selected from saline, glucose, sucrose, or inulin.
- a reconstituting agent selected from saline, glucose, sucrose, or inulin.
- the formulation further comprises glucose as the reconstituting agent.
- a formulation as disclosed herein wherein the formulation comprises (i) a first lipid compound of Formula I as disclosed herein; (ii) a second lipid compound of Formula II as disclosed herein; (iii) a co-lipid; (iv) one or more therapeutic agent; and (v) a reconstituting agent selected from saline, glucose, sucrose, or inulin, wherein the first lipid to the second lipid to the co-lipid is in the mole ratio range of 1:0.1:0.1 to 1:4:4.
- a formulation as disclosed herein wherein the molar ratio of the first lipid to the second lipid is in the range of 1:0.1 to 1:4. In another embodiment of the present disclosure, there is provided a formulation as disclosed herein, wherein the molar ratio of the first lipid to the second lipid is in the range of 1:1 to 1:4. In yet another embodiment of the present disclosure, there is provided a formulation as disclosed herein, wherein the molar ratio of the first lipid to the second lipid is of 1:4.
- a reconstituted formulation comprising the formulation comprising (i) a first lipid compound of Formula I; (ii) a second lipid compound of Formula II; and (iii) a co-lipid with 2-25% (w/w) of a reconstituting agent, wherein the reconstituting agent is selected from saline, glucose, sucrose, or inulin.
- a reconstituted formulation wherein the reconstituting agent is selected from saline, glucose, or sucrose.
- a reconstituted formulation wherein the reconstituting agent is glucose in weight range of 3-10% (w/w). In one another embodiment of the present disclosure, there is provided a reconstituted formulation, wherein the reconstituting agent is 5% (w/w) of glucose.
- a reconstituted formulation comprising the formulation comprising (i) a first lipid compound of Formula I; (ii) a second lipid compound of Formula II; (iii) a co-lipid with 2-25% (w/w) of a reconstituting agent, and one or more therapeutic agent selected from nucleic acids (RNA, mRNA, SiRNA, DNA), locked nucleic acids, small molecules, drugs, proteins, chemotherapeutic agent, a cytotoxin, a steroid, an immunotherapeutic agent, a targeted therapeutic agent, or combinations thereof, wherein the reconstituting agent is selected from saline, glucose, sucrose, or inulin.
- a formulation as disclosed herein wherein the first lipid compound is a targeting lipid which binds to a cell surface receptor; and the cell surface receptor is asialoglycoprotein receptors.
- the formulation is a carrier to deliver the therapeutic agent to a targeted cell, wherein the therapeutic agent selected from nucleic acids (RNA, mRNA, SiRNA, DNA), locked nucleic acids, small molecules, drugs, proteins, chemotherapeutic agent, a cytotoxin, a steroid, an immunotherapeutic agent, a targeted therapeutic agent, or combinations thereof.
- a pharmaceutical composition comprising the formulation (i) a first lipid compound of Formula I as disclosed herein; (ii) a second lipid compound of Formula II as disclosed herein; and (iii) a co-lipid with pharmaceutically acceptable salts thereof.
- a pharmaceutical composition comprising the formulation (i) a first lipid compound of Formula I as disclosed herein; (ii) a second lipid compound of Formula II as disclosed herein; and (iii) a co-lipid with one or more pharmaceutically active compound.
- a pharmaceutical composition as disclosed herein wherein the composition is in the form selected from a tablet, capsule, powder, syrup, solution, aerosol, or suspension.
- a formulation as or the pharmaceutical composition as disclosed herein for use in the manufacture of a medicament for treatment or prevention of a condition or a disorder or a disease mediated by asialoglycoprotein receptor.
- a formulation as or the pharmaceutical composition as disclosed herein for use in the treatment and/or prevention of a condition mediated by asialoglycoprotein receptor.
- a process for preparing the formulation comprising (i) a first lipid compound of Formula I as disclosed herein; (ii) a second lipid compound of Formula II as disclosed herein; and (iii) a co-lipid, the process comprising: mixing stoichiometric ratios of the first lipid compound with the second lipid compound and the co-lipid, wherein the first lipid to the second lipid to the co-lipid is in the mole ratio range of 1:0.1:0.1 to 1:4:4.
- a process for preparing the formulation comprising (i) a first lipid compound of Formula I as disclosed herein; (ii) a second lipid compound of Formula II as disclosed herein; and (iii) a co-lipid, the process comprising: mixing stoichiometric ratios of the first lipid compound with the second lipid compound and the co-lipid in the presence of one or more first solvent selected from chloroform, dichloromethane, tetrahydrofuran, acetonitrile, methanol, ethanol, ethyl acetate,or combinations thereof to obtain the formulation, wherein the first lipid to the second lipid to the co-lipid is in the mole ratio range of 1:0.1:0.1 to 1:4:4.
- X is a bond or selected from -OR 1 -, -O-C
- the compound of Formula I as disclosed herein wherein is selected from [0106]
- the compound of Formula I as disclosed herein wherein the compound is X is a bond or selected from -OR 1 -, -O-C(O)-N(R 1 )-, -N(R 1 )-C(O)-O-, -C(O)N(R 1 )-, - N(R 1 )-C(O)-, or -O-C(O)R 1 -;
- R and R’ are independently selected from C 5-22 alkyl, C 5- 22 alkenyl, C5-22 alkynyl, or -C6-22 aryl;
- R1 and R2 are independently selected from hydrogen, C1-10 alkyl, C2-10 alkenyl, C2-10alkynyl, C3-10 cycloalkyl, or -C6-22 aryl;
- the compound of Formula I as disclosed herein, wherein the compound is X is a bond or selected from - OR 1 -, -O-C(O)-N(R 1 )-, -N(R 1 )-C(O)-O-, -C(O)N(R 1 )-, -N(R 1 )-C(O)-, or -O-C(O)R 1 -;
- R and R’ are independently selected from C5-22 alkyl;
- R1 and R2 are independently selected from hydrogen, or C 1-10 alkyl,;
- m and m’ are independently selected from 5 to 22;
- n is independently selected from 5 to 22 and n’ is selected from 0 to 5.
- the compound of Formula I as disclosed herein, wherein the compound is selected from a) (3S,4R,5S,6S)-6-((4-((ditetradecylamino)methyl)-1H-1,2,3-triazol-1- yl)methyl)tetrahydro-2H-pyran-2,3,4,5-tetraol; b) (3S,4R,5S,6S)-6-((4- ((dihexadecylamino)methyl)-1H-1,2,3-triazol-1-yl)methyl)tetrahydro-2H-pyran- 2,3,4,5-tetraol; and c) (3S,4R,5S,6S)-6-((4-((dioctadecylamino)methyl)-1H-1,2,3- triazol-1-yl)methyl)tetrahydro-2H-pyran-2,3,4,5-tetraol
- second lipid compound of Formula II as disclosed herein, wherein the compound is selected from (a) N,N-dimethyl-2-tetradecanamido-N-(2-tetradecanamidoethyl)ethan-1-aminium chloride; (b) N,N-dimethyl-2-palmitamido-N-(2-palmitamidoethyl)ethan-1-aminium chloride; (c) N,N-dimethyl-2-stearamido-N-(2-stearamidoethyl)ethan-1-aminium chloride; (d) 2-hydroxy-N-methyl-N,N-bis(2-tetradecanamidoethyl)ethan-1-aminium chloride; (e) 2-hydroxy-N-methyl-N,N-bis(2-palmitamidoethyl)ethan-1-aminium chloride; (f) 2-hydroxy-N-methyl-N,N-bis(2-palmitamidoethyl)ethan-1-
- a process for preparing the compound of Formula I as disclosed herein comprising: reacting compound of Formula A with compound of Formula B in the presence of a first reagent and a second solvent to result in a compound of Formula C; and treating the compound of Formula C in the presence of a second reagent at a temperature in the range of -5 to 30° C to obtain the compound of Formula I wherein is selected from C 5-22 alkyl, C 5-22 carbocyclyl, or C 5-22 heterocyclyl, wherein C 5-22 alkyl, C 5-22 carbocyclyl, or C 5-22 heterocyclyl optionally further substituted with one or more substituents selected from hydroxy, halogen, azide, C1-10 alkyl, C1-10 alkylhydroxy, -NH-C(O)-C1-10 alkyl, or C1-10 alkoxy.
- the first reagent is selected from copper sulphate, sodium ascorbate, or combinations thereof; the second solvent is selected from water, tetrahydrofuran, or combinations thereof; and the second reagent is selected from ether, hydrochloric acid, or combinations thereof.
- a process for preparing the compound of Formula I as disclosed herein comprising [0114]
- a process for preparing the compound of Formula II as disclosed herein comprising: reacting compound of Formula D with a compound of Formula E in the presence of a reducing agent at a temperature in the range of 120 to 140 °C to obtain a compound of Formula F; and converting the compound of Formula F in the presence of a third reagent to obtain the compound of Formula II, wherein X is hydrogen or C 1-6 alkyl.
- the reducing agent is selected from sodium borohydride, sodium cyanoborohydride, lithium aluminum hydride, diborane, DIBAL-H, lithium tetrahydridoaluminate, lithium tri-tert- butoxyaluminum hydride, sodium dithionite, sodium hydrogensulfite, sodium hydrosulfite, dodium hydroxymethanesulfinate, sodium hypophosphite, sodium tetrahydroborate, sodium triacetoxyborohydride, triethoxysilane, triethylphosphine, triethylsilane trimethylamine borane, trimethylphoshpine, trimethylphosphite, triphenylphosphine, or combinations thereof; and the third reagent is selected from methyl iodide, ethyl hypochlorite, potassium carbonate, or
- a method of treatment or prevention of a condition, or a disease, or a disorder comprising administering the formulation, or the pharmaceutical composition as disclosed herein, to a subject in need thereof.
- a method introducing one or more therapeutic agent into a cell comprising contacting a eukaryotic cell with the formulation, or the pharmaceutical composition as disclosed herein.
- a method for the treatment or prevention of disease or disorder comprising administering to a subject suffering from disease or disorder a therapeutically effective amount of the formulation or the pharmaceutical composition as disclosed herein, with other clinically relevant cytotoxic agents or non-cytotoxic agents to a subject in need thereof.
- a system for delivering one or more therapeutic agent to a subject in need comprising the formulation, or the pharmaceutical composition as disclosed herein, and one or more therapeutic agent, wherein the weight ratio of the formulation to the therapeutic agent is in the range of 1:1 to 50:1.
- the compounds, or the pharmaceutical composition as disclosed herein for the treatment or prevention of disease or disorder, together with other clinically relevant cytotoxic agents or non-cytotoxic agents.
- the disease or disorder or condition is selected from haemophilia, Niemann-Pick type C2, Factor VII deficiency, ⁇ -1-antitrypsin deficiency, Familial tyrosinemia, liver cancer, liver dysfunction, hepatitis, cirrhosis, liver failure, ascites, gallstones, fatty liver disease, autoimmune hepatitis, primary sclerosing cholangitis, primary biliary cirrhosis (PBC), hemochromatosis, wilson’s disease, or hepatocellular carcinoma.
- haemophilia Niemann-Pick type C2
- Factor VII deficiency ⁇ -1-antitrypsin deficiency
- Familial tyrosinemia liver cancer, liver dysfunction, hepatitis, cirrhosis, liver
- reaction mixture was stirred for 5 minutes then 10 mL of H 2 SO 4 was drop wise added to reaction mixture.
- the reaction mixture was allowed to stirring for 6 h at RT. Progress of the reaction was monitored by TLC.
- reaction mixture was allowed to stir at 50 o C for 5 h. Progress of the reaction was monitored by TLC. After 5 h 50% of compound I was consumed, 0.5 equal in g methanesulfonyl chloride and DMAP was added to the reaction mixture and then allowed to stir at same conditions for 10 h. After completion of the reaction, reaction mixture was quenched with water then added ethyl acetate, washed with NH4Cl given water wash, then organic layer was passed through sodium sulphate, solvent was concentrated and dried under vacuum. Crude compound was purified by column chromatography.
- reaction mixture was allowed to stir at 80 0 C for 48 hrs in reflux condition and the progress of the reaction was monitored by TLC.
- K2CO3 was filtered by using Whatman filter paper and then crude product was concentrated by using rotary evaporator.
- reaction mass was extracted with 10% methanol in DCM and the organic solvent was concentrated under vacuum.
- the crude compound was purified by column chromatography by using 60/120 mesh silica gel and the pure viscous liquid compound was collected at 4% methanol in DCM.
- lipid formulations liposomes
- Liposome was prepared by using thin film hydration method from previously established protocol (Srujan etal., The influence of the structural orientation of amide linkers on the serum compatibility and lung transfection properties of cationic amphiphiles, Biomaterials, 2011;32(22):5231-40).
- lipid formulations were prepared using first lipid of Formula I which is Gal16- compound VI, a second lipid of Formula I which is MeOH16 which is 16MOH of Formula IIB and the co-lipid is selected from DOPE, cholesterol, or combinations thereof.
- lipids were dissolved in chloroform.
- a thin lipid film was developed by using flow of nitrogen gas and then to remove trapped solvent from the thin film was kept for drying under high vacuum for 6 hours.1ml of autoclaved Mill ‘Q’ water was added to vacuum dried lipid thin film and then allowed to overnight for swelling.
- the vials were then vortexes for three minutes for multi-lamellar vesicles (MLVs). MLVs were then sonicated initially in a water bath followed by an ice bath until to get clear solution by using a Branson 450 sonifier at 100% duty cycle and 25 W output power to produce small uni-lamellar vesicles.
- MLVs multi-lamellar vesicles
- lipoplexes The lipid-pDNA complex (lipoplex) was prepared as per previous literature ( Dharmalingam etal., An anti-oxidant, alpha-lipoic acid conjugated oleoyl-sn- phosphatidylcholineas a helper lipid in cationic liposomal formulations, Colloids and surfaces B, Biointerfaces.2017;152:133-42).
- 1 mM liposome were serially diluted in serum free medium in final volume of 25 ⁇ L and it was complexes with fixed amount of pDNA (0.3 ⁇ g/well of 96 well plate, 0.5 ⁇ g/well of 24 well plate and 1 ⁇ g/well of 6 well plate) was diluted in 25 ⁇ L of serum free medium.
- Mixture of liposome and pDNA were shaken by using Spin ‘X’ (minimum shaking) in room temperature for 10 to 15 minutes and then subsequently added to treatment wells.
- Lipoplexes of MeOH16DC Gal 0.25 were prepared with eGFP mRNA at varying lipid to base charge ratios, 4:1 and 8:1, added to HepG2. After 48h, transfection efficiencies were analysed in FACS for eGFP expression. eGFP mRNA expression was as good as commercial transfection reagent LT-1, showed ⁇ 80% transfection efficiency. Lyophilized lipoplexes (Figure 3a) of MeOH16DC Gal 0.25 comprising eGFP mRNA/pDNA and HepG2 cells were added to collagen solution and to form gel in order to mimic in vivo conditions where hepatocytes have collagen as extracellular matrix. GFP expression was observed in different time intervals at day 3, 6, 10, and 14.
- Figure 5c depicts the zeta potential of the targeting lipoplexes to the non-targeting lipoplexes.
- Figures 5b and 5c proved that the targeted lipoplexes exhibit narrowed particle size distribution, which means maximum number of particles of the lipoplex have same particle size ⁇ 200nm.
- the zeta potential close to +25mV reveal positive of the lipoplexes that ensures complete binding of nucleic acids.
- Luciferase encoded pDNA and mRNA lipoplexes preparation was carried out as described above.
- Targeted lipoplexes demonstrated more selective luciferase expression in liver, whereas non-targeted lipoplexes showed expression in liver, lung and spleen as well.
- In vivo gene expression profiles This experiment was performed under protocols (IAEC No.3/2020) approved by Institutional Animal Ethical Committee of Christian Medical College, India.Balb/c mice (10–12 weeks age)
- Figure 7 illustrate luminescence expression seen in various organs after injecting pDNA loaded lipid formulations into mice. Luciferase expression profiles of the major organs collected after post-euthanization.
- Targeted lipoplexes demonstrated selective expression in liver, whereas non-targeted lipoplexes showed in liver, lung and spleen.
- Body weight monitor [0161] To assess any possible toxic effects of liposomal formulations, initially, we have performed the body weight monitoring experiment. Swiss albino mice were an intraperitonially injected with different liposomal treatment groups. Mice body weight was monitored every injection time throughout the treatment period.
- Figure 8 illustrate the luminescence expression with respect to body weight of the mice organs after injecting pDNA loaded lipid formulations.
- Figure 9a illustrate luciferase mRNA expression at varying concentrations from 0.5mg/kg body weight to 4 mg/kg body weight.
- Figure 9b depicts luciferase mRNA expression at different time intervals from 2h to 72h. The mRNA showed higher expression in 6h and retained up to 36h..
- Figure 10 illustrate the luciferase mRNA administered with the targeted liposomes thrice with a gap of one week. The mRNA expression was not compromised despite of multiple administration.
- Figure 11 illustrate body weights of the lipoplexes injected animals were observed for 13 days. There were no major changes in the total body weight of the lipoplexes
- Figure 12 illustrate spleen weights of the lipoplexes injected animals were evaluated after 15 days. There are no major changes in the spleen weight of the targeted lipolplexes, whereas non-targeted lipoplexes showed slight increase in the spleen weights.
- H&E Analysis [0162]
- Figure 13 illustrate the representative images of H&E (hematoxylin and eosin) analysis in mice.
- Hematological parameters The blood parameters for each treatment group of single and seven consecutive doses of different treatment groups were performed.
- Table 1 shows the consolidated hematological parameters obtained for the targeted lipoplexes in comparison to the non-targeted liposomes at single dose and after seven doses.
- Table 1 Serum parameters [0165] The final day of experiment, the blood was collected from each mouse into eppendroff around 500 ⁇ L by retro orbital plexus puncture. The blood collected eppendroff samples were kept horizontally for one hour; further serum was separated by centrifugation at 4000 rpm for 15 mins at 4 o C.
- the supernatant serum was used for determining the Aspartate aminotransferase (AST), Alanine transaminase (ALT), Alkaline phosphatase, Total protein content, Triglycerides levels, Cholesterol levels, Glucose levels, Creatinine levels and Blood urea nitrogen (BUN) levels by using auto analyzer Siemens Dimension XpandPlus Integrated Chemistry System. Cytokine analysis by multiplex cytokine assay [0166] The final day of experiment, the blood was collected from each mouse into eppendroff around 500 ⁇ L by retro orbital plexus puncture. Further serum was separated by centrifugation at 4000 rpm for 15 mins at 4 o C.
- the supernatant serum was used for determining the level of inflammatory cytokines, including IL-1 ⁇ , IL1 ⁇ , IL-6, IL-10, IL-12p70, IL-17A, IL-23, IL-27, CCL2(MCP-1), IFN- ⁇ , IFN- ⁇ , TNF- ⁇ , and GM-CSF in different treatment groups were determined by multiplex cytometric bead- based LEGENDplex immunoassay (Mouse Inflammation Panel; BioLegend, London, UK) according to the manufacturer’s protocol.
- cytometric bead- based LEGENDplex immunoassay Mae Inflammation Panel; BioLegend, London, UK
- the serum samples were 2-fold diluted using dilution buffer and the equal volume diluted serum samples, cytokine standards were incubated with specific capture antibodies beads (25 ⁇ l each) for 2 hours with shaking at approx.800 rpm.
- the capture bead-analyte complexes were spin down at 250 x g and washed using 1x wash buffer.
- 25 ⁇ l of biotinylated detection antibody cocktail was added to beads and incubated for 1 hour with shaking to forming capture bead-analyte-detection antibody sandwiches.
- 25 ⁇ l of Streptavidin- phycoerythrin (SA-PE) subsequently was added to bind to the biotinylated detection antibodies complex.
- Statistical analysis [0168] All the in vitro experiments were done in triplicate and the mean standard deviation was considered for comparisons. All the results were analyzed by ‘‘One-way ANOVA’’ test using Dunnett’s post-test (Graph Pad Prism version 6.04 Software, San Diego, CA.). Advantages of the present disclosure [0169] The present disclosure provides a liposome formulation comprising the first lipid compound of Formula I, the second lipid compound of Formula II and the co- lipid.
- the first lipid compound or the targeted lipids of the present disclosure are novel lipids which are galactosylated lipids which acts as ligand and shows high affinity to asialoglycoprotein receptors. These receptors are over expressed in hepatocytes and when these galactosylated lipids binds to these receptors at the hepatocytes, thereby deliver the therapeutic agent into the hepatocytes.
- the second lipid compound of Formula II are novel cationic lipids that are used in the formation of liposome of the present disclosure.
- the liposome of the present disclosure acts as a vehicle or a carrier to deliver specific therapeutic agent to the hepatocytes.
- the liposome of the present disclosure are also made as stable formulations by subjecting to lyophilization and reconstitution.
- the liposome of the present disclosure deliver therapeutic agent selected from nucleic acids (RNA, mRNA, SiRNA, DNA), locked nucleic acids, small molecules, drugs, proteins, chemotherapeutic agent, a cytotoxin, a steroid, an immunotherapeutic agent, a targeted therapeutic agent, or combinations thereof to the hepatocytes.
- the present disclosure provides a pharmaceutical composition comprising the formulation which are used for treatment or prevention of a condition or a disease mediated by asialoglycoreceptors.
- the formulation or the liposome of the present disclosure are also used for preparing medicament for treating various liver related ailments.
- the present disclosure also provides convenient process for preparing the novel first and second lipid compounds.
Abstract
The present disclosure provides a formulation comprising a first lipid compound of Formula I; a second lipid compound of Formula II and a co-lipid wherein the first lipid to the second lipid to the co-lipid is in the mole ratio range of 1:0.1:0.1 to 1:4:4. The present disclosure also provides a first lipid compound of Formula I, and a second lipid compound of Formula II. The present disclosure further provides processes for preparing the formulation, the lipid compounds and methods thereof.
Description
LIPID FORMULATION FOR DELIVERY OF THERAPEUTIC AGENTS
FIELD OF INVENTION
[0001] The present disclosure relates to a formulation. The present disclosure relates to a lipid formulation comprising first lipid compound, second lipid compound and co- lipids. It further relates to compounds of Formula I and Formula II, their structures and preparation process. In addition, the present disclosure relates to the pharmaceutical composition comprising the formulation for treatment or prevention of disease or a condition or a disorder mediated by asialoglycoprotein receptors.
BACKGROUND OF THE INVENTION [0002] Numerous methods have been developed to facilitate the transfer of genetic material or therapeutic material into specific cells which include viral vectors, non- viral vectors, retroviral vectors or adenovirus vectors, microinjection, electroporation, protoplast fusion, calcium phosphate, and liposomes. The liposomal formulation have been widely studied as they form spherical vesicles to encapsulate the genetic or therapeutic material and are taken up by the cells by endocytosis. Due to their multilamellar vesicular structure, liposomes are ideal candidate for delivery of drug, genetic, therapeutic material into the cells. These liposomes are used to overcome various challenges in drug delivery such as limited solubility, serum stability, circulation half-life, biodistribution, and target tissue selectivity. The drugs which benefit the most from liposomal delivery, are those that are chemically labile, subject to enzymatic degradation and have an intracellular site of action. Hence, there is a considerable interest in exploiting liposomes as carriers of nucleic acids (NAs), either as plasmid vectors for gene therapy applications or to deliver smaller nucleic acid species such as antisense oligonucleotides, ribozymes and, more recently, mRNA, siRNA for the purposes of downregulating target genes.
[0003] The advantages of the liposomal drug delivery is that the pharmacokinetics, biodistribution, and intracellular delivery of the liposome payload are largely determined by the physicochemical properties of the carrier i.e., the lipid formulation.
Additionally, liposomes are used to formulate hydrophobic drugs that would otherwise be difficult to administer in aqueous dosage form. Hydrophobic drugs rapidly exchange into lipoproteins or other lipid-rich environments soon after injection, resulting in comparably uncontrolled pharmacology. Therefore design and formulation of liposome are the key factors which provide all the advantages in use of liposomes as a carrier in a therapeutic procedure. [0004] JP6463810B2 provides compositions and methods for intracellular delivery of mRNA in a liposome transfer vehicle to one or more target cells for the production of therapeutic levels of functional secreted proteins, particularly those resulting from protein and / or enzyme deficiencies. [0005] US20050287202A1 discloses a lipid formula for gene therapy for the introduction of nucleic acids into cells for a desired therapeutic agent. The lipid formula is a polyamine which comprises a carbohydrate moiety. Though there are various liposome formulation are available for gene delivery, there exists a need for specific targeted delivery of therapeutic agents or nucleic acids. And it is also essential that the targeted liposomes are stable and active till the targeted delivery. Hence there is still a dire need in the state of art of lipid formulation for specific delivery. SUMMARY OF THE INVENTION [0006] In first aspect of the present disclosure, there is provided a formulation comprising: i. a first lipid compound of Formula I
wherein
is selected from C5-22 alkyl, C5-22 carbocyclyl, or C5-22 heterocyclyl, wherein C5-22 alkyl, C5-22 carbocyclyl, or C5-22 heterocyclyl optionally further substituted with one or more substituents selected from hydroxy, C1-10 alkyl, C1-10 alkylhydroxy, -NH-C(O)-C1-10 alkyl, or C1-10 alkoxy; and C1-10 alkoxy is optionally further substituted with one or more substituents selected from hydroxy, C1-10 alkyl, C1-10 alkylhydroxy, or -OC(O)R1; X is a bond or selected from -OR1-, -O-C(O)-N(R1)-, -N(R1)-C(O)-O-, - C(O)N(R1)-, -N(R1)-C(O)-, or -O-C(O)R1-; R and R’ are independently selected from C5-22 alkyl, C5-22 alkenyl, C5-22 alkynyl, or -C6-22 aryl; R1 is independently selected from hydrogen, C1-10 alkyl, C2-10 alkenyl, C2- 10alkynyl, C3-10 cycloalkyl, or -C6-22 aryl; m and m’ are independently selected from 1 to 22; and n and n’ are independently selected from 0 to 22; ii. a second lipid compound of Formula II:
Formula II wherein R3, and R4 are independently selected from hydrogen, hydroxy, amino, halo, cyano, or -C6-22 aryl; Y is a bond or selected from -OR7-, -O-C(O)-N(R7)-, -N(R7)-C(O)-O-, - C(O)N(R7)-, -N(R7)-C(O)-, or -O-C(O)R7-; R5, and R6 are independently selected from C5-22 alkyl, C5-22 alkenyl, C5-22 alkynyl, or C6-22 aryl;
R7 is independently selected from hydrogen, C1-10 alkyl, C2-10 alkenyl, C2- 10alkynyl, C3-10 cycloalkyl, or -C6-22 aryl; and p, q, r, and s are independently selected from 1 to 15; and iii. a co-lipid wherein the first lipid to the second lipid to the co-lipid is in the mole ratio range of 1:0.1:0.1 to 1:4:4. [0007] In second aspect of the present disclosure, there is provided a reconstituted formulation comprising the formulation comprising (i) a first lipid compound of Formula I; (ii) a second lipid compound of Formula II; and (iii) a co-lipid, with 2-25% (w/w) of a reconstituting agent, wherein the reconstituting agent is selected from saline, glucose, sucrose, or inulin, wherein the first lipid to the second lipid to the co-lipid is in the mole ratio range of 1:0.1:0.1 to 1:4:4. [0008] In third aspect of the present disclosure, there is provided a pharmaceutical composition comprising the formulation comprising (i) a first lipid compound of Formula I; (ii) a second lipid compound of Formula II; and (iii) a co-lipid with pharmaceutically acceptable salts thereof. [0009] In fourth aspect of the present disclosure, there is provided a pharmaceutical composition comprising the formulation comprising (i) a first lipid compound of Formula I; (ii) a second lipid compound of Formula II; and (iii) a co-lipid with one or more pharmaceutically active compound. [0010] In fifth aspect of the present disclosure, there is provided a formulation comprising (i) a first lipid compound of Formula I; (ii) a second lipid compound of Formula II; and (iii) a co-lipid or a pharmaceutical composition comprising the formulation with pharmaceutically acceptable salts thereof or with one or more pharmaceutically active compound, for use in the treatment and/or prevention of a condition mediated by asialoglycoprotein receptor.
[0011] In sixth aspect of the present disclosure, there is provided a first lipid compound of Formula I or its pharmaceutically acceptable salts, complexes, hydrates, solvates, tautomers, polymorphs, stereoisomers, pharmaceutically active derivatives thereof
Formula I wherein
is selected from C5-22 alkyl, C5-22 carbocyclyl, or C5-22 heterocyclyl, wherein C5-22 alkyl, C5-22 carbocyclyl, or C5-22 heterocyclyl optionally further substituted with one or more substituents selected from hydroxy, C1-10 alkyl, C1-10 alkylhydroxy, -NH-C(O)-C1-10 alkyl, or C1-10 alkoxy; and C1-10 alkoxy is optionally further substituted with one or more substituents selected from hydroxy, C1-10 alkyl, C1-10 alkylhydroxy, or -OC(O)R1; X is a bond or selected from -OR1-, -O-C(O)-N(R1), -N(R1)-C(O)-O-, -C(O)N(R1)-, -N(R1)-C(O)-, or -O-C(O)R1-; R and R’ are independently selected from C5-22 alkyl, C5-22 alkenyl, C5-22 alkynyl, or -C6-22 aryl; R1 is independently selected from hydrogen, C1-10 alkyl, C2-10 alkenyl, C2-10alkynyl, C3-10 cycloalkyl, or -C6-22 aryl; m and m’ are independently selected from 1 to 22; and n and n’ are independently selected from 0 to 22. [0012] In seventh aspect of the present disclosure, there is provided a second lipid compound of Formula II or its pharmaceutically acceptable salts, complexes, hydrates,
solvates, tautomers, polymorphs, stereoisomers, pharmaceutically active derivatives thereof
Formula II wherein R3, and R4 are independently selected from hydrogen, hydroxy, amino, halo, cyano, or -C6-22 aryl; Y is a bond or selected from -OR7-, -O-C(O)-N(R7)-, -N(R7)- C(O)-O-, -C(O)N(R7)-, -N(R7)-C(O)-, or -O-C(O)R7-; R5, and R6 are independently selected from C5-22 alkyl, C5-22 alkenyl, C5-22 alkynyl, or C6-22 aryl; R7 is independently selected from hydrogen, C1-10 alkyl, C2-10 alkenyl, C2-10alkynyl, C3-10 cycloalkyl, or - C6-22 aryl; and p, q, r, and s are independently selected from 1 to 15. [0013] In eighth aspect of the present disclosure, there is provided a process for preparing the compound of Formula I, the process comprising, reacting compound of Formula A with compound of Formula B in the presence of a first reagent and a first solvent to result in a compound of Formula C; and treating the compound of Formula C in the presence of a second reagent at a temperature in the range of -5 to 30° C to obtain the compound of Formula I,
wherein
is selected from C5-22 alkyl, C5-22 carbocyclyl, or C5-22 heterocyclyl, wherein C5-22 alkyl, C5-22 carbocyclyl, or C5-22 heterocyclyl optionally further substituted with one or more substituents selected from hydroxy, halogen, azide, C1-10 alkyl, C1-10 alkylhydroxy, -NH-C(O)-C1-10 alkyl, or C1-10 alkoxy. [0014] In ninth aspect of the present disclosure, there is provided a process for preparing the compound of Formula II, the process comprising: reacting compound of Formula D with a compound of Formula E in the presence of a reducing agent at a temperature in the range of 120 to 140° C to obtain a compound of Formula F; and converting the compound of Formula F in the presence of a third reagent to obtain the compound of Formula II, wherein X is hydrogen or C1-6 alkyl.
[0015] In tenth aspect of the present disclosure, there is provided a method of treatment or prevention of a condition, or a disease, or a disorder, the method comprising administering the formulation, or the pharmaceutical composition, to a subject in need thereof. [0016] In eleventh aspect of the present disclosure, there is provided a method of introducing one or more therapeutic agent into a cell, comprising contacting a eukaryotic cell with the formulation, or the pharmaceutical composition. [0017] In twelfth aspect of the present disclosure, there is provided a method for the treatment or prevention of disease or disorder comprising administering to a subject suffering from disease or disorder a therapeutically effective amount of the formulation or the pharmaceutical composition, with other clinically relevant cytotoxic agents or non-cytotoxic agents to a subject in need thereof.
[0018] In thirteenth aspect of the present disclosure, there is provided a system for delivering one or more therapeutic agent to a subject in need, comprising (a) the formulation, or the pharmaceutical composition; and one or more therapeutic agent; wherein the weight ratio of the formulation to the therapeutic agent is in the range of 1:1 to 50: 1. [0019] In the last aspect of the present disclosure, there is provided a use of the compounds, or the pharmaceutical composition, for the treatment or prevention of disease or disorder, together with other clinically relevant cytotoxic agents or non- cytotoxic agents. [0020] These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter BRIEF DESCRIPTION OF DRAWINGS [0021] The following drawings form a part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein. [0022] Figure 1 illustrate the gene delivery efficacies of the second lipid compounds in HepG2 cells, in an accordance with an implementation of the present disclosure. [0023] Figure 2 illustrate the gene delivery efficacies of the first lipid compounds in HepG2 cells, in an accordance with an implementation of the present disclosure. [0024] Figure 3 illustrate (a) mRNA transfections; and (b) mRNA transfections in 3D, in an accordance with an implementation of the present disclosure. [0025] Figure 4 illustrates the in vivo distribution of the first lipid compounds in the mice, in an accordance with an implementation of the present disclosure.
[0026] Figure 5a depicts the process of obtaining of lyophilized and reconstituted formulation; Figure 5b illustrates particle size distribution of lyophilized and reconstituted lipoplexes; and Figure 5c depicts the zeta potential of the targeting lipoplexes to the non-targeting lipoplexes, in an accordance with an implementation of the present disclosure. [0027] Figure 6 illustrate the (a) lipid formulation of the present disclosure and (b) the non-targeting lipid formulation, complexed with luciferase expressing pDNA and injected intraperitoneal (i.p) into mice, in an accordance with implementation of the present disclosure. [0028] Figure 7 illustrate luminescence expression seen in various organs after injecting pDNA loaded lipid formulations into mice, in accordance with an implementation of the present disclosure. [0029] Figure 8 illustrate the luminescence expression with respect to body weight of the mice organs after injecting pDNA loaded lipid formulations, in an accordance with implementation of the present disclosure. [0030] Figure 9 illustrate the luminescence expression with respect to (a) body weight of the mice organs and (b) number of hours after injecting mRNA loaded lipid formulations, in an accordance with implementation of the present disclosure. [0031] Figure 10 illustrates the luminescence expression with respect to number of weeks after injecting mRNA loaded lipid formulations into mice, in an accordance with implementation of the present disclosure. [0032] Figure 11 illustrates a graph comparing the body weights of mice with respect to number of days after injecting lipid formulation, in an accordance with implementation of the present disclosure. [0033] Figure 12 illustrates a graph showing spleen weights of mice with respect to number of days after injecting lipid formulation, in an accordance with implementation of the present disclosure. [0034] Figure 13 illustrates the representative images of H&E (hematoxylin and eosin) analysis in mice, in an accordance with implementation of the present disclosure.
[0035] Figure 14 illustrates the representative images showing fibrosis in mice, in an accordance with implementation of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION [0036] Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all such steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features. Definitions: [0037] For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are collected here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below. [0038] The articles “a”, “an” and “the” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. [0039] The terms “comprise” and “comprising” are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as “consists of only”. [0040] Throughout this specification, unless the context requires otherwise the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or steps. [0041] The term “including” is used to mean “including but not limited to”, “including” and “including but not limited to” are used interchangeably.
[0042] In the structural formulae given herein and throughout the present disclosure, the following terms have been indicated meaning, unless specifically stated otherwise. [0043] Furthermore, the compounds of Formula I and Formula II can be its derivatives, analogs, complexes, tautomeric forms, stereoisomer’s, diastereomers, geometrical isomers, polymorphs, solvates, or pharmaceutically acceptable salts and compositions. [0044] As used herein, the term "substituted" is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents, for example, include those described hereinabove. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, the heteroatoms such as nitrogen may have hydrogen substituents, and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. [0045] The term “alkyl” refers to straight or branched aliphatic hydrocarbon groups having 1 to 22 carbon atoms, which are attached to the rest of the molecule by a single atom, which may be optionally substituted by one or more substituents. Preferred alkyl groups include, without limitation, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, hexadecyl, tetradecyl, octadecyl, and the like. [0046] The term “alkenyl” refers to straight or branched aliphatic hydrocarbon groups having 2 to 22 carbon atoms with at least one carbon-carbon double bond, which are attached to the rest of the molecule by a single atom, which may be optionally substituted by one or more substituents. Preferred alkenyl groups include, without limitation, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and the like. [0047] The term “alkynyl” refers to straight or branched aliphatic hydrocarbon groups having 2 to 22 carbon atoms with at least one carbon-carbon triple bond, which are attached to the rest of the molecule by a single atom, which may be optionally
substituted by one or more substituents. Preferred alkyl groups include, without limitation, ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like. [0048] The term “aryl” refers to aromatic radicals having 6 to 22 carbon atoms, which may be optionally substituted by one or more substituents. Preferred aryl groups include, without limitation, phenyl, naphthyl, indanyl, biphenyl, and the like. [0049] The term “alkoxy” refers to an alkyl group, as defined above, having an oxygen radical attached thereto. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy and the like. An "ether" is two hydrocarbons covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxy, such as can be represented by one of -O-alkyl, -O- alkenyl, -O-alkynyl. The alkoxy groups may be optionally substituted. [0050] The term “alkylhydroxy” refers to an alkyl group, as defined above, having a hydroxy radical attached thereto. Representative alkylhydroxy groups include - CH2OH, -C2H4OH, - C3H7OH, and the like. [0051] The term “carbocyclyl” refers to a saturated, unsaturated or partially saturated ring having 5 to 22 carbon atoms forming cyclic systems. Carbocyclic groups may be spiral or bridged systems, may be substituted. Carbocyclyl groups may be optionally substituted with one or more heteroatoms. Carbocyclyl may include cycloalkyl, aryl, heteroaryl having 5 to 22 carbon atoms and may be optionally substituted. [0052] The term “heterocyclyl” refers to a heterocyclic ring radical having 5 to 22 carbon atoms which may be optionally substituted by one or more substituents. The heterocyclyl ring radical may be attached to the main structure at any heteroatom or carbon atom that results in the creation of a stable structure. [0053] Furthermore, the term “heterocyclyl” refers to a stable 5 to 22 membered rings radical, which consists of carbon atoms and from one to five heteroatoms selected from nitrogen, phosphorus, oxygen, and sulfur. For purposes of this invention the heterocyclic ring radical may be monocyclic, bicyclic or tricyclic ring systems, and the nitrogen, phosphorus, carbon, or sulfur atoms in the heterocyclic ring radical may be
optionally oxidized to various oxidation states. In addition, the nitrogen atom may be optionally quaternized; and the ring radical may be partially or fully saturated. [0054] The term “cycloalkyl” refers to non-aromatic mono or polycyclic ring system of about 3 to 10 carbon atoms, which may be optionally substituted by one or more substituents. The polycyclic ring denotes hydrocarbon systems containing two or more ring systems with one or more ring carbon atoms in common, i.e., a spiro, fused or bridged structures. Preferred cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, groups. [0055] It is understood that included in the family of compounds of Formula (I) are isomeric forms including diastereoisomers, enantiomers, tautomers, and geometrical isomers in “E” or “Z” configurational isomer or a mixture of ‘E’ and ‘Z’ isomers. It is also understood that some isomeric form such as diastereomers, enantiomers and geometrical isomers can be separated by physical and/or chemical methods and by those skilled in the art. [0056] Compounds disclosed herein may exist as single stereoisomers, racemates and or mixtures of enantiomers and/or diastereomers. All such single stereoisomers, racemates and mixtures thereof are intended to be within the scope of the subject matter described. [0057] Compounds disclosed herein include isotopes of hydrogen, carbon, oxygen, fluorine, chlorine, iodine and sulfur which can be incorporated into the compounds, such as not limited to 2H (D), 3H (T), 11C, 13C, 14C, 15N, 18F, 35S, 36Cl and 125I. Compounds of this invention where in atoms were isotopically labeled for example radioisotopes such as 3H, 13C, 14C, and the like can be used in metabolic studies, kinetic studies and imaging techniques such as positron emission tomography used in understanding the tissue distribution of the drugs. Compounds of the invention where hydrogen is replaced with deuterium may improve the metabolic stability and pharmacokinetics properties of the drug such as in vivo half-life. Compounds of the invention where isotopically labeled 18F can be useful as PET imaging studies.
[0058] The phrase “pharmaceutically acceptable” refers to compounds or compositions that are physiologically tolerable and do not typically produce allergic or similar untoward reaction, including but not limited to gastric upset or dizziness when administered to subjects. [0059] Pharmaceutically acceptable salts forming part of this invention include salts derived from inorganic bases such as like Li, Na, K, Ca, Mg, Fe, Cu, Zn and Mn and ammonium, substituted ammonium salts, aluminum salts and the like.; salts of organic bases such as N, N’-diacetylethylenediamine, glucamine, triethylamine, choline, dicyclohexylamine, benzylamine, trialkylamine, thiamine, guanidine, diethanolamine, ^-phenylethylamine, piperidine, morpholine, pyridine, hydroxyethylpyrrolidine, hydroxyethylpiperidine and the like, salts also include amino acid salts such as glycine, alanine, cystine, cysteine, lysine, arginine, phenylalanine, guanidine etc. Salts may include acid addition salts where appropriate which are sulphates, nitrates, phosphates, perchlorates, borates, hydrohalides, acetates, tartrates, maleates, fumarates, citrates, succinates, palmoates, methanesulphonates, tosylates, benzoates, salicylates, hydroxynaphthoates, benzenesulfonates, ascorbates, glycerophosphates, ketoglutarates and the like. [0060] The term “polymorphs” refers to crystal forms of the same molecule, and different polymorphs may have different physical properties such as, for example, melting temperatures, heats of fusion, solubilities, dissolution rates and/or vibrational spectra as a result of the arrangement or conformation of the molecules in the crystal lattice. [0061] The compounds described herein may also exhibit polymorphism. This invention further includes different polymorphs of the compounds of the present invention. The term polymorph refers to a particular crystalline state of a substance, having particular physical properties such as X-ray diffraction, IR spectra, melting point and the like.
[0062] The compounds described herein can also be prepared in any solid or liquid physical form, for example, the compound can be in a crystalline form, in amorphous form and have any particle size. Furthermore, the compound particles may be micronized or nanosized, or may be agglomerated, particulate granules, powders, oils, oily suspensions or any other form of solid or liquid physical forms. [0063] The term “complexes” as used herein, can be interchangeably used as "coordination complex," or "metal coordination complex," and the like. It refers to a complex of an organic compound with a metal that can be empirically differentiated from a simple metal salt of the organic compound based on physiochemical and/or spectroscopic properties, with a coordination complex typically having enhanced covalency as compared to a salt. Without limitation "complexes" as used herein also involves a combination of coordinate covalent bonds and/or ionic bonds. As used herein, the term "complexes" also includes molecules that lack an ionic component (e.g., such as a neutral coordination complex prior to deprotonation, where pKa of the coordination complex falls within a physiologically acceptable range). [0064] The term “solvate” refers to a compound formed by the interaction of a solvent and a solute. In the present disclosure, the term solvate refers to a compound formed by the interaction of solvent and the compounds of Formula I of the present disclosure. [0065] The term “hydrate” refers to a solvate wherein the solvent is water. [0066] The term “receptor” refers to a region of tissue, or a molecule in a cell membrane, which responds specifically to a particular neurotransmitter, hormone, antigen, or other substance. [0067] The term "effective amount" means an amount of a compound or composition which is sufficient enough to significantly and positively modify the symptoms and/or conditions to be treated (e.g., provide a positive clinical response). The effective amount of an active ingredient for use in a pharmaceutical composition will vary with the particular condition being treated, the severity of the condition, the duration of the
treatment, the nature of concurrent therapy, the particular active ingredient(s) being employed, the particular pharmaceutically-acceptable excipient(s)/carrier(s) utilized, the route of administration, and like factors within the knowledge and expertise of the attending physician. [0068] The term “first lipid compound” refers to the compounds of Formula I as disclosed herein and are the targeting lipids of the present disclosure. These lipids are sensitive towards selective receptors and binds to the selective receptors. In the present disclosure, the first lipid compounds or the targeting lipids specifically binds to asialoglycoreceptors. [0069] The term “second lipid compound” refers to the compounds of Formula II as disclosed herein and are the cationic lipids of the present disclosure. These lipids possess positive charge and may exists in salt form. These are used to interact with negatively charged species in a cell and hence are capable of delivering specific therapeutic agent into the cells. [0070] The term “co-lipid” refers to lipid compounds which are binding sites for intra and intercellular proteins. These lipids serves as a primary cellular component. [0071] The term “asialoglycoreceptors” refers to a specific receptor present in the hepatic cells or liver cells [0072] The term “liposomes” refers to the formulation comprising the targeting lipid, cationic lipid with co-lipids. These mixture of lipids form a spherical vesicle which can act as a delivery vehicle for administration of nutrients and or any therapeutic agents. Liposomes may also act as pharmaceutically effective compound by itself. Liposomes may encapsulate therapeutic agents such as nucleic acids (RNA, mRNA, siRNA, DNA), locked nucleic acids, small molecules, drugs, proteins, chemotherapeutic agent, a cytotoxin, a steroid, an immunotherapeutic agent, a targeted therapeutic agent, or combinations thereof. [0073] The term “lipoplex” refers to a complex of nucleic acids mixed with lipids that spontaneously forms a vesicle containing therapeutic agent. In the present
disclosure, the lipoplexes are formed by mixing pDNA or mRNA with the lipid formulation or liposome of the present disclosure. [0074] The term “reconstituted formulation” refers to a stabilized liposomal formulation which are obtained by mixing dried liposome with a reconstituting agent. The term “reconstituting agent” refers to a buffer which has physiological pH and isotonicity. In the present disclosure, the reconstituted formulation comprises the formulation of the present disclosure and the reconstituting agent selected from saline, glucose, sucrose, or inulin [0075] The term “one or more pharmaceutically active compound” refers to other pharmaceutically active compound which can be used in combinatorial with the pharmaceutical composition of the present disclosure. The other pharmaceutically active compound may include but not limited to hydrophobic drugs including quercetin, 7-amino-4-methylcoumarin, paclitaxel, atorvastatin, and so on. [0076] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference. [0077] As discussed in the background, a formulation of liposome plays a significant role in bringing out all the essential advantageous features of the liposome as a carrier in a therapeutic method. A well-designed liposomal delivery system are capable of reducing the toxicity and increasing the potency of gene/nucleic acid/ drugs by optimizing gene/nucleic acid /drug delivery to target tissues. There are various properties such as stability, size, charge, hydrophobicity, interaction with serum proteins, and interaction with target/nontarget cell surfaces which needs to considered while designing the liposome. And when specific to a targeted delivery, the compatibility and binding property towards the targeted cell is crucial property to be considered. Accordingly, the present disclosure provides novel targeting lipids and
cationic lipids in specific to asialoglycoreceptors. The present disclosure provides a lipid formulation or liposome comprising the targeting lipids, cationic lipids and co- lipids. The present disclosure also provides process for preparing the lipids and the liposomes. The present disclosure further provides liposome for delivery of nucleic acids such as RNA, mRNA, SiRNA, DNA and locked nucleic acids, small molecules, drugs, proteins, chemotherapeutic agent, a cytotoxin, a steroid, an immunotherapeutic agent, or a targeted therapeutic agent to the hepatocytes for treating liver related diseases, condition or disorder. [0078] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for exemplification only. Functionally equivalent products, compositions, and methods are clearly within the scope of the disclosure, as described herein. A term once described, the same meaning applies for it, throughout the patent. [0079] In an embodiment of the present disclosure, there is provided a formulation comprising:. i. a first lipid compound of Formula I;
Formula I wherein
is selected from C5-22 alkyl, C5-22 carbocyclyl, or C5-22 heterocyclyl, wherein C5-22 alkyl, C5-22 carbocyclyl, or C5-22 heterocyclyl optionally further substituted with one or more substituents selected from hydroxy, C1-10 alkyl, C1-10 alkylhydroxy, -NH-C(O)-C1-10 alkyl, or C1-10 alkoxy;
and C1-10 alkoxy is optionally further substituted with one or more substituents selected from hydroxy, C1-10 alkyl, C1-10 alkylhydroxy, or -OC(O)R1; X is a bond or selected from -OR1-, -O-C(O)-N(R1)-, -N(R1)-C(O)-O-, - C(O)N(R1)-, -N(R1)-C(O)-, or -O-C(O)R1-; R and R’ are independently selected from C5-22 alkyl, C5-22 alkenyl, C5-22 alkynyl, or -C6-22 aryl; R1 is independently selected from hydrogen, C1-10 alkyl, C2-10 alkenyl, C2- 10alkynyl, C3-10 cycloalkyl, or -C6-22 aryl; m and m’ are independently selected from 1 to 22; and n and n’ are independently selected from 0 to 22; ii. a second lipid compound of Formula II:
Formula II wherein R3, and R4 are independently selected from hydrogen, hydroxy, amino, halo, cyano, or -C6-22 aryl; Y is a bond or selected from -OR7-, -O-C(O)-N(R7)-, -N(R7)-C(O)-O-, - C(O)N(R7)-, -N(R7)-C(O)-, or -O-C(O)R7-; R5, and R6 are independently selected from C5-22 alkyl, C5-22 alkenyl, C5-22 alkynyl, or C6-22 aryl; R7 is independently selected from hydrogen, C1-10 alkyl, C2-10 alkenyl, C2- 10alkynyl, C3-10 cycloalkyl, or -C6-22 aryl; and p, q, r, and s are independently selected from 1 to 15; and iii. a co-lipid
wherein the first lipid to the second lipid to the co-lipid is in the mole ratio range of 1:0.1:0.1 to 1:4:4. [0080] In an embodiment of the present disclosure, there is provided a formulation comprising: i. a first lipid compound of Formula I as disclosed herein; wherein is selected from C5-12 carbocyclyl, or C5-12 heterocyclyl, wherein C5-12 carbocyclyl, or C5-12 heterocyclyl optionally further substituted with one or more substituents selected from hydroxy, or C1-10 alkyl; X is a bond; R and R’ are independently selected from C7-18 alkyl; m and m’ are independently selected from 1 to 12; and n and n’ are independently selected from 0 to 12; ii. a second lipid compound of Formula II, wherein R3, and R4 are independently selected from hydrogen, hydroxy, amino, halo, cyano, or -C6-22 aryl; Y is a bond or selected from -OR7-, -O-C(O)-N(R7)-, -N(R7)-C(O)-O-, - C(O)N(R7)-, -N(R7)-C(O)-, or -O-C(O)R7-; R5, and R6 are independently selected from C5-22 alkyl, C5-22 alkenyl, C5-22 alkynyl, or C6-22 aryl; R7 is independently selected from hydrogen, C1-10 alkyl, C2-10 alkenyl, C2- 10alkynyl, C3-10 cycloalkyl, or -C6-22 aryl; and p, q, r, and s are independently selected from 1 to 15; and iii) a co-lipid wherein the first lipid to the second lipid to the co-lipid is in the mole ratio range of 1:0.1:0.1 to 1:4:4. [0081] In an embodiment of the present disclosure, there is provided a formulation comprising:.
i. a first lipid compound of Formula I as disclosed herein; wherein
is selected from C5-12 carbocyclyl, or C5-12 heterocyclyl, wherein C5-12 carbocyclyl, or C5-12 heterocyclyl optionally further substituted with one or more substituents selected from hydroxy, or C1-10 alkyl; X is a bond; R and R’ are independently selected from C7-18 alkyl; m and m’ are independently selected from 1 to 12; and n and n’ are independently selected from 0 to 12; ii. a second lipid compound of Formula II, wherein R3, and R4 are independently selected from hydrogen, or hydroxy; Y is a bond or selected from -C(O)N(R7), or -N(R7)-C(O)-; R5, and R6 are independently selected from C5-22 alkyl; R7 is independently selected from hydrogen, C1-10 alkyl; and p, q, r, and s are independently selected from 1 to 5; and; and iii) a co-lipid wherein the first lipid to the second lipid to the co-lipid is in the mole ratio range of 1:0.1:0.1 to 1:4:4. [0082] In an embodiment of the present disclosure, there is provided a formulation comprising: i. a first lipid compound of Formula I as disclosed herein; wherein
is selected from C5-10 heterocyclyl, wherein C5-10 heterocyclyl optionally further substituted with one or more of hydroxy; X is a bond; R and R’ are independently selected from C10-15 alkyl; m and m’ are independently selected from 1 to 2; and n and n’ are independently selected from 0 to 2; iii. a second lipid compound of Formula II, wherein R3, and R4 are independently selected from hydrogen, or hydroxy; Y is a bond or -N(R7)-C(O)-; R5, and R6 are
independently selected from C10-18 alkyl; R7 is hydrogen; and p, q, r, and s are independently selected from 1 to 5; and; and iii) a co-lipid wherein the first lipid to the second lipid to the co-lipid is in the mole ratio range of 1:0.1:0.1 to 1:4:4. [0083] In an embodiment of the present disclosure, there is provided a formulation as disclosed herein, wherein the formulation comprises (i) a first lipid compound of Formula I; (ii) a second lipid compound of Formula II; (iii) a co-lipid; and (iv) one or more therapeutic agent, wherein the first lipid to the second lipid to the co-lipid is in the mole ratio range of 1:0.5:0.5 to 1:4:4. In another embodiment of the present disclosure, there is provided a formulation as disclosed herein, wherein the first lipid to the second lipid to the co-lipid is in the mole ratio range of 1:1:1 to 1:4:4. In yet another embodiment of the present disclosure, there is provided a formulation as disclosed herein, wherein the first lipid to the second lipid to the co-lipid is in the mole ratio of 1:4:4. [0084] In an embodiment of the present disclosure, there is provided a formulation as disclosed herein, wherein the formulation comprises one or more therapeutic agent. [0085] In an embodiment of the present disclosure, there is provided a formulation as disclosed herein, wherein the formulation comprises (i) a first lipid compound of Formula I as disclosed herein; (ii) a second lipid compound of Formula II as disclosed herein; (iii) a co-lipid; and (iv) one or more therapeutic agent, wherein the first lipid to the second lipid to the co-lipid is in the mole ratio range of 1:0.1:0.1 to 1:4:4. [0086] In an embodiment of the present disclosure, there is provided a formulation as disclosed herein, wherein the co-lipid is selected from steroids, cholesterol, sitostanol, sitosterol, ß-sitosterol-d6, campesterol, campesterol-d6, desmosterol, desmosterol-d6, 7-dehydrodesmosterol, zymosterol, zymosterol-d5, zymostenol, zymostenol-d7, diosgenin, stigmasterol, lathosterol, lathosterol-d7, lanosterol, lanosterol-d6, lanostenol, dihydrolanosterol-d7, 14-demethyl-lanosterol, 14-demethyl-lanosterol-d6, 14-demethyl-14-dehydrolanosterol (FF-MAS), cholesterol sulfate,
dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulphate (DHEA sulphate), phosphatidylethanolamines, dilauroylphosphatidylethanolamine (DLPE), dimirystoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylethanolamine (DOPE), phosphatidylcholine, dilauroylphosphatidylcholine (DLPC), dimirystoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC) dioleoylphosphatidylcholine (DOPC), egg phosphatidylcholine (Egg PC), hydro egg phosphatidylcholine (Hydro egg PC), egg lyso phosphatidylcholine (Egg , Lyso PC), soy phosphatidylcholine (Soy PC), hydro soy phosphatidylcholine (Hydro Soy PC), soy lyso phosphatidylcholine (Soy Lyso PC), heart phosphatidylcholine (Heart PC), brain phosphatidylcholine (Brain PC), liver phosphatidylcholine (Liver PC), or combinations thereof. In another embodiment of the present disclosure, wherein the co-lipid is selected from steroids, cholesterol, dioleoylphosphatidylethanolamine (DOPE), phosphatidylcholine, dilauroylphosphatidylcholine (DLPC), dimirystoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC) dioleoylphosphatidylcholine (DOPC), or combinations thereof. In yet another embodiment of the present disclosure, wherein the co-lipid is selected from cholesterol (Chol), dioleoylphosphatidylethanolamine (DOPE), or combinations thereof. [0087] In an embodiment of the present disclosure, there is provided a formulation as disclosed herein, wherein the therapeutic agent is selected from nucleic acids (RNA, mRNA, SiRNA, DNA), locked nucleic acids, small molecules, drugs, proteins, chemotherapeutic agent, a cytotoxin, a steroid, an immunotherapeutic agent, a targeted therapeutic agent, or combinations thereof. In another embodiment of the present disclosure, wherein the therapeutic agent is selected from nucleic acids such as RNA, mRNA, SiRNA, DNA, or locked nucleic acids. [0088] In an embodiment of the present disclosure, there is provided a formulation as disclosed herein, wherein the formulation comprises (i) a first lipid compound of
Formula I as disclosed herein; (ii) a second lipid compound of Formula II as disclosed herein; (iii) a co-lipid; and (iv) one or more therapeutic agent, wherein the first lipid to the second lipid to the co-lipid is in the mole ratio range of 1:0.1:0.1 to 1:4:4; and the therapeutic agent is selected from nucleic acids (RNA, mRNA, SiRNA, DNA), locked nucleic acids, small molecules, drugs, proteins, chemotherapeutic agent, a cytotoxin, a steroid, an immunotherapeutic agent, a targeted therapeutic agent, or combinations thereof. [0089] In an embodiment of the present disclosure, there is provided a formulation as disclosed herein, wherein the formulation further comprises a reconstituting agent selected from saline, glucose, sucrose, or inulin. In another embodiment of the present disclosure, wherein the formulation further comprises glucose as the reconstituting agent. [0090] In an embodiment of the present disclosure, there is provided a formulation as disclosed herein, wherein the formulation comprises (i) a first lipid compound of Formula I as disclosed herein; (ii) a second lipid compound of Formula II as disclosed herein; (iii) a co-lipid; (iv) one or more therapeutic agent; and (v) a reconstituting agent selected from saline, glucose, sucrose, or inulin, wherein the first lipid to the second lipid to the co-lipid is in the mole ratio range of 1:0.1:0.1 to 1:4:4. [0091] In an embodiment of the present disclosure, there is provided a formulation as disclosed herein, wherein the molar ratio of the first lipid to the second lipid is in the range of 1:0.1 to 1:4. In another embodiment of the present disclosure, there is provided a formulation as disclosed herein, wherein the molar ratio of the first lipid to the second lipid is in the range of 1:1 to 1:4. In yet another embodiment of the present disclosure, there is provided a formulation as disclosed herein, wherein the molar ratio of the first lipid to the second lipid is of 1:4. [0092] In an embodiment of the present disclosure, there is provided a reconstituted formulation comprising the formulation comprising (i) a first lipid compound of Formula I; (ii) a second lipid compound of Formula II; and (iii) a co-lipid with 2-25%
(w/w) of a reconstituting agent, wherein the reconstituting agent is selected from saline, glucose, sucrose, or inulin. [0093] In an embodiment of the present disclosure, there is provided a reconstituted formulation, wherein the reconstituting agent is selected from saline, glucose, or sucrose. In another embodiment of the present disclosure, there is provided a reconstituted formulation, wherein the reconstituting agent is glucose in weight range of 3-10% (w/w). In one another embodiment of the present disclosure, there is provided a reconstituted formulation, wherein the reconstituting agent is 5% (w/w) of glucose. [0094] In an embodiment of the present disclosure, there is provided a reconstituted formulation comprising the formulation comprising (i) a first lipid compound of Formula I; (ii) a second lipid compound of Formula II; (iii) a co-lipid with 2-25% (w/w) of a reconstituting agent, and one or more therapeutic agent selected from nucleic acids (RNA, mRNA, SiRNA, DNA), locked nucleic acids, small molecules, drugs, proteins, chemotherapeutic agent, a cytotoxin, a steroid, an immunotherapeutic agent, a targeted therapeutic agent, or combinations thereof, wherein the reconstituting agent is selected from saline, glucose, sucrose, or inulin. [0095] In an embodiment of the present disclosure, there is provided a formulation as disclosed herein, wherein the first lipid compound is a targeting lipid which binds to a cell surface receptor; and the cell surface receptor is asialoglycoprotein receptors. [0096] In an embodiment of the present disclosure, there is provided a formulation as disclosed herein, wherein the formulation is a carrier to deliver the therapeutic agent to a targeted cell, wherein the therapeutic agent selected from nucleic acids (RNA, mRNA, SiRNA, DNA), locked nucleic acids, small molecules, drugs, proteins, chemotherapeutic agent, a cytotoxin, a steroid, an immunotherapeutic agent, a targeted therapeutic agent, or combinations thereof. [0097] In an embodiment of the present disclosure, there is provided a pharmaceutical composition comprising the formulation (i) a first lipid compound of Formula I as disclosed herein; (ii) a second lipid compound of Formula II as disclosed herein; and (iii) a co-lipid with pharmaceutically acceptable salts thereof.
[0098] In an embodiment of the present disclosure, there is provided a pharmaceutical composition comprising the formulation (i) a first lipid compound of Formula I as disclosed herein; (ii) a second lipid compound of Formula II as disclosed herein; and (iii) a co-lipid with one or more pharmaceutically active compound. [0099] In an embodiment of the present disclosure, there is provided a pharmaceutical composition as disclosed herein, wherein the composition is in the form selected from a tablet, capsule, powder, syrup, solution, aerosol, or suspension. [0100] In an embodiment of the present disclosure, there is provided a formulation as or the pharmaceutical composition as disclosed herein, for use in the manufacture of a medicament for treatment or prevention of a condition or a disorder or a disease mediated by asialoglycoprotein receptor. [0101] In an embodiment of the present disclosure, there is provided a formulation as or the pharmaceutical composition as disclosed herein, for use in the treatment and/or prevention of a condition mediated by asialoglycoprotein receptor. [0102] In an embodiment of the present disclosure, there is provided a process for preparing the formulation comprising (i) a first lipid compound of Formula I as disclosed herein; (ii) a second lipid compound of Formula II as disclosed herein; and (iii) a co-lipid, the process comprising: mixing stoichiometric ratios of the first lipid compound with the second lipid compound and the co-lipid, wherein the first lipid to the second lipid to the co-lipid is in the mole ratio range of 1:0.1:0.1 to 1:4:4. [0103] In an embodiment of the present disclosure, there is provided a process for preparing the formulation comprising (i) a first lipid compound of Formula I as disclosed herein; (ii) a second lipid compound of Formula II as disclosed herein; and (iii) a co-lipid, the process comprising: mixing stoichiometric ratios of the first lipid compound with the second lipid compound and the co-lipid in the presence of one or more first solvent selected from chloroform, dichloromethane, tetrahydrofuran, acetonitrile, methanol, ethanol, ethyl acetate,or combinations thereof to obtain the formulation, wherein the first lipid to the second lipid to the co-lipid is in the mole ratio range of 1:0.1:0.1 to 1:4:4.
[0104] In an embodiment of the present disclosure, there is provided a first lipid compound of Formula I or its pharmaceutically acceptable salts, complexes, hydrates, solvates, tautomers, polymorphs, stereoisomers, pharmaceutically active derivatives thereof as disclosed herein,
Formula I wherein
is selected from C5-22 alkyl, C5-22 carbocyclyl, or C5-22 heterocyclyl, wherein C5-22 alkyl, C5-22 carbocyclyl, or C5-22 heterocyclyl optionally further substituted with one or more substituents selected from hydroxy, C1-10 alkyl, C1-10 alkylhydroxy, -NH-C(O)-C1-10 alkyl, or C1-10 alkoxy; and C1-10 alkoxy is optionally further substituted with one or more substituents selected from hydroxy, C1-10 alkyl, C1-10 alkylhydroxy, or -OC(O)R1; X is a bond or selected from -OR1-, -O-C(O)- N(R1)-, -N(R1)-C(O)-O-, -C(O)N(R1)-, -N(R1)-C(O)-, or -O-C(O)R1-; R and R’ are independently selected from C5-22 alkyl, C5-22 alkenyl, C5-22 alkynyl, or -C6-22 aryl; R1 is independently selected from hydrogen, C1-10 alkyl, C2-10 alkenyl, C2-10alkynyl, C3- 10 cycloalkyl, or -C6-22 aryl; m and m’ are independently selected from 1 to 22; and n and n’ are independently selected from 0 to 22. [0105] In an embodiment of the present disclosure, there is provided the compound of Formula I as disclosed herein, wherein is selected from
[0106] In an embodiment of the present disclosure, there is provided the compound of Formula I as disclosed herein, wherein the compound is
X is a bond or selected from -OR1-, -O-C(O)-N(R1)-, -N(R1)-C(O)-O-, -C(O)N(R1)-, - N(R1)-C(O)-, or -O-C(O)R1-; R and R’ are independently selected from C5-22 alkyl, C5- 22 alkenyl, C5-22 alkynyl, or -C6-22 aryl; R1 and R2 are independently selected from hydrogen, C1-10 alkyl, C2-10 alkenyl, C2-10alkynyl, C3-10 cycloalkyl, or -C6-22 aryl; m and m’ are independently selected from 1 to 22; and n and n’ are independently selected from 0 to 22. [0107] In an embodiment of the present disclosure, there is provided the compound of Formula I as disclosed herein, wherein the compound is X is a bond or selected from - OR1-, -O-C(O)-N(R1)-, -N(R1)-C(O)-O-, -C(O)N(R1)-, -N(R1)-C(O)-, or -O-C(O)R1-; R and R’ are independently selected from C5-22 alkyl; R1 and R2 are independently selected from hydrogen, or C1-10 alkyl,; m and m’ are independently selected from 5 to 22; n is independently selected from 5 to 22 and n’ is selected from 0 to 5. [0108] In an embodiment of the present disclosure, there is provided the compound of Formula I as disclosed herein, wherein the compound is selected from a) (3S,4R,5S,6S)-6-((4-((ditetradecylamino)methyl)-1H-1,2,3-triazol-1- yl)methyl)tetrahydro-2H-pyran-2,3,4,5-tetraol; b) (3S,4R,5S,6S)-6-((4- ((dihexadecylamino)methyl)-1H-1,2,3-triazol-1-yl)methyl)tetrahydro-2H-pyran-
2,3,4,5-tetraol; and c) (3S,4R,5S,6S)-6-((4-((dioctadecylamino)methyl)-1H-1,2,3- triazol-1-yl)methyl)tetrahydro-2H-pyran-2,3,4,5-tetraol. [0109] In an embodiment of the present disclosure, there is provided a second lipid compound of Formula II or its pharmaceutically acceptable salts, complexes, hydrates, solvates, tautomers, polymorphs, stereoisomers, pharmaceutically active derivatives thereof as disclosed herein,
Formula II wherein R3, and R4 are independently selected from hydrogen, hydroxy, amino, halo, cyano, or -C6-22 aryl; Y is a bond or selected from -OR7-, -O-C(O)-N(R7)-, -N(R7)- C(O)-O-, -C(O)N(R7)-, -N(R7)-C(O)-, or -O-C(O)R7-; R5, and R6 are independently selected from C5-22 alkyl, C5-22 alkenyl, C5-22 alkynyl, or C6-22 aryl; R7 is independently selected from hydrogen, C1-10 alkyl, C2-10 alkenyl, C2-10alkynyl, C3-10 cycloalkyl, or - C6-22 aryl; and p, q, r, and s are independently selected from 1 to 15. [0110] In an embodiment of the present disclosure, there is provide second lipid compound of Formula II as disclosed herein, wherein the compound is selected from (a) N,N-dimethyl-2-tetradecanamido-N-(2-tetradecanamidoethyl)ethan-1-aminium chloride; (b) N,N-dimethyl-2-palmitamido-N-(2-palmitamidoethyl)ethan-1-aminium chloride; (c) N,N-dimethyl-2-stearamido-N-(2-stearamidoethyl)ethan-1-aminium chloride; (d) 2-hydroxy-N-methyl-N,N-bis(2-tetradecanamidoethyl)ethan-1-aminium chloride; (e) 2-hydroxy-N-methyl-N,N-bis(2-palmitamidoethyl)ethan-1-aminium chloride; (f) 2-hydroxy-N-methyl-N,N-bis(2-stearamidoethyl)ethan-1-aminium chloride; (g) 1-(bis(2-hydroxyethyl)(2-tetradecanamidoethyl)-l4-azanyl)-2- tetradecanamidoethan-1-ylium chloride; (h) 1-(bis (2-hydroxyethyl) (2-
palmitamidoethyl)-l4-azanyl)-2-palmitamidoethan-1-ylium chloride; and (i) 1-(bis (2- hydroxyethyl) (2-stearamidoethyl)-l4-azanyl)-2-stearamidoethan-1-ylium chloride. [0111] In an embodiment of the present disclosure, there is provided a process for preparing the compound of Formula I as disclosed herein, the process comprising: reacting compound of Formula A with compound of Formula B in the presence of a first reagent and a second solvent to result in a compound of Formula C; and treating the compound of Formula C in the presence of a second reagent at a temperature in the range of -5 to 30° C to obtain the compound of Formula I
wherein
is selected from C5-22 alkyl, C5-22 carbocyclyl, or C5-22 heterocyclyl, wherein C5-22 alkyl, C5-22 carbocyclyl, or C5-22 heterocyclyl optionally further substituted with one or more substituents selected from hydroxy, halogen, azide, C1-10 alkyl, C1-10 alkylhydroxy, -NH-C(O)-C1-10 alkyl, or C1-10 alkoxy. [0112] In an embodiment of the present disclosure, there is provided a process for preparing the compound of Formula I as disclosed herein, wherein the first reagent is selected from copper sulphate, sodium ascorbate, or combinations thereof; the second solvent is selected from water, tetrahydrofuran, or combinations thereof; and the second reagent is selected from ether, hydrochloric acid, or combinations thereof.
[0113] In an embodiment of the present disclosure, there is provided a process for preparing the compound of Formula I as disclosed herein, the process comprising
[0114] In an embodiment of the present disclosure, there is provided a process for preparing the compound of Formula II as disclosed herein, the process comprising: reacting compound of Formula D with a compound of Formula E in the presence of a reducing agent at a temperature in the range of 120 to 140 °C to obtain a compound of Formula F; and converting the compound of Formula F in the presence of a third reagent to obtain the compound of Formula II,
wherein X is hydrogen or C1-6 alkyl. [0115] In an embodiment of the present disclosure, there is provided a process for preparing the compound of Formula II as disclosed herein, wherein the reducing agent is selected from sodium borohydride, sodium cyanoborohydride, lithium aluminum hydride, diborane, DIBAL-H, lithium tetrahydridoaluminate, lithium tri-tert- butoxyaluminum hydride, sodium dithionite, sodium hydrogensulfite, sodium
hydrosulfite, dodium hydroxymethanesulfinate, sodium hypophosphite, sodium tetrahydroborate, sodium triacetoxyborohydride, triethoxysilane, triethylphosphine, triethylsilane trimethylamine borane, trimethylphoshpine, trimethylphosphite, triphenylphosphine, or combinations thereof; and the third reagent is selected from methyl iodide, ethyl hypochlorite, potassium carbonate, or combinations thereof. [0116] In an embodiment of the present disclosure as disclosed herein, there is provided a method of treatment or prevention of a condition, or a disease, or a disorder, the method comprising administering the formulation, or the pharmaceutical composition as disclosed herein, to a subject in need thereof. [0117] In an embodiment of the present disclosure, there is provided a method introducing one or more therapeutic agent into a cell, comprising contacting a eukaryotic cell with the formulation, or the pharmaceutical composition as disclosed herein. [0118] In an embodiment of the present disclosure, there is provided a method for the treatment or prevention of disease or disorder comprising administering to a subject suffering from disease or disorder a therapeutically effective amount of the formulation or the pharmaceutical composition as disclosed herein, with other clinically relevant cytotoxic agents or non-cytotoxic agents to a subject in need thereof. [0119] In an embodiment of the present disclosure, there is provided a system for delivering one or more therapeutic agent to a subject in need, comprising the formulation, or the pharmaceutical composition as disclosed herein, and one or more therapeutic agent, wherein the weight ratio of the formulation to the therapeutic agent is in the range of 1:1 to 50:1. [0120] In an embodiment of the present disclosure, there is provided use of the compounds, or the pharmaceutical composition as disclosed herein, for the treatment or prevention of disease or disorder, together with other clinically relevant cytotoxic agents or non-cytotoxic agents. [0121] In an embodiment of the present disclosure, there is provided a method as disclosed herein, wherein the disease or disorder or condition is selected from
haemophilia, Niemann-Pick type C2, Factor VII deficiency, α-1-antitrypsin deficiency, Familial tyrosinemia, liver cancer, liver dysfunction, hepatitis, cirrhosis, liver failure, ascites, gallstones, fatty liver disease, autoimmune hepatitis, primary sclerosing cholangitis, primary biliary cirrhosis (PBC), hemochromatosis, wilson’s disease, or hepatocellular carcinoma. [0122] Although the subject matter has been described in considerable detail with reference to certain examples and implementations thereof, other implementations are possible. EXAMPLES [0123] The following examples provide the details about the synthesis, activities, and applications of the compounds of the present disclosure. It should be understood the following is representative only, and that the invention is not limited by the details set forth in these examples. Preparation of the first lipid compound of Formula I [0124] The first lipid compounds of Formula I are prepared by Scheme 1 depicted below. In a specific example, Scheme 1a and 1b represent the preparation process of the compounds of Formula I.
Scheme 1
Scheme 1c Reagents: a) ZnCl2-H2SO4, Acetone, RT, 6hrs; b) Tosyl chloride, Pyridine, 50 oC, 15hrs; c) NaN3, DMF, 120 oC, 16hrs; d) K2CO3, EtOAc, 80 oC, 48hrs; e) CuSO4.5H2O, Sodium ascorbate, Water:THF (1:1 v/v), RT, 16hrs; f) Ether-HCl, 0 oC to RT, 24hrs Synthesis of ((5S,5aR,8aR,8bS)-2,2,7,7-tetramethyltetrahydro-5H- bis([1f,3]dioxolo)[4,5-b:4',5'-d]pyran-5-yl)methanol (I): [0125] Galactose (5g, 1eq, 27.75mmoles) was taken in a 160 mL of acetone further ZnCl2 (4.53g, 1.2 eq, 33.3 mmoles) was added. The reaction mixture was stirred for 5 minutes then 10 mL of H2SO4 was drop wise added to reaction mixture. The reaction mixture was allowed to stirring for 6 h at RT. Progress of the reaction was monitored by TLC. After completion of the reaction, reaction mixture was neutralized with aqueous NaHCO3 solution, then solid was filtered, filtrate was concentrated, crude product was extracted with ethyl acetate, given water wash then dried, and then crude
compound was purified by using 60/120 mesh silica gel column chromatography. The pure compound was collected at 2.5% methanol in DCM (3.27 g, 45% yield, TLC: Rf =0.5 in 5% methanol in DCM). ESI-MS m/z: Calculated mass 260.13 for C12H20O6, found mass 279.05 [M+H2O]+ 1H NMR (400MHz, CDCl3): δ 1.34 (s, 6H), 1.46 (s, 3.09H), 1.54 (s1.54H), 3.84-3.87 (m, 2.08H), 4.29 (s, 0.97H), 4.33-4.35 (s, 1.04 H), 4.61-4.62 (s, 0.96H), 5.57-5.58 (s, 0.90H), 7.54 (s, 1H) Synthesis of (5R,5aS,8aR,8bS)-2,2,7,7-tetramethyl-5- ((methylsulfonyl)methyl)tetrahydro-5H-bis([1,3]dioxolo)[4,5-b:4',5'-d]pyran (II): [0126] Compound I (500mg, 1eq, 1.92 mmoles) was taken in a 2mL of pyridine then DMAP (23mg, 0.1 eq, 0.19mmoles) and methanesulfonyl chloride (621mg, 1.7 eq, 3.26 mmoles) was added to reaction mixture. The reaction mixture was allowed to stir at 50oC for 5 h. Progress of the reaction was monitored by TLC. After 5 h 50% of compound I was consumed, 0.5 equal in g methanesulfonyl chloride and DMAP was added to the reaction mixture and then allowed to stir at same conditions for 10 h. After completion of the reaction, reaction mixture was quenched with water then added ethyl acetate, washed with NH4Cl given water wash, then organic layer was passed through sodium sulphate, solvent was concentrated and dried under vacuum. Crude compound was purified by column chromatography. The pure compound was collected at 20% ethyl acetate in hexane (728mg, 91% yield, TLC: Rf = 0.5 in 10% ethyl acetate in hexane) ESI-MS m/z: Calculated mass 322.11 for C13H22O7S, found [M+H]+ 1H NMR (400MHz, CDCl3): δ 1.34 (s, 6H), 1.46 (s, 3H), 1.55 (s, 2.96H), 3.14 (s, 3.09), 4.12 (m, 1.12H), 4.26 (m, 1.04H), 4.38 (m, 3.04H), 4.64 (m, 0.98H), 5.55 (0.96H).
Synthesis of (5S,5aR,8aR,8bS)-5-(azidomethyl)-2,2,7,7-tetramethyltetrahydro- 5H-bis([1,3]dioxolo)[4,5-b:4',5'-d]pyran (III): [0127] Compound II (338mg, 1eq, 0.815 mmoles) was taken in 4mL DMF solvent further stirred for 5 minutes then NaN3(212mg, 4eq, 3.26 mmoles) was added to reaction mixture and then reaction was allowed to stir at 120oC for 16 hrs. Progress of the reaction was monitored by TLC after completion of the reaction water was added to reaction mixture then extracted with ethyl acetate (3 times, each time with 5ml solvent) and then solvent was concentrated, dried under vacuum at 35oC, after that yellow liquid compound was stored at 4oC (210 mg, 90% yield, TLC: Rf = 0.5 in 20% ethyl acetate in hexane). ESI-MS m/z: Calculated mass 285.132 for C12H19N3O5, found [M+H]+ Synthesis of N-(prop-2-yn-1-yl)-N-tetradecyltetradecan-1-amine (IV): [0128] Tetradecyl bromide (10.08g, 4eq, 36.35 mmoles) compound was dissolved in ethyl acetate, and then added propargyl amine (500mg, 1eq, 9.09mmoles) and K2CO3(5.01g, 4eq, 36.30 mmoles) was added to reaction mixture. The reaction mixture was allowed to stir at 800C for 48 hrs in reflux condition and the progress of the reaction was monitored by TLC. After completion of the reaction, K2CO3 was filtered by using Whatman filter paper and then crude product was concentrated by using rotary evaporator. Crude compound was purified by column chromatography by using 60/120 mesh silica gel and the pure yellow liquid compound was collected at 15% ethyl acetate in hexane (1.6g, 40% yield, TLC: Rf = 0.4 in 10% ethyl acetate in hexane). Synthesis of N-hexadecyl-N-(prop-2-yn-1-yl)hexadecan-1-amine: [0129] This compound was synthesized by a similar procedure as described for compound IV. (2.06 g, 45% yield, TLC: Rf = 0.4 in 10% ethyl acetate in hexane) ESI-MS m/z: Calculated mass 503.94 for C35H69N, found mass 504.45 [M+H]+
1H NMR (400MHz, CDCl3): δ 0.89 (t, 6H), 1.26 (m, 51.89), 1.44 (m, 4.05H), 2.14 (s, 0.90H), 2.43-2.46 (m, 3.82H), 3.40 (s, 1.88). Synthesis of N-octadecyl-N-(prop-2-yn-1-yl)octadecan-1-amine [0130] This compound was synthesized by a similar procedure as described for compound IV. (2.06 g, 45% yield, TLC: Rf = 0.4 in 10% ethyl acetate in hexane) Synthesis of N-tetradecyl-N-((1-(((5S,5aR,8aR,8bS)-2,2,7,7- tetramethyltetrahydro-5H-bis([1,3]dioxolo)[4,5-b:4',5'-d]pyran-5-yl)methyl)-1H- 1,2,3-triazol-4-yl)methyl)tetradecan-1-amine (V): [0131] Compound III (79 mg, 1.25eq, 0.279 mmoles) and compound IV (100mg, 1eq, 0.223 mmoles) were dissolved in a THF: Water (1:1 v/v ratio) solution and then CuSO4 (5.4 mg, 0.1mg, 0.022 mmoles) and sodium ascorbate (3mg, 0.1eq, 0.19mmoles) was added to the reaction mixture and then allowed to stir at RT for 16hrs, progression of the reaction was monitored by TLC. After completion of the reaction, the reaction mass was extracted with 10% methanol in DCM and the organic solvent was concentrated under vacuum. The crude compound was purified by column chromatography by using 60/120 mesh silica gel and the pure viscous liquid compound was collected at 4% methanol in DCM. (120 mg, 74% yield, TLC: Rf = 0.5 in 4% methanol in DCM) Synthesis of N-hexadecyl-N-((1-(((5S,5aR,8aR,8bS)-2,2,7,7- tetramethyltetrahydro-5H-bis([1,3]dioxolo)[4,5-b:4',5'-d]pyran-5-yl)methyl)-1H- 1,2,3-triazol-4-yl)methyl)hexadecan-1-amine: [0132] This compound was synthesized by a similar procedure as described for compound V. (190mg, 81% yield, TLC: Rf = 0.5 in 4% methanol in DCM) ESI-MS m/z: Calculated mass 788.68 for C47H88N4O5, found [M+H]+ Synthesis of N-octadecyl-N-((1-(((5S,5aR,8aR,8bS)-2,2,7,7- tetramethyltetrahydro-5H-bis([1,3]dioxolo)[4,5-b:4',5'-d]pyran-5-yl)methyl)-1H- 1,2,3-triazol-4-yl)methyl)octadecan-1-amine:
[0133] This compound was synthesized by a similar procedure as described for compound V. (90mg, 79% yield, TLC: Rf = 0.5 in 4% methanol in DCM) Synthesis of (3S,4R,5S,6S)-6-((4-((ditetradecylamino)methyl)-1H-1,2,3-triazol-1- yl)methyl)tetrahydro-2H-pyran-2,3,4,5-tetraol (VI): [0134] Compound V (120mg, 1eq, 0.163 mmoles) was taken in round bottom flask after that cooled it to 0oC and then added 2.5 ml of HCl-ether solution and then reaction mixture was allowed to stir at RT for 24 hrs, after that reaction mass was concentrated then washed with ether and dried under vacuum, product was obtained. (80mg, 71% yield, TLC: Rf = 0.5 in 5% methanol in chloroform) Synthesis of (3S,4R,5S,6S)-6-((4-((dihexadecylamino)methyl)-1H-1,2,3-triazol-1- yl)methyl)tetrahydro-2H-pyran-2,3,4,5-tetraol: [0135] This compound was synthesized by a similar procedure as described for compound VI. (192mg, 84% yield, TLC: Rf = 0.5 in 5% methanol in chloroform) ESI-MS m/z: Calculated mass 708.61 for C41H80N4O5, found [M+H]+ 1H NMR (400MHz, CDCl3): δ 0.88 (t, 6H), 1.26 (m, 34.71H), 1.45 (m, 1.15H), 2.06 (m, 1.04H), 2.17 (s, 2.02H), 3.49 (s, 0.81H). Synthesis of (3S,4R,5S,6S)-6-((4-((dioctadecylamino)methyl)-1H-1,2,3-triazol-1- yl)methyl)tetrahydro-2H-pyran-2,3,4,5-tetraol: [0136] This compound was synthesized by a similar procedure as described for compound VI. (155mg, 86% yield, TLC: Rf = 0.5 in 5% methanol in chloroform) Preparation of the second lipid compound of Formula II [0137] The second lipid compounds of Formula II are prepared by the Scheme 2 depicted below. In specific example, Scheme 2a represent the preparation process of the compounds of Formula II.
Scheme 2a Reagents: a) CAL-B, Fatty acid, Toluene, 80oC, 96h; b) CH3I, Excess, RT, 12h; c) Amberlite IRA 400 Cl , MeOH and CHCl3; d) 2-chloroethanol, Excess, 80oC, Reflux, 24h; e) CAL-B, Fatty acid, Toluene, 80oC, 96h; f) 2-chloroethanol, Excess, 80oC, Reflux, 12h Synthesis of Secondary amines N,N'-(azanediylbis(ethane-2,1-diyl))ditetradecanamide (13SA): [0138] In a 100 mL single-neck round-bottom flask, diethylenetriamine (1 eq, 0.1 g,0.97 mmol) and dodecanoic acid (2.3 eq, 0.446 g, 2.32 mmol) were dissolved in dry toluene. CAL-B enzyme (10% w/w) was added and the reaction mixture was stirred at 50oC for 96 h. The reaction mixture was filtered off to remove the enzyme. To the filtrate CAL-B enzyme (10% w/w) was added again and reaction continued at 50oC for
a further period of 48 h. The reaction mixture was filtered and concentrated. The residue upon column chromatographic purification (using 60-120 mesh size silica gel and 5:95 methanol: chloroform as eluent) afforded intermediate IA as a white solid (0.29 g, 65% yield, Rf= 0.2, 5:95 methanol: chloroform, v/v). 1H NMR of (CDCl3, 400 MHz): δ/ppm = 0.88 [t, 6H, CH3-(CH 2)12-]; 1.27 [m, 40H, CH3-(CH2)10-CH2-CH2-]; 1.65 [m, 4H, CH3-(CH 2)10-CH2-CH2-];2.20[t, 4H, CH3-(CH 2)10-CH2-CH2-]; 2.78[t,4H, -CO-NH-CH2-CH2-NH-CH2-CH2-NH-CO-]; 3.36[t, 4H, - CO-NH-CH2-CH2-NH-CH2-CH2-NH-CO-]; 6.05[t, 2H, -CO-NH-CH2-CH2-NH-CH2- CH2-NH-CO-], ESI-MS m/z: calcd523.89 (for C32H65N3O2), found 525[M+H]+ N,N'-(azanediylbis(ethane-2,1-diyl))dipalmitamide (15SA): [0139] This compound was synthesized by a similar procedure as described for Intermediate IA compound (0.4 g, 71% yield, Rf=0.3, 5:95 methanol : chloroform, v/v). 1H NMR of (CDCl3, 400 MHz): δ = 0.88 [t, 6H, CH3-(CH 2)14-]; 1.27 [m, 48H, CH3- (CH2)12-CH2-CH2-]; 1.65 [m, 4H, CH3-(CH 2)12-CH2-CH2-]; 2.20[t, 4H, CH3-(CH 2)12- CH2-CH2-]; 2.78[t,4H, -CO-NH-CH2-CH2-NH-CH2-CH2-NH-CO-]; 3.36[t, 4H, -CO- NH-CH2-CH2-NH-CH2-CH2-NH-CO-]; 6.05[t, 2H, -CO-NH-CH2-CH2-NH-CH2-CH2- NH-CO-], ESI-MS m/z: calcd580.00 (for C36H73N3O2), found 581[M+H]+ N,N'-(azanediylbis(ethane-2,1-diyl))distearamide(17SA): [0140] This compound was synthesized by a similar procedure as described for Intermediate IA compound (0.417 g, 74% yield, Rf=0.4, 5:95 methanol:chloroform, v/v). 1H NMR of (CDCl3, 400 MHz): δ/ppm = 0.88 [t, 6H, CH3-(CH 2)16-]; 1.27 [m, 56H, CH3-(CH2)14-CH2-CH2-]; 1.65 [m, 4H, CH3-(CH 2)14-CH2-CH2-]; 2.20[t, 4H, CH3-(CH 2)14-CH2-CH2-]; 2.50 2.78[t,4H, -CO-NH-CH2-CH2-NH-CH2-CH2-NH-CO-]; 3.36[t, 4H, -CO-NH-CH2-CH2-NH-CH2-CH2-NH-CO-]; 6.05[t, 2H, -CO-NH-CH2-CH2-NH- CH2-CH2-NH-CO-], ESI-MS m/z: calcd636.11 (for C40H81N3O2), found 637 [M]+
Synthesis of Tertiary amines N,N'-((methylazanediyl)bis(ethane-2,1-diyl))ditetradecanamide (13TA): [0141] This compound was synthesized by a similar procedure as described for Intermediate IA compound (0.317 g, 69% yield, Rf=0.2, 5:95 methanol:chloroform, v/v). 1H NMR of (CDCl3, 400 MHz): δ = 0.87 [t, 6H, CH3-(CH 2)12-]; 1.25 [m, 40H, CH3- (CH2)10-CH2-CH2-]; 1.61 [m, 4H, CH3-(CH 2)10-CH2-CH2-]; 2.19 [t, 4H, CH3-(CH 2)10- CH2-CH2-]; 2.24 [s, 3H, , -CO-NH-CH2-CH2-NCH3-CH2-CH2-NH-CO-]; 2.50 [t,4H, - CO-NH-CH2-CH2-NH-CH2-CH2-NH-CO-];3.34 [t, 4H, -CO-NH-CH2-CH2-NH-CH2- CH2-NH-CO-]; 6.05[t, 2H, -CO-NH-CH2-CH2-NH-CH2-CH2-NH-CO-], ESI-MS m/z: calcd537.92 (for C33H67N3O2), found 539 [M+H]+ N,N'-((methylazanediyl)bis(ethane-2,1-diyl))dipalmitamide (15TA): [0142] This compound was synthesized by a similar procedure as described for Intermediate IA compound (0.365 g, 72% yield, Rf=0.3, 5:95 methanol:chloroform, v/v). 1H NMR of (CDCl3, 400 MHz): δ/ppm = 0.87 [t, 6H, CH3-(CH 2)14-]; 1.25 [m, 40H, CH3-(CH2)12-CH2-CH2-]; 1.61 [m, 4H, CH3-(CH 2)12-CH2-CH2-]; 2.19 [t, 4H, CH3-(CH 2)12-CH2-CH2-]; 2.24 [s, 3H, , -CO-NH-CH2-CH2-NCH3-CH2-CH2-NH-CO-]; 2.50 [t,4H, -CO-NH-CH2-CH2-NH-CH2-CH2-NH-CO-]; 3.34 [t, 4H, -CO-NH-CH2-CH2- NH-CH2-CH2-NH-CO-]; 6.05[t, 2H, -CO-NH-CH2-CH2-NH-CH2-CH2-NH-CO-], ESI-MS m/z: calcd594.03 (for C37H75N3O2) N,N'-((methylazanediyl)bis(ethane-2,1-diyl))distearamide (17TA): [0143] This compound was synthesized by a similar procedure as described for Intermediate IA compound (0.411 g, 74% yield, Rf=0.4, 5:95 methanol:chloroform, v/v). 1H NMR of (CDCl3, 400 MHz): δ/ppm = 0.87 [t, 6H, CH3-(CH 2)16-]; 1.25 [m, 40H, CH3-(CH2)14-CH2-CH2-]; 1.61 [m, 4H, CH3-(CH 2)14-CH2-CH2-]; 2.19 [t, 4H, CH3-(CH 2)14-CH2-CH2-]; 2.24 [s, 3H, , -CO-NH-CH2-CH2-NCH3-CH2-CH2-NH-CO-]; 2.50
[t,4H, -CO-NH-CH2-CH2-NH-CH2-CH2-NH-CO-]; 3.34 [t, 4H, -CO-NH-CH2-CH2- NH-CH2-CH2-NH-CO-]; 6.05[t, 2H, -CO-NH-CH2-CH2-NH-CH2-CH2-NH-CO-] ESI-MS m/z: calcd650.13 (for C41H83N3O2), found 651[M]+ Synthesis of Quaternary amines
Formula IIa N,N-dimethyl-2-tetradecanamido-N-(2-tetradecanamidoethyl)ethan-1-aminium chloride (13DME): [0144] Intermediate IA compound (1eq, 0.1g, 0.190 mmoles) was stirred with excess methyl iodide (1 mL) and K2CO3 (2eq, 0.52g, 0.376 mmoles) for 12 h at room temperature. After that the reaction mixture was filtered, concentrated and the residue was purified by column chromatographic purification (using 60 to 120 mesh size silica gel and 6% methanol in chloroform was used as eluent) to give white solid compound which was then passed through chloride ion exchange chromatography (using Amberlite IRA-400Cl resin and methanol as eluent) yielded compound 16DME as a white solid (0.94 g, 90% yield, Rf = 0.3 in 10% methanol in chloroform, v/v) 1H NMR of (CDCl3, 400 MHz): δ/ppm = 0.87 [t, 6H, CH3-(CH 2)12-]; 1.24 [m, 40H, CH3-(CH2)10-CH2-CH2-]; 1.60 [m, 4H, CH3-(CH 2)10-CH2-CH2-]; 2.27 [t, 4H, CH3-(CH 2)10-CH2-CH2-]; 3.29 [S, 6H, -CO-NH-CH2-CH2-N+ (CH3)2-CH2-CH2-NH-CO-]; 3.79 [t, 8H, -CO-NH-CH2-CH2-N+ (CH3)2-CH2-CH2-NH-CO-]; 7.53[t, 2H, -NH-CO-CH2- CH2-N+ (CH3)2-CH2-CH2-CO-NH-]; ESI-MS m/z: calcd552.95 (for C34H70N3O2)+, found 553[M]+ ESI-HRMS: 552.5458 (calculated mass for C34 H70 O2 N3 = 552.5463)
HPLC- Purity: 98.55% N,N-dimethyl-2-palmitamido-N-(2-palmitamidoethyl)ethan-1-aminium chloride (15DME): [0145] This compound was synthesized by a similar procedure as described for 13DME compound (0.88 g, 85% yield, Rf= 0.4, 10:90 methanol:chloroform, v/v). 1H NMR of (CDCl3, 400 MHz): δ/ppm = 0.87 [t, 6H, CH3-(CH 2)14-]; 1.24 [m, 48H, CH3-(CH2)12-CH2-CH2-]; 1.60 [m, 4H, CH3-(CH 2)12-CH2-CH2-];2.27 [t, 4H, CH3-(CH 2)12-CH2-CH2-]; 3.29 [S, 6H, -CH2-CH2-N+ (CH3)2-CH2-CH2-]; 3.79 [t, 8H, -CO-CH2- CH2-N+ (CH3)2-CH2-CH2-CO-]; 7.53[t, 2H, -NH-CO-CH2-CH2-N+ (CH3)2-CH2-CH2- CO-NH-], ESI-MS m/z: calcd609.06 (for C38H78N3O2+), found 609[M]+ ESI-HRMS: 608.6081 (calculated mass forC38 H78 O2 N3 = 608.6089) HPLC- Purity: 98.25% N,N-dimethyl-2-stearamido-N-(2-stearamidoethyl)ethan-1-aminium chloride (17DME): [0146] This compound was synthesized by a similar procedure as described for 13DME compound (0.92 g, 89% yield, Rf=0.5, 10:90 methanol:chloroform, v/v). 1H NMR of (CDCl3, 400 MHz): δ/ppm = 0.87 [t, 6H, CH3-(CH 2)16-]; 1.24 [m, 56H, CH3-(CH2)14-CH2-CH2-]; 1.60 [m, 4H, CH3-(CH 2)14-CH2-CH2-];2.27 [t, 4H, CH3-(CH 2)14-CH2-CH2-]; 3.29 [S, 6H, -CH2-CH2-N+(CH3)2-CH2-CH2-]; 3.79 [t, 8H, -CO-CH2- CH2-N+ (CH3)2-CH2-CH2-CO-]; 7.53[t, 2H, -NH-CO-CH2-CH2-N+ (CH3)2-CH2-CH2- CO-NH-], ESI-MS m/z: calcd665.17 (for C42H86N3O2+), found 665[M]+ ESI-HRMS: 664.6711(calculated mass for C42 H86 O2 N3 = 664.6715) HPLC- Purity: 98.22%
Formula IIb 2-hydroxy-N-methyl-N,N-bis(2-tetradecanamidoethyl)ethan-1-aminium chloride (13MOH): [0147] Intermediate IIA compound (1eq, 0.1 g, 0.1858 mmoles) was stirred with excess 2-chloroethanol (1 mL) and K2CO3 (2eq, 0.51g, 0.3717 mmoles) for 12 h at 80oC reflux condition. After that the reaction mixture was filtered, concentrated and the residue was purified by column chromatographic purification (using 60 to 120 mesh size silica gel and 6% methanol in chloroform was used as eluent) to give white solid compound yielded 13MOH as a white solid (0.81 g, 75% yield, Rf = 0.4 in 10% methanol in chloroform, v/v) 1H NMR of (CD3OD, 400 MHz): δ/ppm = 0.88 [t, 6H, CH3-(CH 2)12-]; 1.23 [m, 40H, CH3-(CH2)10-CH2-CH2-]; 1.54 [m, 4H, CH3-(CH 2)10-CH2-CH2-]; 2.18 [t, 4H, CH3-(CH 2)10-CH2-CH2-]; 3.24[S,3H, -CO-NH-CH2-CH2-N(CH3)(CH2CH2OH)-CH2CH2-NH- CO-]; 3.34 [t, 2H, -CO-NH-CH2-CH2-N(CH3)(CH2CH2OH)-CH2CH2-NH-CO-];3.48 [t, 4H, -CO-NH-CH2-CH2-N+ (CH3)(CH2CH2OH)-CH2CH2-NH-CO-];3.59 [t, 4H, - CO-NH-CH2-CH2-N+ (CH3)(CH2CH2OH)-CH2CH2-NH-CO-]; 3.81[[t, 2H, -CO-NH- CH2-CH2-N+ (CH3)(CH2CH2OH)-CH2CH2-NH-CO-]; 3.96 [S,1H, , -CO-NH-CH2- CH2-N+ (CH3)(CH2CH2OH)-CH2CH2-NH-CO-]; ESI-MS m/z: calcd 582.98 (for C37H77ClN3O3 +), found 583[M]+ ESI-HRMS: 582.556(calculated mass forC35 H72 O3 N3 = 582.557) HPLC- Purity: 98.25%
2-hydroxy-N-methyl-N,N-bis(2-palmitamidoethyl)ethan-1-aminium chloride (15MOH): [0148] This compound was synthesized by a similar procedure as described for 13MOH compound (0.82 g, 77% yield, Rf=0.5, 10:90 methanol: chloroform, v/v). 1H NMR of (CD3OD, 400 MHz): δ/ppm = 0.88 [t, 6H, CH3-(CH 2)12-]; 1.23 [m, 40H, CH3-(CH2)10-CH2-CH2-]; 1.54 [m, 4H, CH3-(CH 2)10-CH2-CH2-]; 2.18 [t, 4H, CH3-(CH 2)10-CH2-CH2-]; 3.24[S,3H, -CO-NH-CH2-CH2-N+ (CH3)(CH2CH2OH)-CH2CH2-NH- CO-]; 3.34 [t, 2H, -CO-NH-CH2-CH2-N+ (CH3)(CH2CH2OH)-CH2CH2-NH-CO-]; 3.48 [t, 4H, -CO-NH-CH2-CH2-N+(CH3)(CH2CH2OH)-CH2CH2-NH-CO-]; 3.59 [t, 4H, -CO-NH-CH2-CH2-N+ (CH3)(CH2CH2OH)-CH2CH2-NH-CO-]; 3.81 [[t, 2H, -CO- NH-CH2-CH2-N+ (CH3)(CH2CH2OH)-CH2CH2-NH-CO-]; 3.96 [S,1H, , -CO-NH- CH2-CH2-N+ (CH3)(CH2CH2OH)-CH2CH2-NH-CO-]; ESI-MS m/z: calcd 674.54 (for C39H80ClN3O3+), found 639[M]+ ESI-HRMS: 638.6191(calculated mass forC39 H80 O3 N3 = 638.6194 HPLC- Purity: 98.50% 2-hydroxy-N-methyl-N,N-bis(2-stearamidoethyl)ethan-1-aminium chloride (17MOH): [0149] This compound was synthesized by a similar procedure as described for 13MOH compound (0.76 g, 72% yield, Rf=0.6, 10:90 methanol:chloroform, v/v). 1H NMR of (CD3OD, 400 MHz): δ/ppm = 0.88 [t, 6H, CH3-(CH 2)12-]; 1.23 [m, 40H, CH3-(CH2)10-CH2-CH2-]; 1.54 [m, 4H, CH3-(CH 2)10-CH2-CH2-]; 2.18 [t, 4H, CH3-(CH 2)10-CH2-CH2-]; 3.24[S,3H, -CO-NH-CH2-CH2-N+ (CH3)(CH2CH2OH)-CH2CH2-NH- CO-]; 3.34 [t, 2H, -CO-NH-CH2-CH2-N+ (CH3)(CH2CH2OH)-CH2CH2-NH-CO-]; 3.48 [t, 4H, -CO-NH-CH2-CH2-N+ (CH3)(CH2CH2OH)-CH2CH2-NH-CO-]; 3.59 [t, 4H, -CO-NH-CH2-CH2-N+ (CH3)(CH2CH2OH)-CH2CH2-NH-CO-]; 3.81[[t, 2H, -CO- NH-CH2-CH2-N+ (CH3)(CH2CH2OH)-CH2CH2-NH-CO-]; 3.96 [S,1H, , -CO-NH- CH2-CH2-N+ (CH3)(CH2CH2OH)-CH2CH2-NH-CO-]; ESI-MS m/z: calcd 695.20 (for C43H88ClN3O3+), found 695[M]+ ESI-HRMS: 694.6809 (calculated mass for C43H88ClN3O3 + : 695.20)
HPLC- Purity: 98.29%
1-(bis(2-hydroxyethyl)(2-tetradecanamidoethyl)-l4-azanyl)-2- tetradecanamidoethan-1-ylium chloride (13DOH): [0150] Intermediate IA compound (1eq, 0.1g, 0.1904 mmoles) was stirred with excess 2-chloroethanol (1 mL) and K2CO3 (2eq, 0.52 g, 0.3809 mmoles) for 24 h at 80oC reflux conditon. After that the reaction mixture was filtered, concentrated and the residue was purified by column chromatographic purification (using 60 to 120 mesh size silica gel and 6% methanol in chloroform was used as eluent) to give white solid compound yielded 13MOH as a white solid (0.75 g, 65% yield, Rf = 0.3 in 10% methanol in chloroform, v/v) 1H NMR of (CD3OD, 400 MHz): δ/ppm =0.84 [t, 6H, CH3-(CH 2)12-]; 1.24 [m, 40H, CH3-(CH2)10-CH2-CH2-]; 1.54 [m, 4H, CH3-(CH 2)10-CH2-CH2-]; 2.16 [t, 4H, CH3-(CH 2)10-CH2-CH2-]; 3.25 [t, 4H, -CO-NH-CH2-CH2-N+ (CH2-CH2-OH)2-CH2-CH2-NH- CO-]; 3.55 [t, 4H, -CO-NH-CH2-CH2-N+ (CH2-CH2-OH)2-CH2-CH2-NH-CO-]; 3.61 [t, 4H, -CO-NH-CH2-CH2-N+ (CH2-CH2-OH)2-CH2-CH2-NH-CO-]; 3.99 [m, 4H, -CO- NH-CH2-CH2-N+ (CH2-CH2-OH)2-CH2-CH2-NH-CO-]; 4.53 [S, 2H, -CO-NH-CH2- CH2-N+ (CH2-CH2-OH)2-CH2-CH2-NH-CO-]; ESI-MS m/z: calcd613.01 (for C36H73ClN3O4+), found 613[M]+ ESI-HRMS: 612.5661(calculated mass forC36H73ClN3O4 += 612.5674) HPLC- Purity: 97.99%
1-(bis (2-hydroxyethyl) (2-palmitamidoethyl)-l4-azanyl)-2-palmitamidoethan-1- ylium chloride (15DOH): [0151] This compound was synthesized by a similar procedure as described for 13DOH compound (0.79 g, 69% yield, Rf=0.4, 10:90 methanol:chloroform, v/v). 1H NMR of (CD3OD, 400 MHz): δ/ppm =0.84 [t, 6H, CH3-(CH 2)12-]; 1.24 [m, 40H, CH3-(CH2)10-CH2-CH2-]; 1.54 [m, 4H, CH3-(CH 2)10-CH2-CH2-]; 2.16 [t, 4H, CH3-(CH 2)10-CH2-CH2-]; 3.25 [t, 4H, -CO-NH-CH2-CH2-N+ (CH2-CH2-OH)2-CH2-CH2-NH- CO-]; 3.55 [t, 4H, -CO-NH-CH2-CH2-N+ (CH2-CH2-OH)2-CH2-CH2-NH-CO-]; 3.61 [t, 4H, -CO-NH-CH2-CH2-N+ (CH2-CH2-OH)2-CH2-CH2-NH-CO-]; 3.99 [m, 4H, -CO- NH-CH2-CH2-N+ (CH2-CH2-OH)2-CH2-CH2-NH-CO-]; 4.53 [S, 2H, -CO-NH-CH2- CH2-N+ (CH2-CH2-OH)2-CH2-CH2-NH-CO-]; ESI-MS m/z: calcd669.11 (for C40H81ClN3O4 +), found 669[M]+ ESI-HRMS: 668.6290(calculated mass forC40H81ClN3O4+= 668.6300) HPLC- Purity: 98.03% 1-(bis (2-hydroxyethyl) (2-stearamidoethyl)-l4-azanyl)-2-stearamidoethan-1- ylium chloride (17DOH): [0152] This compound was synthesized by a similar procedure as described for 13DOH compound (0.80 g, 71% yield, Rf=0.5, 10:90 methanol:chloroform, v/v). 1H NMR of (CD3OD, 400 MHz): δ/ppm =0.84 [t, 6H, CH3-(CH 2)12-]; 1.24 [m, 40H, CH3-(CH2)10-CH2-CH2-]; 1.54 [m, 4H, CH3-(CH 2)10-CH2-CH2-]; 2.16 [t, 4H, CH3-(CH 2)10-CH2-CH2-]; 3.25 [t, 4H, -CO-NH-CH2-CH2-N (CH2-CH2-OH)2-CH2-CH2-NH- CO-]; 3.55 [t, 4H, -CO-NH-CH2-CH2-N (CH2-CH2-OH)2-CH2-CH2-NH-CO-]; 3.61 [t, 4H, -CO-NH-CH2-CH2-N (CH2-CH2-OH)2-CH2-CH2-NH-CO-]; 3.99 [m, 4H, - CO-NH-CH2-CH2-N (CH2-CH2-OH)2-CH2-CH2-NH-CO-]; 4.53 [S, 2H, -CO-NH- CH2-CH2-N (CH2-CH2-OH)2-CH2-CH2-NH-CO-]; ESI-MS m/z: calcd725.22 (for C44H89ClN3O4 +), found 725[M]+ ESI-HRMS: 724.6914 (calculated mass forC44H89ClN3O4 +=724.6926) HPLC- Purity: 98.05%
Co-lipids: [0153] The co-lipids of the present disclosure are selected from from steroids, cholesterol, sitostanol, sitosterol, ß-sitosterol-d6, campesterol, campesterol-d6, desmosterol, desmosterol-d6, 7-dehydrodesmosterol, zymosterol, zymosterol-d5, zymostenol, zymostenol-d7, diosgenin, stigmasterol, lathosterol, lathosterol-d7, lanosterol, lanosterol-d6, lanostenol, dihydrolanosterol-d7, 14-demethyl-lanosterol, 14-demethyl-lanosterol-d6, 14-demethyl-14-dehydrolanosterol (FF-MAS), cholesterol sulfate, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulphate (DHEA sulphate), phosphatidylethanolamines, dilauroylphosphatidylethanolamine (DLPE), dimirystoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylethanolamine (DOPE), phosphatidylcholine, dilauroylphosphatidylcholine (DLPC), dimirystoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC) dioleoylphosphatidylcholine (DOPC), egg phosphatidylcholine (Egg PC), hydro egg phosphatidylcholine (Hydro egg PC), egg lyso phosphatidylcholine (Egg , Lyso PC), soy phosphatidylcholine (Soy PC), hydro soy phosphatidylcholine (Hydro Soy PC), soy lyso phosphatidylcholine (Soy Lyso PC), heart phosphatidylcholine (Heart PC), brain phosphatidylcholine (Brain PC), liver phosphatidylcholine (Liver PC), or combinations thereof. Preparation of lipid formulations (liposomes) [0154] Liposome was prepared by using thin film hydration method from previously established protocol (Srujan etal., The influence of the structural orientation of amide linkers on the serum compatibility and lung transfection properties of cationic amphiphiles, Biomaterials, 2011;32(22):5231-40). In the present disclosure, lipid formulations were prepared using first lipid of Formula I which is Gal16- compound VI, a second lipid of Formula I which is MeOH16 which is 16MOH of Formula IIB and the co-lipid is selected from DOPE, cholesterol, or combinations thereof.
[0155] The mole ratios of (Non-targeted liposomes) NTL liposomes were prepared in thin film hydration method using 1:0.5:0.5, molar ratios of MeOH16:DOPE:Chol. Targeted liposomes (TL liposomes) were prepared with the above mentioned protocol using 1:0.5:0.5:0.25, molar ratios of MeOH16:DOPE:Chol:Gal16 (Compound VI-) (3S,4R,5S,6S)-6-((4-((ditetradecylamino)methyl)-1H-1,2,3-triazol-1- yl)methyl)tetrahydro-2H-pyran-2,3,4,5-tetraol). In a glass vial, the appropriate mole ratios of lipids were dissolved in chloroform. A thin lipid film was developed by using flow of nitrogen gas and then to remove trapped solvent from the thin film was kept for drying under high vacuum for 6 hours.1ml of autoclaved Mill ‘Q’ water was added to vacuum dried lipid thin film and then allowed to overnight for swelling. The vials were then vortexes for three minutes for multi-lamellar vesicles (MLVs). MLVs were then sonicated initially in a water bath followed by an ice bath until to get clear solution by using a Branson 450 sonifier at 100% duty cycle and 25 W output power to produce small uni-lamellar vesicles. 1mM liposome (with respect to MeOH16 cationic lipid) was used for all in vitro studies, for cellular localization 0.1 mole ratio of Rho-PE were used and 2 mM liposome (with respect to MeOH16 cationic lipid) was used for all in vivo studies, for in vivo imaging studies 100µg of DiR (Cat no D12731) were used. Preparation of lipoplexes The lipid-pDNA complex (lipoplex) was prepared as per previous literature ( Dharmalingam etal., An anti-oxidant, alpha-lipoic acid conjugated oleoyl-sn- phosphatidylcholineas a helper lipid in cationic liposomal formulations, Colloids and surfaces B, Biointerfaces.2017;152:133-42). Screening in vitro transfection efficiencies of library of lipids: [0156] Screening of liposomes of the amide lipid analogues in HepG2 cells were done using eGFP pDNA as reporter gene.1mM Liposomes were prepared using respective amide lipid (Selection for second lipid), cholesterol and dioleoyl phopshatidyl ethylamine. 104 cells were seeded in 96 well plate. After 16h, the lipoplexes were added and 48h post addition of lipoplexes, cells were subjected to FACS analysis for
their eGFP expression. (n=3). Among all the liposomes of lipids screened, MeOH16 found to be efficient in delivering pDNA into HepG2 cells (Figure 1). Transfections of formulations with galactosylated lipid: [0157] Formulations comprising 1mM ofMeOH16 each (Selected as second lipid from screening from Figure 1), cholesterol and dioleoyl phopshatidyl ethylamine and varying concentrations of first lipid of 0.25 and 0.5 mM and 104 cells were seeded in 96 well plate with the formulations. After 16h, the lipoplexes were added and 48h post addition of lipoplexes, cells were subjected to FACS analysis for their eGFP expression. (n=3) Increasing the first lipid concentration from 0.25 mM to 0.5 mM decreased the transfections as seen in Figure 2. This might be due to higher hydration effect of galactose ligand leading to decrease the interaction of cationic lipid with nucleic acids. Overall, the targeted liposomes are as efficient as commercial transfection reagent LT-1. Transfections with mRNA in 2D and 3D conditions [0158] Briefly, for in vitro experiments 1 mM liposomes were used and the charge ratios of cationic lipid to pDNA were maintained as 1:1, 2:1, 4:1 and 8:1. 1 mM liposome were serially diluted in serum free medium in final volume of 25µL and it was complexes with fixed amount of pDNA (0.3 µg/well of 96 well plate, 0.5 µg/well of 24 well plate and 1µg/well of 6 well plate) was diluted in 25 µL of serum free medium. Mixture of liposome and pDNA were shaken by using Spin ‘X’ (minimum shaking) in room temperature for 10 to 15 minutes and then subsequently added to treatment wells. For in vivo experiments to deliver pDNA, 25µg of pDNA and 75µL of 2mM liposome were complexes in 500µL of 5% glucose, these lipoplex were lyophilized and then reconstituted in 150µL of 5% glucose. To deliver mRNA, 5µg of mRNA and 75µL of 2mM liposome were complexes in 500µL of 5% glucose, these lipoplex were lyophilized and then reconstituted in 150µL of 5% glucose. Figure 3 illustrate (a) mRNA transfections; and (b) mRNA transfections in 3D for the lipid formulations of the present disclosure. Lipoplexes of MeOH16DC Gal 0.25 were prepared with eGFP mRNA at varying lipid to base charge ratios, 4:1 and 8:1, added
to HepG2. After 48h, transfection efficiencies were analysed in FACS for eGFP expression. eGFP mRNA expression was as good as commercial transfection reagent LT-1, showed~80% transfection efficiency. Lyophilized lipoplexes (Figure 3a) of MeOH16DC Gal 0.25 comprising eGFP mRNA/pDNA and HepG2 cells were added to collagen solution and to form gel in order to mimic in vivo conditions where hepatocytes have collagen as extracellular matrix. GFP expression was observed in different time intervals at day 3, 6, 10, and 14. Surprisingly, strong fluorescence was observed with galactosylated liposomal formulations at day 3 and the expression slowly decreased up to 14 days (Figure 3b). And Figure 4 illustrates the in vivo distribution of the first lipid compound in the mice. Characterization of selected liposomes and lipoplexes by Dynamic light scattering (DLS) technique [0159] The Hydrodynamic diameter (HDD), Zeta potentials and Polydispersity index (PDI) of liposomes (50ul liposome +950ul medium) and lipoplexes (50µl lipoplex +950ul medium) were analyzed in Milli ‘Q’ water and serum free media by using photon correlation spectroscopy and electophoretic mobility on an Anton-Paar Litesizer 500 instrument. Figure 5a depicts the process of obtaining of lyophilized and reconstituted formulation. Figure 5b illustrates particle size distribution of lyophilized and reconstituted lipoplexes. Figure 5c depicts the zeta potential of the targeting lipoplexes to the non-targeting lipoplexes. Figures 5b and 5c proved that the targeted lipoplexes exhibit narrowed particle size distribution, which means maximum number of particles of the lipoplex have same particle size ~200nm. And the zeta potential close to +25mV reveal positive of the lipoplexes that ensures complete binding of nucleic acids. In vivo imaging [0160] Luciferase encoded pDNA and mRNA lipoplexes preparation was carried out as described above. For each mouse (25µg of pDNA or 5µg of mRNA and 75µL of
2mM liposome) 150µL volume of lipoplex were injected BALB/c male mice intraperitonially (i.p). One hour before of imaging 150 mg/kg D-luciferin was injected i.p. and luminescence was measured using PerkinElmer's IVIS® optical imaging system. After 2D and 3D imaging mice were sacrificed and isolated the organs and imaged. Figure 6 illustrate the (a) targeting lipoplexes and (b) the non-targeting lipoplexes, complexed with luciferase expressing pDNA and injected intraperitoneal (i.p) into mice. Targeted lipoplexes demonstrated more selective luciferase expression in liver, whereas non-targeted lipoplexes showed expression in liver, lung and spleen as well. In vivo gene expression profiles :This experiment was performed under protocols (IAEC No.3/2020) approved by Institutional Animal Ethical Committee of Christian Medical College, India.Balb/c mice (10–12 weeks age) Figure 7 illustrate luminescence expression seen in various organs after injecting pDNA loaded lipid formulations into mice. Luciferase expression profiles of the major organs collected after post-euthanization. Targeted lipoplexes demonstrated selective expression in liver, whereas non-targeted lipoplexes showed in liver, lung and spleen. Body weight monitor [0161] To assess any possible toxic effects of liposomal formulations, initially, we have performed the body weight monitoring experiment. Swiss albino mice were an intraperitonially injected with different liposomal treatment groups. Mice body weight was monitored every injection time throughout the treatment period. Figure 8 illustrate the luminescence expression with respect to body weight of the mice organs after injecting pDNA loaded lipid formulations. Figure 9a illustrate luciferase mRNA expression at varying concentrations from 0.5mg/kg body weight to 4 mg/kg body weight. Figure 9b depicts luciferase mRNA expression at different time intervals from 2h to 72h. The mRNA showed higher expression in 6h and retained up to 36h.. Figure 10 illustrate the luciferase mRNA administered with the targeted liposomes thrice with
a gap of one week. The mRNA expression was not compromised despite of multiple administration. Figure 11 illustrate body weights of the lipoplexes injected animals were observed for 13 days. There were no major changes in the total body weight of the lipoplexes Figure 12 illustrate spleen weights of the lipoplexes injected animals were evaluated after 15 days. There are no major changes in the spleen weight of the targeted lipolplexes, whereas non-targeted lipoplexes showed slight increase in the spleen weights. H&E Analysis [0162] Figure 13 illustrate the representative images of H&E (hematoxylin and eosin) analysis in mice. Histology of Targeted lipoplex treated mice was found to be similar when compared to the normal mice. No morphological changes were observed even after 7 administrations. H&E staining of tissues from major organs of the lipoplexes treated animals. Cellularity of the treated group similar compared to saline treated animals which indicated that treatment did not show any negative impact on the major organs. Fibrosis Analysis [0163] Masson trichome staining was done on tissues of treated animals in order to find occurrence of any fibrosis due to the administration of targeted lipoplexes. Figure 14 illustrate the representative images showed that there is no significant change in the staining of fibrous tissue that indicated that the multiple administrations of lipoplexes did not induce fibrosis. Hematological parameters [0164] The blood parameters for each treatment group of single and seven consecutive doses of different treatment groups were performed. The blood sample (~300 µL) from each mouse was collected into eppendroff containing dipotassium EDTA (5%) by retro orbital plexus puncture at the final day of experiment. The hematological analysis estimated were as follows: RBC (red blood cells) count, WBC (white blood cells)
count, PLT (platelets/ thrombocytes) count, MPV (Mean platelet volume) HGB (haemoglobin), MCV (mean corpuscular volume), HCT (haematocrit value), MCH (mean corpuscular haemoglobin), MCHC (mean corpuscular haemoglobin concentration), , differential leucocytes (lymphocytes, monocytes, eosinophils, neutrophils). All the parameters were estimated using automated Hematology analyzer (Siemens ADVIA 2120i Hematology Systems). Table 1 shows the consolidated hematological parameters obtained for the targeted lipoplexes in comparison to the non-targeted liposomes at single dose and after seven doses. Table 1
Serum parameters [0165] The final day of experiment, the blood was collected from each mouse into eppendroff around 500 μL by retro orbital plexus puncture. The blood collected eppendroff samples were kept horizontally for one hour; further serum was separated by centrifugation at 4000 rpm for 15 mins at 4oC. The supernatant serum was used for determining the Aspartate aminotransferase (AST), Alanine transaminase (ALT), Alkaline phosphatase, Total protein content, Triglycerides levels, Cholesterol levels, Glucose levels, Creatinine levels and Blood urea nitrogen (BUN) levels by using auto analyzer Siemens Dimension XpandPlus Integrated Chemistry System. Cytokine analysis by multiplex cytokine assay [0166] The final day of experiment, the blood was collected from each mouse into eppendroff around 500 μL by retro orbital plexus puncture. Further serum was separated by centrifugation at 4000 rpm for 15 mins at 4oC. The supernatant serum was used for determining the level of inflammatory cytokines, including IL-1α, IL1β, IL-6, IL-10, IL-12p70, IL-17A, IL-23, IL-27, CCL2(MCP-1), IFN-β, IFN-γ, TNF-α, and GM-CSF in different treatment groups were determined by multiplex cytometric bead- based LEGENDplex immunoassay (Mouse Inflammation Panel; BioLegend, London, UK) according to the manufacturer’s protocol. Briefly, the serum samples were 2-fold diluted using dilution buffer and the equal volume diluted serum samples, cytokine standards were incubated with specific capture antibodies beads (25 µl each) for 2 hours with shaking at approx.800 rpm. The capture bead-analyte complexes were spin down at 250 x g and washed using 1x wash buffer. 25 µl of biotinylated detection antibody cocktail was added to beads and incubated for 1 hour with shaking to forming capture bead-analyte-detection antibody sandwiches. 25 µl of Streptavidin- phycoerythrin (SA-PE) subsequently was added to bind to the biotinylated detection antibodies complex. After washing and spin down, the beads were diluted in 150 µl of 1x wash buffer. Then, fluorescent signal intensities of each bead were analyzed using
a BD Aria (BD Biosciences, San José, USA). The concentration of serum cytokines was calculated in LEGENDplex analysis software (BioLegend, San Diego, USA) using FCS file generated on BD Aria. Immunohistochemistry [0167] At the end of the experiment, we sacrificed the mice and isolated the organs, followed by stored in 10% neutral phosphate buffered formalin. Following fixation, samples were dehydrated and embedded in paraffin. Five micrometer microtome sections of the inflamed skin were then stained with hematoxylin and eosin or masson’s tricome. The Olympus BX43 light microscope equipped with computer-controlled digital camera was used to visualize the images on the slides. Statistical analysis: [0168] All the in vitro experiments were done in triplicate and the mean standard deviation was considered for comparisons. All the results were analyzed by ‘‘One-way ANOVA’’ test using Dunnett’s post-test (Graph Pad Prism version 6.04 Software, San Diego, CA.). Advantages of the present disclosure [0169] The present disclosure provides a liposome formulation comprising the first lipid compound of Formula I, the second lipid compound of Formula II and the co- lipid. The first lipid compound or the targeted lipids of the present disclosure are novel lipids which are galactosylated lipids which acts as ligand and shows high affinity to asialoglycoprotein receptors. These receptors are over expressed in hepatocytes and when these galactosylated lipids binds to these receptors at the hepatocytes, thereby deliver the therapeutic agent into the hepatocytes. The second lipid compound of Formula II are novel cationic lipids that are used in the formation of liposome of the present disclosure. The liposome of the present disclosure acts as a vehicle or a carrier to deliver specific therapeutic agent to the hepatocytes. The liposome of the present disclosure are also made as stable formulations by subjecting to lyophilization and reconstitution. The liposome of the present disclosure deliver therapeutic agent selected
from nucleic acids (RNA, mRNA, SiRNA, DNA), locked nucleic acids, small molecules, drugs, proteins, chemotherapeutic agent, a cytotoxin, a steroid, an immunotherapeutic agent, a targeted therapeutic agent, or combinations thereof to the hepatocytes. The present disclosure provides a pharmaceutical composition comprising the formulation which are used for treatment or prevention of a condition or a disease mediated by asialoglycoreceptors. The formulation or the liposome of the present disclosure are also used for preparing medicament for treating various liver related ailments. The present disclosure also provides convenient process for preparing the novel first and second lipid compounds.
Claims
I/We claim: 1. A formulation comprising: i. a first lipid compound of Formula I;
Formula I wherein
is selected from C5-22 alkyl, C5-22 carbocyclyl, or C5-22 heterocyclyl, wherein C5-22 alkyl, C5-22 carbocyclyl, or C5-22 heterocyclyl optionally further substituted with one or more substituents selected from hydroxy, C1-10 alkyl, C1-10 alkylhydroxy, -NH-C(O)-C1-10 alkyl, or C1-10 alkoxy; and C1-10 alkoxy is optionally further substituted with one or more substituents selected from hydroxy, C1-10 alkyl, C1-10 alkylhydroxy, or -OC(O)R1; X is a bond or selected from -OR1-, -O-C(O)-N(R1)-, -N(R1)-C(O)-O-, -C(O)N(R1)- , -N(R1)-C(O)-, or -O-C(O)R1-; R and R’ are independently selected from C5-22 alkyl, C5-22 alkenyl, C5-22 alkynyl, or -C6-22 aryl; R1 is independently selected from hydrogen, C1-10 alkyl, C2-10 alkenyl, C2-10alkynyl, C3-10 cycloalkyl, or -C6-22 aryl; m and m’ are independently selected from 1 to 22; and n and n’ are independently selected from 0 to 22; ii.a second lipid compound of Formula II:
Formula II wherein R3, and R4 are independently selected from hydrogen, hydroxy, amino, halo, cyano, or -C6-22 aryl; Y is a bond or selected from -OR7-, -O-C(O)-N(R7)-, -N(R7)-C(O)-O-, -C(O)N(R7)- , -N(R7)-C(O)-, or -O-C(O)R7-; R5, and R6 are independently selected from C5-22 alkyl, C5-22 alkenyl, C5-22 alkynyl, or C6-22 aryl; R7 is independently selected from hydrogen, C1-10 alkyl, C2-10 alkenyl, C2-10alkynyl, C3-10 cycloalkyl, or -C6-22 aryl; and p, q, r, and s are independently selected from 1 to 15; and iii.a co-lipid wherein the first lipid to the second lipid to the co-lipid is in the mole ratio range of 1:0.1:0.1 to 1:4:4.
2.The formulation as claimed in claim 1, wherein the formulation comprises one or more therapeutic agent.
3.The formulation as claimed in claim 1, wherein the co-lipid is selected from steroids, cholesterol, sitostanol, sitosterol, ß-sitosterol-d6, campesterol, campesterol-d6, desmosterol, desmosterol-d6, 7-dehydrodesmosterol, zymosterol, zymosterol-d5, zymostenol, zymostenol-d7, diosgenin, stigmasterol, lathosterol, lathosterol-d7, lanosterol, lanosterol-d6, lanostenol, dihydrolanosterol-d7, 14-demethyl-lanosterol, 14-demethyl-lanosterol-d6, 14-demethyl-14-dehydrolanosterol (FF-MAS),
cholesterol sulfate, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulphate (DHEA sulphate), phosphatidylethanolamines, dilauroylphosphatidylethanolamine (DLPE), dimirystoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylethanolamine (DOPE), phosphatidylcholine, dilauroylphosphatidylcholine (DLPC), dimirystoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC) dioleoylphosphatidylcholine (DOPC), egg phosphatidylcholine (Egg PC), hydro egg phosphatidylcholine (Hydro egg PC), egg lyso phosphatidylcholine (Egg , Lyso PC), soy phosphatidylcholine (Soy PC), hydro soy phosphatidylcholine (Hydro Soy PC), soy lyso phosphatidylcholine (Soy Lyso PC), heart phosphatidylcholine (Heart PC), brain phosphatidylcholine (Brain PC), liver phosphatidylcholine (Liver PC), or combinations thereof.
4.The formulation as claimed in claim 2, wherein the therapeutic agent is selected from nucleic acids (RNA, mRNA, SiRNA, DNA), locked nucleic acids, small molecules, drugs, proteins, chemotherapeutic agent, a cytotoxin, a steroid, an immunotherapeutic agent, a targeted therapeutic agent, or combinations thereof.
5.The formulation as claimed in claim 1, wherein the formulation further comprises a reconstituting agent selected from saline, glucose, sucrose, or inulin.
6.The formulation as claimed in claim 1, wherein the molar ratio of the first lipid to the second lipid is in the range of 1:0.1 to 1:4.
7.A reconstituted formulation comprising: the formulation as claimed in claim 1 with 2-25% (w/w) of a reconstituting agent, wherein the reconstituting agent is selected from saline, glucose, sucrose, or inulin.
8.The formulation as claimed in claim 7, wherein reconstituted formulation comprises a therapeutic agent selected from nucleic acids (RNA, mRNA, SiRNA, DNA), locked nucleic acids, small molecules, drugs, proteins, chemotherapeutic agent, a
cytotoxin, a steroid, an immunotherapeutic agent, a targeted therapeutic agent, or combinations thereof.
9.The formulation as claimed in claim 1, wherein the first lipid compound is a targeting lipid which binds to a cell surface receptor; and the cell surface receptor is asialoglycoprotein receptors.
10.The formulation as claimed in any one of the claims 1 to 9, wherein the formulation is a carrier to deliver the therapeutic agent to a targeted cell.
11.A pharmaceutical composition comprising the formulation as claimed in claim 1 with pharmaceutically acceptable salts thereof.
12.A pharmaceutical composition comprising the formulation as claimed in claim 1 with one or more pharmaceutically active compound.
13.The pharmaceutical composition as claimed in claims 11 and 12, wherein the composition is in the form selected from a tablet, capsule, powder, syrup, solution, aerosol, or suspension.
14.A formulation as claimed in any of claims 1 to 10 or the pharmaceutical composition as claimed in claims 11 and 12 for use in the manufacture of a medicament for treatment or prevention of a condition or a disorder or a disease mediated by asialoglycoprotein receptor.
15.A formulation as claimed in any of claims 1 to 10 or the pharmaceutical composition as claimed in claims 11 and 12 for use in the treatment and/or prevention of a condition mediated by asialoglycoprotein receptor.
16.A process for preparing the formulation as claimed in claim 1, the process comprising: mixing stoichiometric ratios of the first lipid compound with the second lipid compound and the co-lipid, wherein the first lipid to the second lipid to the co- lipid is in the mole ratio range of 1:0.1:0.1 to 1:4:4.
17.The process as claimed in claim 16, wherein mixing stoichiometric ratios of the first lipid compound with the second lipid compound and the co-lipid is carried out in the presence of one or more first solvent selected from chloroform,
dichloromethane, tetrahydrofuran, acetonitrile, methanol, ethanol, ethyl acetate, or combinations thereof.
18.A first lipid compound of Formula I or its pharmaceutically acceptable salts, complexes, hydrates, solvates, tautomers, polymorphs, stereoisomers, pharmaceutically active derivatives thereof:
Formula I wherein
is selected from C5-22 alkyl, C5-22 carbocyclyl, or C5-22 heterocyclyl, wherein C5-22 alkyl, C5-22 carbocyclyl, or C5-22 heterocyclyl optionally further substituted with one or more substituents selected from hydroxy, C1-10 alkyl, C1-10 alkylhydroxy, -NH-C(O)-C1-10 alkyl, or C1-10 alkoxy; and C1-10 alkoxy is optionally further substituted with one or more substituents selected from hydroxy, C1-10 alkyl, C1-10 alkylhydroxy, or -OC(O)R1; X is a bond or selected from -OR1-, -O-C(O)-N(R1)-, -N(R1)-C(O)-O-, -C(O)N(R1)- , -N(R1)-C(O)-, or -O-C(O)R1-; R and R’ are independently selected from C5-22 alkyl, C5-22 alkenyl, C5-22 alkynyl, or -C6-22 aryl; R1 is independently selected from hydrogen, C1-10 alkyl, C2-10 alkenyl, C2-10alkynyl, C3-10 cycloalkyl, or -C6-22 aryl; m and m’ are independently selected from 1 to 22; and n and n’ are independently selected from 0 to 22.
20.The compound as claimed in claim 18, wherein the compound is
X is a bond or selected from -OR1-, -O-C(O)-N(R1)-, -N(R1)-C(O)-O-, -C(O)N(R1)- , -N(R1)-C(O)-, or -O-C(O)R1-; R and R’ are independently selected from C5-22 alkyl, C5-22 alkenyl, C5-22 alkynyl, or -C6-22 aryl; R1 and R2 are independently selected from hydrogen, C1-10 alkyl, C2-10 alkenyl, C2- 10alkynyl, C3-10 cycloalkyl, or -C6-22 aryl; m and m’ are independently selected from 1 to 22; and n and n’ are is independently selected from 0 to 22.
21.The compound of Formula I as claimed in claim 18, wherein the compound is selected from i. (3S,4R,5S,6S)-6-((4-((ditetradecylamino)methyl)-1H-1,2,3-triazol-1- yl)methyl)tetrahydro-2H-pyran-2,3,4,5-tetraol; ii.(3S,4R,5S,6S)-6-((4-((dihexadecylamino)methyl)-1H-1,2,3-triazol-1- yl)methyl)tetrahydro-2H-pyran-2,3,4,5-tetraol; and iii. (3S,4R,5S,6S)-6-((4-((dioctadecylamino)methyl)-1H-1,2,3-triazol-1- yl)methyl)tetrahydro-2H-pyran-2,3,4,5-tetraol.
22.A second lipid compound of Formula II or its pharmaceutically acceptable salts, complexes, hydrates, solvates, tautomers, polymorphs, stereoisomers, pharmaceutically active derivatives thereof,
Formula II wherein R3, and R4 are independently selected from hydrogen, hydroxy, amino, halo, cyano, or -C6-22 aryl; Y is a bond or selected from -OR7-, -O-C(O)-N(R7)-, -N(R7)-C(O)-O-, -C(O)N(R7)- , -N(R7)-C(O)-, or -O-C(O)R7-; R5, and R6 are independently selected from C5-22 alkyl, C5-22 alkenyl, C5-22 alkynyl, or C6-22 aryl; R7 is independently selected from hydrogen, C1-10 alkyl, C2-10 alkenyl, C2-10alkynyl, C3-10 cycloalkyl, or -C6-22 aryl; and p, q, r, and s are independently selected from 1 to 15.
23.The compound of Formula II as claimed in claim 22, the compound is wherein
are independently selected from hydrogen, hydroxy, amino, halo, cyano, or -C6-22 aryl; R5, and R6 are independently selected from C5-22 alkyl, C5-22 alkenyl, C5-22 alkynyl, or -C6-22 aryl; and p, q, r, and s are independently selected from 1 to 15.
24.The compound of Formula I as claimed in claim 22, wherein the compound is selected from i. N,N-dimethyl-2-tetradecanamido-N-(2-tetradecanamidoethyl)ethan-1-aminium chloride; ii. N,N-dimethyl-2-palmitamido-N-(2-palmitamidoethyl)ethan-1-aminium chloride; iii. N,N-dimethyl-2-stearamido-N-(2-stearamidoethyl)ethan-1-aminium chloride; iv. 2-hydroxy-N-methyl-N,N-bis(2-tetradecanamidoethyl)ethan-1-aminium chloride; v. 2-hydroxy-N-methyl-N,N-bis(2-palmitamidoethyl)ethan-1-aminium chloride; vi. 2-hydroxy-N-methyl-N,N-bis(2-stearamidoethyl)ethan-1-aminium chloride; vii. 1-(bis(2-hydroxyethyl)(2-tetradecanamidoethyl)-l4-azanyl)-2- tetradecanamidoethan-1-ylium chloride; viii. 1-(bis (2-hydroxyethyl) (2-palmitamidoethyl)-l4-azanyl)-2-palmitamidoethan-1- ylium chloride; and ix. 1-(bis (2-hydroxyethyl) (2-stearamidoethyl)-l4-azanyl)-2-stearamidoethan-1- ylium chloride.
25.A process for preparing the compound of Formula I, the process comprising: reacting compound of Formula A with compound of Formula B in the presence of a first reagent and a second solvent to result in a compound of Formula C; and treating the compound of Formula C in the presence of a second reagent at a temperature in the range of -5 to 30° C to obtain the compound of Formula I,
wherein
is selected from C5-22 alkyl, C5-22 carbocyclyl, or C5-22 heterocyclyl, wherein C5-22 alkyl, C5-22 carbocyclyl, or C5-22 heterocyclyl optionally further substituted with one or more substituents selected from hydroxy, halogen, azide, C1- 10 alkyl, C1-10 alkylhydroxy, -NH-C(O)-C1-10 alkyl, or C1-10 alkoxy.
26.The process as claimed in claim 25, wherein the first reagent is selected from copper sulphate, sodium ascorbate, or combinations thereof; the second solvent is selected from water, tetrahydrofuran, or combinations thereof; and the second reagent is selected from ether, hydrochloric acid, or combinations thereof.
27.A process for preparing the compound of Formula II, the process comprising: (i) reacting compound of Formula D with a compound of Formula E in the presence of a reducing agent at a temperature in the range of 120 to 140 °C to obtain a compound of Formula F; (ii) converting the compound of Formula F in the presence of a third reagent to obtain the compound of Formula II
28.The process as claimed in claim 27, wherein the reducing agent is selected from sodium borohydride, sodium cyanoborohydride, lithium aluminum hydride, diborane, DIBAL-H, lithium tetrahydridoaluminate, lithium tri-tert- butoxyaluminum hydride, sodium dithionite, sodium hydrogensulfite, sodium hydrosulfite, dodium hydroxymethanesulfinate, sodium hypophosphite, sodium tetrahydroborate, sodium triacetoxyborohydride, triethoxysilane, triethylphosphine, triethylsilane trimethylamine borane, trimethylphoshpine, trimethylphosphite, triphenylphosphine, or combinations thereof; and the third reagent is selected from methyl iodide, ethyl hypochlorite, potassium carbonate, or combinations thereof.
29.A method of treatment or prevention of a condition, or a disease, or a disorder, the method comprising administering the formulation as claimed in any one of the claims 1 to 10, or the pharmaceutical composition as claimed in any one of the claims 11 to 12, to a subject in need thereof.
30. A method of introducing one or more therapeutic agent into a cell, comprising contacting a eukaryotic cell with the formulation as claimed in any one of the claims 1 to 10, or the pharmaceutical composition as claimed in any one of the claims 11 to 12.
31.A method for the treatment or prevention of disease or disorder comprising administering to a subject suffering from disease or disorder a therapeutically effective amount of the formulation according to any one of the claims 1 – 10 or the pharmaceutical composition as claimed in any one of the claims 11 and 12, with
other clinically relevant cytotoxic agents or non-cytotoxic agents to a subject in need thereof.
32.A system for delivering one or more therapeutic agent to a subject in need, comprising: a. the formulation as claimed in any one of the claims 1 to 10, or the pharmaceutical composition as claimed in any one of the claims 11 to 12; and b. one or more therapeutic agent, wherein the weight ratio of the formulation to the therapeutic agent is in the range of 1:1 to 50:1.
33.Use of the compounds as claimed in any one of the claims 1 – 10, or the pharmaceutical composition as claimed in any one of the claims 11 and 12, for the treatment or prevention of disease or disorder, together with other clinically relevant cytotoxic agents or non-cytotoxic agents.
34.The method as claimed in any one of the claims 29-31, wherein the disease or disorder or condition is selected from haemophilia, Niemann-Pick type C2, Factor VII deficiency, α-1-antitrypsin deficiency, Familial tyrosinemia, liver cancer, liver dysfunction, hepatitis, cirrhosis, liver failure, ascites, gallstones, fatty liver disease, autoimmune hepatitis, primary sclerosing cholangitis, primary biliary cirrhosis (PBC), hemochromatosis, wilson’s disease, or hepatocellular carcinoma.
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