EP1203084A1 - Stickstoffmonoxid-synthase-quelle in mikrokapseln - Google Patents
Stickstoffmonoxid-synthase-quelle in mikrokapselnInfo
- Publication number
- EP1203084A1 EP1203084A1 EP00948182A EP00948182A EP1203084A1 EP 1203084 A1 EP1203084 A1 EP 1203084A1 EP 00948182 A EP00948182 A EP 00948182A EP 00948182 A EP00948182 A EP 00948182A EP 1203084 A1 EP1203084 A1 EP 1203084A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- microcapsules
- cells
- nos
- ecdysone
- human
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
- 102000008299 Nitric Oxide Synthase Human genes 0.000 title claims abstract description 63
- 108010021487 Nitric Oxide Synthase Proteins 0.000 title claims abstract description 63
- 239000003094 microcapsule Substances 0.000 claims abstract description 164
- 239000004098 Tetracycline Substances 0.000 claims abstract description 50
- 229960002180 tetracycline Drugs 0.000 claims abstract description 50
- 229930101283 tetracycline Natural products 0.000 claims abstract description 50
- 235000019364 tetracycline Nutrition 0.000 claims abstract description 50
- 150000003522 tetracyclines Chemical class 0.000 claims abstract description 50
- UPEZCKBFRMILAV-JNEQICEOSA-N Ecdysone Natural products O=C1[C@H]2[C@@](C)([C@@H]3C([C@@]4(O)[C@@](C)([C@H]([C@H]([C@@H](O)CCC(O)(C)C)C)CC4)CC3)=C1)C[C@H](O)[C@H](O)C2 UPEZCKBFRMILAV-JNEQICEOSA-N 0.000 claims abstract description 49
- UPEZCKBFRMILAV-UHFFFAOYSA-N alpha-Ecdysone Natural products C1C(O)C(O)CC2(C)C(CCC3(C(C(C(O)CCC(C)(C)O)C)CCC33O)C)C3=CC(=O)C21 UPEZCKBFRMILAV-UHFFFAOYSA-N 0.000 claims abstract description 49
- UPEZCKBFRMILAV-JMZLNJERSA-N ecdysone Chemical compound C1[C@@H](O)[C@@H](O)C[C@]2(C)[C@@H](CC[C@@]3([C@@H]([C@@H]([C@H](O)CCC(C)(C)O)C)CC[C@]33O)C)C3=CC(=O)[C@@H]21 UPEZCKBFRMILAV-JMZLNJERSA-N 0.000 claims abstract description 49
- 108091026890 Coding region Proteins 0.000 claims abstract description 46
- 238000004519 manufacturing process Methods 0.000 claims abstract description 25
- 108091033319 polynucleotide Proteins 0.000 claims abstract description 17
- 102000040430 polynucleotide Human genes 0.000 claims abstract description 17
- 239000002157 polynucleotide Substances 0.000 claims abstract description 17
- 241001465754 Metazoa Species 0.000 claims abstract description 11
- 108010057988 ecdysone receptor Proteins 0.000 claims description 26
- 238000000034 method Methods 0.000 claims description 26
- 239000013598 vector Substances 0.000 claims description 24
- 230000002950 deficient Effects 0.000 claims description 15
- 101001124991 Homo sapiens Nitric oxide synthase, inducible Proteins 0.000 claims description 14
- 108010034634 Repressor Proteins Proteins 0.000 claims description 7
- 102000009661 Repressor Proteins Human genes 0.000 claims description 7
- 239000000833 heterodimer Substances 0.000 claims description 6
- 239000003937 drug carrier Substances 0.000 claims description 5
- 101000974009 Homo sapiens Nitric oxide synthase, brain Proteins 0.000 claims description 4
- 101001124309 Homo sapiens Nitric oxide synthase, endothelial Proteins 0.000 claims description 4
- 239000003085 diluting agent Substances 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 239000003814 drug Substances 0.000 claims description 3
- 239000008194 pharmaceutical composition Substances 0.000 claims description 3
- 102000034527 Retinoid X Receptors Human genes 0.000 claims 2
- 108010038912 Retinoid X Receptors Proteins 0.000 claims 2
- 229920000936 Agarose Polymers 0.000 claims 1
- NIXOWILDQLNWCW-UHFFFAOYSA-N acrylic acid group Chemical group C(C=C)(=O)O NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 claims 1
- 229940102838 methylmethacrylate Drugs 0.000 claims 1
- 229920002627 poly(phosphazenes) Polymers 0.000 claims 1
- 238000002560 therapeutic procedure Methods 0.000 claims 1
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 abstract description 116
- 230000002159 abnormal effect Effects 0.000 abstract description 4
- 210000004027 cell Anatomy 0.000 description 200
- 101150061166 tetR gene Proteins 0.000 description 35
- 229920000615 alginic acid Polymers 0.000 description 31
- LQGNCUXDDPRDJH-UHFFFAOYSA-N 3'-GMP Natural products C1C(O)C(O)CC2(C)C(C(O)CC3(C(C(C)(O)C(O)CCC(C)C)CCC33O)C)C3=CC(=O)C21 LQGNCUXDDPRDJH-UHFFFAOYSA-N 0.000 description 29
- LRJUYAVTHIEHAI-UHFFFAOYSA-N Muristeron A Natural products C1C(O)C(O)CC2(C)C(C(O)CC3(C(C(C)(O)C(O)CCC(C)C)CCC33O)C)C3=CC(=O)C21O LRJUYAVTHIEHAI-UHFFFAOYSA-N 0.000 description 29
- LRJUYAVTHIEHAI-LHBNDURVSA-N Muristerone Chemical compound C1[C@@H](O)[C@@H](O)C[C@]2(C)[C@@H]([C@H](O)C[C@@]3([C@@H]([C@@](C)(O)[C@H](O)CCC(C)C)CC[C@]33O)C)C3=CC(=O)[C@@]21O LRJUYAVTHIEHAI-LHBNDURVSA-N 0.000 description 29
- 235000010443 alginic acid Nutrition 0.000 description 26
- 239000013612 plasmid Substances 0.000 description 25
- 239000000243 solution Substances 0.000 description 25
- 229940072056 alginate Drugs 0.000 description 24
- 239000000463 material Substances 0.000 description 23
- 108090000623 proteins and genes Proteins 0.000 description 23
- 230000014509 gene expression Effects 0.000 description 21
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 description 19
- 239000000126 substance Substances 0.000 description 19
- 230000000694 effects Effects 0.000 description 14
- 239000002299 complementary DNA Substances 0.000 description 13
- 102000011779 Nitric Oxide Synthase Type II Human genes 0.000 description 12
- 108010076864 Nitric Oxide Synthase Type II Proteins 0.000 description 12
- 239000000499 gel Substances 0.000 description 12
- 102000055708 human NOS2 Human genes 0.000 description 12
- 230000004044 response Effects 0.000 description 11
- 238000006467 substitution reaction Methods 0.000 description 11
- PJYYBCXMCWDUAZ-JJJZTNILSA-N 2,3,14,20,22-pentahydroxy-(2β,3β,5β,22R)-Cholest-7-en-6-one Chemical compound C1[C@@H](O)[C@@H](O)C[C@]2(C)[C@@H](CC[C@@]3([C@@H]([C@@](C)(O)[C@H](O)CCC(C)C)CC[C@]33O)C)C3=CC(=O)[C@@H]21 PJYYBCXMCWDUAZ-JJJZTNILSA-N 0.000 description 10
- PJYYBCXMCWDUAZ-YKDQUOQBSA-N Ponasterone A Natural products O=C1[C@H]2[C@@](C)([C@@H]3C([C@@]4(O)[C@@](C)([C@H]([C@@](O)([C@@H](O)CCC(C)C)C)CC4)CC3)=C1)C[C@H](O)[C@H](O)C2 PJYYBCXMCWDUAZ-YKDQUOQBSA-N 0.000 description 10
- 238000001727 in vivo Methods 0.000 description 10
- 229920001184 polypeptide Polymers 0.000 description 10
- 108090000765 processed proteins & peptides Proteins 0.000 description 10
- 102000004196 processed proteins & peptides Human genes 0.000 description 10
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 9
- 241000699666 Mus <mouse, genus> Species 0.000 description 9
- 206010028980 Neoplasm Diseases 0.000 description 9
- 239000006285 cell suspension Substances 0.000 description 9
- 238000005538 encapsulation Methods 0.000 description 9
- 238000002474 experimental method Methods 0.000 description 9
- 239000012091 fetal bovine serum Substances 0.000 description 9
- 230000006698 induction Effects 0.000 description 9
- 230000001105 regulatory effect Effects 0.000 description 9
- 239000000725 suspension Substances 0.000 description 9
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 8
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 8
- 238000006595 Griess deamination reaction Methods 0.000 description 8
- 239000002609 medium Substances 0.000 description 8
- -1 peroxynitrite anion Chemical class 0.000 description 8
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 7
- 238000012217 deletion Methods 0.000 description 7
- 230000037430 deletion Effects 0.000 description 7
- 238000009792 diffusion process Methods 0.000 description 7
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 7
- 210000005260 human cell Anatomy 0.000 description 7
- 238000002513 implantation Methods 0.000 description 7
- 210000002540 macrophage Anatomy 0.000 description 7
- 210000004962 mammalian cell Anatomy 0.000 description 7
- 239000012528 membrane Substances 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 125000003729 nucleotide group Chemical group 0.000 description 7
- 239000002953 phosphate buffered saline Substances 0.000 description 7
- 229920000729 poly(L-lysine) polymer Polymers 0.000 description 7
- 239000008159 sesame oil Substances 0.000 description 7
- 235000011803 sesame oil Nutrition 0.000 description 7
- 239000011734 sodium Substances 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 238000013459 approach Methods 0.000 description 6
- 230000004888 barrier function Effects 0.000 description 6
- 210000004379 membrane Anatomy 0.000 description 6
- 239000002773 nucleotide Substances 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 235000018102 proteins Nutrition 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 5
- 241000701022 Cytomegalovirus Species 0.000 description 5
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 5
- 241000238631 Hexapoda Species 0.000 description 5
- 241000699670 Mus sp. Species 0.000 description 5
- 238000000636 Northern blotting Methods 0.000 description 5
- 108010084455 Zeocin Proteins 0.000 description 5
- 150000001413 amino acids Chemical class 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 229910001424 calcium ion Inorganic materials 0.000 description 5
- 150000001768 cations Chemical class 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 208000029742 colonic neoplasm Diseases 0.000 description 5
- 239000012634 fragment Substances 0.000 description 5
- 238000011068 loading method Methods 0.000 description 5
- 108020004999 messenger RNA Proteins 0.000 description 5
- CWCMIVBLVUHDHK-ZSNHEYEWSA-N phleomycin D1 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC[C@@H](N=1)C=1SC=C(N=1)C(=O)NCCCCNC(N)=N)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C CWCMIVBLVUHDHK-ZSNHEYEWSA-N 0.000 description 5
- 238000001890 transfection Methods 0.000 description 5
- 210000004881 tumor cell Anatomy 0.000 description 5
- 238000001262 western blot Methods 0.000 description 5
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 4
- 206010009944 Colon cancer Diseases 0.000 description 4
- 206010033128 Ovarian cancer Diseases 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 235000001014 amino acid Nutrition 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 4
- 239000011575 calcium Substances 0.000 description 4
- 239000001110 calcium chloride Substances 0.000 description 4
- 229910001628 calcium chloride Inorganic materials 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000000284 extract Substances 0.000 description 4
- 239000000710 homodimer Substances 0.000 description 4
- 238000002955 isolation Methods 0.000 description 4
- 230000007246 mechanism Effects 0.000 description 4
- 235000015097 nutrients Nutrition 0.000 description 4
- 229910052760 oxygen Inorganic materials 0.000 description 4
- 239000001301 oxygen Substances 0.000 description 4
- 229920000642 polymer Polymers 0.000 description 4
- 108091008146 restriction endonucleases Proteins 0.000 description 4
- 239000003270 steroid hormone Substances 0.000 description 4
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 230000001225 therapeutic effect Effects 0.000 description 4
- 229920001169 thermoplastic Polymers 0.000 description 4
- 238000011144 upstream manufacturing Methods 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 3
- 102000006538 Nitric Oxide Synthase Type I Human genes 0.000 description 3
- 108010008858 Nitric Oxide Synthase Type I Proteins 0.000 description 3
- 102000008052 Nitric Oxide Synthase Type III Human genes 0.000 description 3
- 108010075520 Nitric Oxide Synthase Type III Proteins 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- 206010061535 Ovarian neoplasm Diseases 0.000 description 3
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 3
- 229930006000 Sucrose Natural products 0.000 description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 3
- 102000004142 Trypsin Human genes 0.000 description 3
- 108090000631 Trypsin Proteins 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 239000011324 bead Substances 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 239000013592 cell lysate Substances 0.000 description 3
- 230000000295 complement effect Effects 0.000 description 3
- 238000004132 cross linking Methods 0.000 description 3
- 235000013681 dietary sucrose Nutrition 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 210000002889 endothelial cell Anatomy 0.000 description 3
- 235000011187 glycerol Nutrition 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 230000009931 harmful effect Effects 0.000 description 3
- 102000055707 human NOS1 Human genes 0.000 description 3
- 102000055702 human NOS3 Human genes 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 239000003112 inhibitor Substances 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- 239000010410 layer Substances 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 238000011580 nude mouse model Methods 0.000 description 3
- 229920002239 polyacrylonitrile Polymers 0.000 description 3
- 108020003175 receptors Proteins 0.000 description 3
- 102000005962 receptors Human genes 0.000 description 3
- 150000003384 small molecules Chemical class 0.000 description 3
- 235000015424 sodium Nutrition 0.000 description 3
- 229910052708 sodium Inorganic materials 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 229960004793 sucrose Drugs 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 238000013518 transcription Methods 0.000 description 3
- 230000035897 transcription Effects 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- 238000012384 transportation and delivery Methods 0.000 description 3
- 239000012588 trypsin Substances 0.000 description 3
- 230000004614 tumor growth Effects 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- SGKRLCUYIXIAHR-AKNGSSGZSA-N (4s,4ar,5s,5ar,6r,12ar)-4-(dimethylamino)-1,5,10,11,12a-pentahydroxy-6-methyl-3,12-dioxo-4a,5,5a,6-tetrahydro-4h-tetracene-2-carboxamide Chemical compound C1=CC=C2[C@H](C)[C@@H]([C@H](O)[C@@H]3[C@](C(O)=C(C(N)=O)C(=O)[C@H]3N(C)C)(O)C3=O)C3=C(O)C2=C1O SGKRLCUYIXIAHR-AKNGSSGZSA-N 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- ZIIQCSMRQKCOCT-UHFFFAOYSA-N 2-acetamido-3-methyl-3-nitrososulfanylbutanoic acid Chemical compound CC(=O)NC(C(O)=O)C(C)(C)SN=O ZIIQCSMRQKCOCT-UHFFFAOYSA-N 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 125000001433 C-terminal amino-acid group Chemical group 0.000 description 2
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 2
- 201000009030 Carcinoma Diseases 0.000 description 2
- 229920001661 Chitosan Polymers 0.000 description 2
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 101000756632 Homo sapiens Actin, cytoplasmic 1 Proteins 0.000 description 2
- 241000701024 Human betaherpesvirus 5 Species 0.000 description 2
- 108010050904 Interferons Proteins 0.000 description 2
- 102000014150 Interferons Human genes 0.000 description 2
- RHGKLRLOHDJJDR-BYPYZUCNSA-N L-citrulline Chemical compound NC(=O)NCCC[C@H]([NH3+])C([O-])=O RHGKLRLOHDJJDR-BYPYZUCNSA-N 0.000 description 2
- 241000699660 Mus musculus Species 0.000 description 2
- 229910002651 NO3 Inorganic materials 0.000 description 2
- 101150005851 NOS gene Proteins 0.000 description 2
- RHGKLRLOHDJJDR-UHFFFAOYSA-N Ndelta-carbamoyl-DL-ornithine Natural products OC(=O)C(N)CCCNC(N)=O RHGKLRLOHDJJDR-UHFFFAOYSA-N 0.000 description 2
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 2
- SNIOPGDIGTZGOP-UHFFFAOYSA-N Nitroglycerin Chemical compound [O-][N+](=O)OCC(O[N+]([O-])=O)CO[N+]([O-])=O SNIOPGDIGTZGOP-UHFFFAOYSA-N 0.000 description 2
- 208000012868 Overgrowth Diseases 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- 108010039918 Polylysine Chemical group 0.000 description 2
- 241000283984 Rodentia Species 0.000 description 2
- 241000714474 Rous sarcoma virus Species 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 102000004243 Tubulin Human genes 0.000 description 2
- 108090000704 Tubulin Proteins 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- DZBUGLKDJFMEHC-UHFFFAOYSA-N acridine Chemical group C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 2
- 238000002399 angioplasty Methods 0.000 description 2
- 230000000890 antigenic effect Effects 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 239000001768 carboxy methyl cellulose Substances 0.000 description 2
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 2
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 235000010980 cellulose Nutrition 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 229960002173 citrulline Drugs 0.000 description 2
- 235000013477 citrulline Nutrition 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000001461 cytolytic effect Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- XEYBHCRIKKKOSS-UHFFFAOYSA-N disodium;azanylidyneoxidanium;iron(2+);pentacyanide Chemical compound [Na+].[Na+].[Fe+2].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].[O+]#N XEYBHCRIKKKOSS-UHFFFAOYSA-N 0.000 description 2
- 231100000673 dose–response relationship Toxicity 0.000 description 2
- 229960003722 doxycycline Drugs 0.000 description 2
- 239000003623 enhancer Substances 0.000 description 2
- 238000001952 enzyme assay Methods 0.000 description 2
- 230000001605 fetal effect Effects 0.000 description 2
- 210000002950 fibroblast Anatomy 0.000 description 2
- 230000003176 fibrotic effect Effects 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 239000000017 hydrogel Substances 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 230000002163 immunogen Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 239000000411 inducer Substances 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 230000002757 inflammatory effect Effects 0.000 description 2
- 230000028709 inflammatory response Effects 0.000 description 2
- 238000001802 infusion Methods 0.000 description 2
- 229940079322 interferon Drugs 0.000 description 2
- 238000007912 intraperitoneal administration Methods 0.000 description 2
- 210000003292 kidney cell Anatomy 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 229920000609 methyl cellulose Polymers 0.000 description 2
- 239000001923 methylcellulose Substances 0.000 description 2
- 235000010981 methylcellulose Nutrition 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 229940049954 penicillin Drugs 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 230000004962 physiological condition Effects 0.000 description 2
- 231100000614 poison Toxicity 0.000 description 2
- 229920003229 poly(methyl methacrylate) Polymers 0.000 description 2
- 229920002401 polyacrylamide Polymers 0.000 description 2
- 229920000656 polylysine Chemical group 0.000 description 2
- 239000004926 polymethyl methacrylate Substances 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 230000001698 pyrogenic effect Effects 0.000 description 2
- 208000037803 restenosis Diseases 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 2
- 229940083618 sodium nitroprusside Drugs 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 230000000638 stimulation Effects 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 229960005322 streptomycin Drugs 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 229920001059 synthetic polymer Polymers 0.000 description 2
- 239000006188 syrup Substances 0.000 description 2
- 235000020357 syrup Nutrition 0.000 description 2
- 239000003440 toxic substance Substances 0.000 description 2
- 230000005909 tumor killing Effects 0.000 description 2
- 230000035899 viability Effects 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- ODKSFYDXXFIFQN-FXLFCPKBSA-N (2S)-2-amino-5-(diaminomethylideneamino)(214C)pentanoic acid Chemical compound N[14C@@H](CCCNC(N)=N)C(=O)O ODKSFYDXXFIFQN-FXLFCPKBSA-N 0.000 description 1
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 description 1
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 1
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 108010039627 Aprotinin Proteins 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 101100329226 Bacillus anthracis cpfC1 gene Proteins 0.000 description 1
- VGGGPCQERPFHOB-MCIONIFRSA-N Bestatin Chemical compound CC(C)C[C@H](C(O)=O)NC(=O)[C@@H](O)[C@H](N)CC1=CC=CC=C1 VGGGPCQERPFHOB-MCIONIFRSA-N 0.000 description 1
- VGGGPCQERPFHOB-UHFFFAOYSA-N Bestatin Natural products CC(C)CC(C(O)=O)NC(=O)C(O)C(N)CC1=CC=CC=C1 VGGGPCQERPFHOB-UHFFFAOYSA-N 0.000 description 1
- 229920002261 Corn starch Polymers 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 1
- 241000255601 Drosophila melanogaster Species 0.000 description 1
- LVGKNOAMLMIIKO-UHFFFAOYSA-N Elaidinsaeure-aethylester Natural products CCCCCCCCC=CCCCCCCCC(=O)OCC LVGKNOAMLMIIKO-UHFFFAOYSA-N 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 206010019280 Heart failures Diseases 0.000 description 1
- 208000031226 Hyperlipidaemia Diseases 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 101150109636 Inos gene Proteins 0.000 description 1
- 102100037850 Interferon gamma Human genes 0.000 description 1
- 102000006992 Interferon-alpha Human genes 0.000 description 1
- 108010047761 Interferon-alpha Proteins 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- IAJILQKETJEXLJ-SQOUGZDYSA-N L-guluronic acid Chemical compound O=C[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O IAJILQKETJEXLJ-SQOUGZDYSA-N 0.000 description 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- GDBQQVLCIARPGH-UHFFFAOYSA-N Leupeptin Natural products CC(C)CC(NC(C)=O)C(=O)NC(CC(C)C)C(=O)NC(C=O)CCCN=C(N)N GDBQQVLCIARPGH-UHFFFAOYSA-N 0.000 description 1
- 108010074338 Lymphokines Proteins 0.000 description 1
- 102000008072 Lymphokines Human genes 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 241001599018 Melanogaster Species 0.000 description 1
- 241000713869 Moloney murine leukemia virus Species 0.000 description 1
- 241000204031 Mycoplasma Species 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 1
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 1
- 239000004695 Polyether sulfone Substances 0.000 description 1
- 229920002367 Polyisobutene Polymers 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 239000004372 Polyvinyl alcohol Substances 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 108010029485 Protein Isoforms Proteins 0.000 description 1
- 102000001708 Protein Isoforms Human genes 0.000 description 1
- 108091034057 RNA (poly(A)) Proteins 0.000 description 1
- 208000001647 Renal Insufficiency Diseases 0.000 description 1
- 241000700584 Simplexvirus Species 0.000 description 1
- 235000021355 Stearic acid Nutrition 0.000 description 1
- RYYWUUFWQRZTIU-UHFFFAOYSA-N Thiophosphoric acid Chemical group OP(O)(S)=O RYYWUUFWQRZTIU-UHFFFAOYSA-N 0.000 description 1
- 108020004440 Thymidine kinase Proteins 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- 229940122618 Trypsin inhibitor Drugs 0.000 description 1
- 101710162629 Trypsin inhibitor Proteins 0.000 description 1
- 102000018594 Tumour necrosis factor Human genes 0.000 description 1
- 108050007852 Tumour necrosis factor Proteins 0.000 description 1
- BZHJMEDXRYGGRV-UHFFFAOYSA-N Vinyl chloride Chemical compound ClC=C BZHJMEDXRYGGRV-UHFFFAOYSA-N 0.000 description 1
- 230000003187 abdominal effect Effects 0.000 description 1
- 210000003815 abdominal wall Anatomy 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 239000000783 alginic acid Substances 0.000 description 1
- 229960001126 alginic acid Drugs 0.000 description 1
- 150000004781 alginic acids Chemical class 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 230000003466 anti-cipated effect Effects 0.000 description 1
- 239000002260 anti-inflammatory agent Substances 0.000 description 1
- 230000009833 antibody interaction Effects 0.000 description 1
- 229960004405 aprotinin Drugs 0.000 description 1
- 239000012736 aqueous medium Substances 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 210000001367 artery Anatomy 0.000 description 1
- 239000003855 balanced salt solution Substances 0.000 description 1
- 210000002469 basement membrane Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- AEMOLEFTQBMNLQ-UHFFFAOYSA-N beta-D-galactopyranuronic acid Natural products OC1OC(C(O)=O)C(O)C(O)C1O AEMOLEFTQBMNLQ-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 238000002306 biochemical method Methods 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000013060 biological fluid Substances 0.000 description 1
- 229930189065 blasticidin Natural products 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
- CJZGTCYPCWQAJB-UHFFFAOYSA-L calcium stearate Chemical compound [Ca+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O CJZGTCYPCWQAJB-UHFFFAOYSA-L 0.000 description 1
- 239000008116 calcium stearate Substances 0.000 description 1
- 235000013539 calcium stearate Nutrition 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 229920006217 cellulose acetate butyrate Polymers 0.000 description 1
- 229920001727 cellulose butyrate Polymers 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 201000010897 colon adenocarcinoma Diseases 0.000 description 1
- 230000030944 contact inhibition Effects 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 239000008120 corn starch Substances 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- AEMOLEFTQBMNLQ-YBSDWZGDSA-N d-mannuronic acid Chemical compound O[C@@H]1O[C@@H](C(O)=O)[C@H](O)[C@@H](O)[C@H]1O AEMOLEFTQBMNLQ-YBSDWZGDSA-N 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- NHNBFGGVMKEFGY-OUBTZVSYSA-N dioxido(oxo)azanium Chemical compound [O-][15N+]([O-])=O NHNBFGGVMKEFGY-OUBTZVSYSA-N 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
- MOTZDAYCYVMXPC-UHFFFAOYSA-N dodecyl hydrogen sulfate Chemical class CCCCCCCCCCCCOS(O)(=O)=O MOTZDAYCYVMXPC-UHFFFAOYSA-N 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 150000002058 ecdysones Chemical class 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- LVGKNOAMLMIIKO-QXMHVHEDSA-N ethyl oleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC LVGKNOAMLMIIKO-QXMHVHEDSA-N 0.000 description 1
- 229940093471 ethyl oleate Drugs 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 239000011536 extraction buffer Substances 0.000 description 1
- 238000001125 extrusion Methods 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 239000007888 film coating Substances 0.000 description 1
- 238000009501 film coating Methods 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 229960002963 ganciclovir Drugs 0.000 description 1
- IRSCQMHQWWYFCW-UHFFFAOYSA-N ganciclovir Chemical compound O=C1NC(N)=NC2=C1N=CN2COC(CO)CO IRSCQMHQWWYFCW-UHFFFAOYSA-N 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 238000001879 gelation Methods 0.000 description 1
- 238000001476 gene delivery Methods 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 150000002334 glycols Chemical class 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 231100000086 high toxicity Toxicity 0.000 description 1
- 239000012510 hollow fiber Substances 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 229940027941 immunoglobulin g Drugs 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- ZPNFWUPYTFPOJU-LPYSRVMUSA-N iniprol Chemical compound C([C@H]1C(=O)NCC(=O)NCC(=O)N[C@H]2CSSC[C@H]3C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(N[C@H](C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC=4C=CC=CC=4)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC=4C=CC=CC=4)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC2=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC=2C=CC=CC=2)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H]2N(CCC2)C(=O)[C@@H](N)CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N2[C@@H](CCC2)C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N2[C@@H](CCC2)C(=O)N3)C(=O)NCC(=O)NCC(=O)N[C@@H](C)C(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@H](C(=O)N1)C(C)C)[C@@H](C)O)[C@@H](C)CC)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 ZPNFWUPYTFPOJU-LPYSRVMUSA-N 0.000 description 1
- 239000000201 insect hormone Substances 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 239000011229 interlayer Substances 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 230000000622 irritating effect Effects 0.000 description 1
- 239000000644 isotonic solution Substances 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 201000006370 kidney failure Diseases 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 239000012633 leachable Substances 0.000 description 1
- 235000010445 lecithin Nutrition 0.000 description 1
- 239000000787 lecithin Substances 0.000 description 1
- 229940067606 lecithin Drugs 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- GDBQQVLCIARPGH-ULQDDVLXSA-N leupeptin Chemical compound CC(C)C[C@H](NC(C)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C=O)CCCN=C(N)N GDBQQVLCIARPGH-ULQDDVLXSA-N 0.000 description 1
- 108010052968 leupeptin Proteins 0.000 description 1
- 229960004393 lidocaine hydrochloride Drugs 0.000 description 1
- YECIFGHRMFEPJK-UHFFFAOYSA-N lidocaine hydrochloride monohydrate Chemical compound O.[Cl-].CC[NH+](CC)CC(=O)NC1=C(C)C=CC=C1C YECIFGHRMFEPJK-UHFFFAOYSA-N 0.000 description 1
- 229920005684 linear copolymer Polymers 0.000 description 1
- 239000007937 lozenge Substances 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Substances [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000029052 metamorphosis Effects 0.000 description 1
- YACKEPLHDIMKIO-UHFFFAOYSA-N methylphosphonic acid Chemical compound CP(O)(O)=O YACKEPLHDIMKIO-UHFFFAOYSA-N 0.000 description 1
- 230000000394 mitotic effect Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- OHDXDNUPVVYWOV-UHFFFAOYSA-N n-methyl-1-(2-naphthalen-1-ylsulfanylphenyl)methanamine Chemical compound CNCC1=CC=CC=C1SC1=CC=CC2=CC=CC=C12 OHDXDNUPVVYWOV-UHFFFAOYSA-N 0.000 description 1
- 229920001206 natural gum Polymers 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000000269 nucleophilic effect Effects 0.000 description 1
- 238000013392 nude mouse xenograft model Methods 0.000 description 1
- 235000021048 nutrient requirements Nutrition 0.000 description 1
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 1
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- 239000004006 olive oil Substances 0.000 description 1
- 235000008390 olive oil Nutrition 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000002611 ovarian Effects 0.000 description 1
- 239000007800 oxidant agent Substances 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 238000005192 partition Methods 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 229920001277 pectin Polymers 0.000 description 1
- 239000001814 pectin Substances 0.000 description 1
- 235000010987 pectin Nutrition 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 210000003200 peritoneal cavity Anatomy 0.000 description 1
- 210000004303 peritoneum Anatomy 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 239000008024 pharmaceutical diluent Substances 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 230000009894 physiological stress Effects 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920002492 poly(sulfone) Polymers 0.000 description 1
- 229920006393 polyether sulfone Polymers 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920000098 polyolefin Polymers 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 229940068965 polysorbates Drugs 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 235000019422 polyvinyl alcohol Nutrition 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 229920001592 potato starch Polymers 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000001007 puffing effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002510 pyrogen Substances 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004043 responsiveness Effects 0.000 description 1
- 239000012723 sample buffer Substances 0.000 description 1
- 201000000980 schizophrenia Diseases 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 235000010413 sodium alginate Nutrition 0.000 description 1
- 239000000661 sodium alginate Substances 0.000 description 1
- 229940005550 sodium alginate Drugs 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 235000010288 sodium nitrite Nutrition 0.000 description 1
- 229920003109 sodium starch glycolate Polymers 0.000 description 1
- 229940079832 sodium starch glycolate Drugs 0.000 description 1
- 239000008109 sodium starch glycolate Substances 0.000 description 1
- 210000004872 soft tissue Anatomy 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 229940032147 starch Drugs 0.000 description 1
- 239000008117 stearic acid Substances 0.000 description 1
- 108020003113 steroid hormone receptors Proteins 0.000 description 1
- 102000005969 steroid hormone receptors Human genes 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 238000009495 sugar coating Methods 0.000 description 1
- FDDDEECHVMSUSB-UHFFFAOYSA-N sulfanilamide Chemical compound NC1=CC=C(S(N)(=O)=O)C=C1 FDDDEECHVMSUSB-UHFFFAOYSA-N 0.000 description 1
- 229940124530 sulfonamide Drugs 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 238000012385 systemic delivery Methods 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 235000012222 talc Nutrition 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 239000004416 thermosoftening plastic Substances 0.000 description 1
- 230000036962 time dependent Effects 0.000 description 1
- 238000000844 transformation Methods 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 239000002753 trypsin inhibitor Substances 0.000 description 1
- 230000005747 tumor angiogenesis Effects 0.000 description 1
- 229950009811 ubenimex Drugs 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/06—Antihyperlipidemics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/12—Antihypertensives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K2035/126—Immunoprotecting barriers, e.g. jackets, diffusion chambers
- A61K2035/128—Immunoprotecting barriers, e.g. jackets, diffusion chambers capsules, e.g. microcapsules
Definitions
- This invention relates to microencapsulated recombinant cells for in vivo delivery of therapeutic substances.
- NO nitric oxide
- nitric oxide can have beneficial effects in a number of disease states.
- NO can be a double edged sword: the high levels of NO produced by macrophages and endothelial cells can have a tumor killing effect, whereas low and medium doses of NO may actually promote tumor growth, upregulation of p53 and tumor angiogenesis. Control of NO levels in vivo. however, is difficult to achieve.
- a well established pharmacological NO-based therapeutic approach is to add NO in some form.
- One way to do this is simply to add authentic NO solution.
- NO-donors which can release NO continuously in a biological environment.
- Compounds such as glycerin trinitrate (GTN), sodium nitroprusside (SNP) and S-nitroso-N-acetyl-DL- penicillamine (SNAP) are widely used therapeutically.
- GTN glycerin trinitrate
- SNP sodium nitroprusside
- SNAP S-nitroso-N-acetyl-DL- penicillamine
- NO release is often not linear.
- NO-donors may also give rise to related NO species that have different biological properties from native NO. For example, NO may react with oxygen derived radicals to produce toxic substances such as the peroxynitrite anion (ONOO-).
- nitric oxide synthases inducible nitric oxide synthase (iNOS), neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS), in the presence of the insect steroid hormone ecdysone or an analog thereof.
- iNOS inducible nitric oxide synthase
- nNOS neuronal nitric oxide synthase
- eNOS endothelial nitric oxide synthase
- cell lines which are capable of producing NO in a dose- and time-dependent manner by treatment of the cells with ecdysone or an analog thereof or tetracycline or an analog thereof.
- Our cell-based NO "switches” produce biologically active inducible NOS capable of generating NO.
- microencapsulated cells of the invention were implanted into mice. They survived and upon removal from the host mice were still capable of induction by muristerone A and of NO production.
- microencapsulated cells of the invention may be used as artificial endothelial cells or as artificial macrophage cells in the treatment of human conditions related to abnormal NO metabolism or conditions where the administration of nitric oxide is required.
- This approach has advantages over NO- donor and gene therapy approaches as 100% of the cells introduced into a target region will be capable of generating NO in a highly regulated way.
- a promoter which is responsive to ecdysone or an analog thereof and which is operably linked to a coding sequence for a nitric oxide synthase (NOS) or a functional variant thereof; or
- a promoter operably linked to one or more tetracycline operator site sequences and a coding sequence in that order, wherein the coding sequence encodes a nitric oxide synthase (NOS) or a functional variant thereof.
- NOS nitric oxide synthase
- the invention also provides: microcapsules of the invention for use in a method of treatment of the human or animal body; - use of microcapsules of the invention for the manufacture of a medicament for use in the treatment of a condition associated with deficient NO production; a pharmaceutical composition comprising microcapsules of the invention and a pharmaceutically acceptable carrier or diluent; - a method of delivering microcapsules to a host comprising administering microcapsules of the invention to the host; a method of treating a host suffering from a condition associated with deficient NO production, which method comprises administering ecdysone or an analog thereof to a host which harbours microcapsules of the invention.
- - a product containing microcapsules of the invention and ecdysone or an analog thereof as a combined preparation for simultaneous, separate or sequential use in the treatment of condition associated with deficient NO production;
- a polynucleotide construct comprising: (a) a promoter operably linked to a coding sequence, wherein the promoter is responsive to ecdysone or an analog thereof and the coding sequence encodes a nitric oxide synthase (NOS) or a functional variant thereof; or (b) a promoter operably linked to one or more tetracycline operator site sequences and a coding sequence in that order, wherein the coding sequence encodes a nitric oxide synthase (NOS) or a functional variant thereof; a vector which incorporates a polynucleotide construct of the invention; a process for preparing microcapsules comprising encapsulating cells of the invention.
- Figure 1 (a) shows the plasmid map for pIND-hiNOS-f (human iNOS);
- Figure 1(b) shows the plasmid map for pIND-hnNOS-f (human nNOS);
- Figure 1 (c) shows the plasmid map for p-IND-heNOS-f (human eNOS).
- Figure 1(d) shows the plasmid map of pTet-hiNOS-f (human iNOS).
- FIG. 2(a) shows generation of NO by EcR293 clone 1 1, following treatment with muristerone A.
- EcR293 clone 11 cells were grown with varying concentrations of muristerone A and at different time intervals supernatants were taken and the Griess reaction was used to measure the nitrite concentration.
- Figure 2(b) shows
- Northern and Western blots of carried out on extracts from cells treated with either 1 ⁇ M or lO ⁇ M muristerone A. Extracts were also taken from cells grown in the absence of muristerone A.
- Northern blots filters were probed with a human iNOS cDNA and hybridisation with human ⁇ -actin was used as a loading control.
- Western blots filters were probed with a polyclonal antibody raised against the 7
- Figure 3 shows generation of NO in a panel of cell lines transfected with pTet-hiNOS-f. Cells were treated with 1 ⁇ g/ml tetracycline for 24h and NOS activity was assessed by assaying for accumulated nitrite using the Griess reaction.
- FIG. 4 shows the general strategy used for microencapsulation of cells.
- EcR293 clone 11 cells are mixed with Na + alginate in the barrel of a syringe.
- the resulting solution is expelled dropwise through the syringe needle into a solution of CaCl 2 giving rise to polymer beads containing EcR293 clone 1 1 cells.
- the microencapsuled are hardened by mixing with poly-L-lysine.
- the microcapsules are ready for in vitro culture or in vivo injection/transplantation. Alternatively, microcapsules may be stored in DMEM medium supplemented with 10% fetal bovine serum.
- FIG. 5 shows generation of NO by encapsulated EcR293 clone 11 cells, following treatment with muristerone A.
- EcR293 clone 11 cells were encapsulated with alginate-poly-L-lysine-alginate. Cells were induced to produce NO at a controlled level by the application of lO ⁇ M muristerone A. When muristerone A was removed the production of NO fell. On re-application of lO ⁇ M muristerone A the level of NO production again increased.
- Figure 6 shows the results of experiments carried out with microencapsulated EcR293 clone 11 cells.
- A shows the results of subcutaneous injection of human colon cancer DLD-1 cells (lxl 0 6 per mouse) with microencapsulated human EcR293 clone 11 cells (5xl0 6 per mouse) either under induction conditions (ponasterone A lmg per mouse, ⁇ ) or sesame-seed oil only ( ⁇ ). Each point represents mean ⁇ S.D. for 5 mice.
- Steroid hormones are small hydrophobic molecules that can diffuse through the plasma membrane of cells where they can bind reversibly to specific steroid- hormone-receptor proteins in the cytoplasm or nucleus.
- the binding of hormone activates the receptor, enabling it to bind with high affinity to specific DNA sequences that act as trans c ⁇ ptional enhancers This binding mcreases the level of trans c ⁇ pti on from certain nearby genes
- a pulse of the insect steroid hormone ecdysone triggers metamorphosis m Drosoph ⁇ a melanogaster, showing effects such as chromosomal puffing within minutes of hormone addition Mediating this response is the functional ecdysone receptor, which is a heterodimer of the ecdysone receptor (EcR) and the product of the ultraspiracle gene (USP)
- Insect hormone responsiveness can be lecreated m cultured mammalian cells by cotransfection of a cell with a functional ecdysone receptor (a heterodimer of EcR and USP) and an ecdysone responsive construct and treatment of the cell with ecdysone or an analog theieof
- a tetiacyclme responsive system can be created in cultured mammalian cells by cotransfection of a cell with a plasmid encoding a tetracyclme repressor protein (tetR) and a plasmid containing a tetracyclme responsive element linked to a piomoter
- the piomoter sequence is used to dnve heterologous gene expression
- the tetracyclme responsive element comp ⁇ ses particular DNA sequences called tetracyclme operator sites, which can bind a homodimer of tetR If those sequences are positioned between a promoter and a coding sequence in a construct, the presence of tetR bound to a tetracycline operator site will prevent the promoter driving expression of the coding sequence
- tetracyclme when tetracyclme is added to cells the tetracyclme binds to tetR homodimers leading to a conformational change m
- the invention provides polynucleotide constructs which are responsive to ecdysone or an analog thereof
- the invention also provides polynucleotide constructs which are responsive to tetracycline or an analog thereof
- the ecdysonse responsive constructs comp ⁇ se a promoter operably lmked to a coding sequence, wherein the promoter is responsive to ecdysone or an analog thereof and the coding sequence codes for a NOS or a functional variant or fragment thereof.
- the tetracycline responsive constructs comprise a promoter operably linked to one or more teracycline operator site sequences and a coding sequence in that order, wherein the coding sequence codes for a NOS or a functional variant or fragment thereof.
- the constructs may comprise DNA or RNA. They may also include within them synthetic or modified nucleotides. A number of different types of modification to polynucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule.
- the constructs described herein may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or lifespan of constructs of the invention. Constructs of the invention may be produced recombinantly, synthetically, or by any means available to those of skill in the art.
- operably linked refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.
- a regulatory sequence such as a promoter
- operably linked to a coding sequence is positioned in such a way that expression of the coding sequence is achieved under conditions compatible with the regulatory sequence.
- a promoter for use in an ecdysone-reponsive construct of the invention may be any promoter which can drive the transcription of a coding sequence to which it is operably linked in the presence of the steroid hormone ecdysone or an analog thereof.
- the promoter may be a naturally occurring promoter from a Drosophila melanogaster or other insect ecdysone-responsive gene.
- the promoter may be a non-naturally occuring promoter.
- a non-naturally occuring promoter may be used which comprises a minimal promoter and an ecdysone-responsive element (EcRE).
- An EcRE is a nucleotide sequence to which a functional ecdysone receptor can bind in the presence of ecdysone.
- Suitable minimal promoters include the minimal heat shock promoter.
- An ecdysone-responsive promoter may comp ⁇ se more than one EcRE, for example 2 to 10 elements oi more preferably 4 to 6 elements The sequence of an EcRE will depend on the exact functional ecdysone receptor used If a modified functional ecdysone receptor is used (see below) it may be approp ⁇ ate to use a modified EcRE (see No et a , Proc Natl Acad Sci USA, 93 3346-3351) The
- EcRE(s) and minimal promoter sequences do not have to be immediately adjacent Because EcREs function as transc ⁇ ptional enhancers, they can be placed some distance upstream, for example from 1, 10 or 25 nucleotides to 30, 40, 50, 100, 500 oi lOOOkb upstream of a minimal promoter EcREs could even be placed further than lkb upstream of a minimal promoter Generally, if multiple copes of an EcRE are used, the mutiple copies will be arranged m an array, one after the other
- Constiucts of the invention may be responsive to ecdysone [ ⁇ 2 ⁇ , 3 ⁇ , 5 ⁇ , 22R)- 2 3,14,22.25-pentahydroxycholest-7-en-6-one] or an analog thereof Suitable analogs of ecdysone for use m the invention include muristerone A [2 ⁇ , 3 ⁇ ,5 ⁇ 1 l ⁇ , 14 20, 22-heptahyd ⁇ oxy-5 ⁇ 7-cholesten-6-one] or ponasterone A [ (2 ⁇ 3 ⁇ , 5 ⁇ , 22R)-
- a promoter for use m an tetracyclme-reponsive construct of the invention may be any promoter which can dnve the transcnption of a coding sequence to which it is opeiably linked m the presence of the antibiotic tetracyclme oi an analog thereof
- promoter will depend on the host cell to be used for expression of the coding sequence Typically, expression m mammalian cells, for example human cells will be required and thus a mammlian promoter will be preferred Mammalian promoters, such as ⁇ -actm promoters, may be used Tissue- specific promoters may be used Viral promoters may also be used, for example the Moloney munne leukaemia virus long terminal repeat (MMLV LTR), the rous sarcoma virus (RSV) LTR piomoter, the SV40 promoter, the human cytomegalovirus (CMV) IE piomoter, adenovirus, HSV promoters (such as the HSV IE promoters), or HPV piomoters, particularly the HP V upstream regulatory region (URR) Viral promoters are readily available in the art Constitutive promoters, for example the CMV promoter, are prefeired.
- MMLV LTR Moloney munne leukaemia virus long
- Tetracycline-responsive constructs comprise one or more tetracycline operator site (Tet0 2 ) sequences, situated between the promoter and coding sequence. For example, two. three, four or even up to ten Tet0 2 sequences may be used. Typically, if more than one Tet0 2 site is used those sites will be arranged in the form of an array. However, other intervening nucleotide sequences may be situated between individual Tet0 2 sites. For example one, two, three, four, five, up to ten or up to 15 nucleotides may intervene between any two Tet0 2 sites.
- Tet0 2 tetracycline operator site
- Tet0 2 sequence is 5'-TCCCTATCAGTGATAGAGA-3' (Hillen and Berens, 1994. Annu. Rev. Microbiol. 48, 345-369; Hillen et ai, 1983, J. Mol. Biol.
- Tet0 2 sequence or a functional variant thereof is capable of being bound by a homodimer of tetR or a functional variant thereof.
- a functional variant of the Tet0 2 sequence is a sequence which is similar to that of the Tet0 2 sequence and which remains capable of binding a homodimer of tetR or a functional variant thereof.
- the binding affinity of tetR for a functional variant of the Tet0 2 sequence may be substantially the same as that of tetR for the Tet0 2 sequence.
- tetR may have a binding affinity for a functional variant of the Tet0 2 sequence which is greater or less than that of tetR for the Tet0 2 sequence.
- a functional variant of Tet0 2 typically comprises a sequence substantially similar to that of the TetO, sequence.
- a functional variant of TetO will generally have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%) or at least 99% sequence identity to the Tet0 2 sequence, calculated over the full length of those sequences.
- a functional variant of the Tet0 2 sequence may be a modified version of that sequence obtained by, for example, nucleotide substitution or deletion. Up to 1, up to 2, up to 3, up to 4, up to 5, up to 6 or more nucleotide substitutions or deletions or combinations thereof may be made to the TetO, sequence to produce a functional variant of that sequence.
- Constructs of the invention may be responsive to the antibiotic tetracycline or an analog thereof.
- Tetracycline binds to tetR homodimers, such that the tetR:tetracylcine complex dissociates from the Tet0 2 sequence.
- the association constant of tetracycline to tetR is 3 x 10 9 M "1 .
- Preferred analogs of tetracycline will have an association constant substantially similar to or greater than that of tetracycline for tetR.
- Suitable analogs of tetracycline include doxycycline. Doxycycline exhibits similar dose response and induction characteristics with constructs of the invention, but has a longer half-life than tetracyclme (48 hours vs. 24 hours respectively).
- the coding sequence used in both ecdysone- and tetracycline-responsive contructs of the invention can be any sequence which encodes a NOS or a functional valiant thereof
- the phrase "nitric oxide synthase" is intended to include all naturally occuring forms of iNOS, nNOS and eNOS as well as variants which retain NOS activity, for example variants produced by mutagenesis techniques.
- the coding sequence encodes a NOS of mammlian origin for example rodent (including rat and mouse) or human.
- the coding sequence encode the human iNOS (GenBank accession number: X73029, Coding sequence 226-3687), human nNOS (GenBank accession number: U17327, Coding sequence 686-4990) or human eNOS (GenBank accession number: M95296, Coding sequence 21-3632) or a functional variant of any one of those enzymes.
- a functional variant of a NOS is any polypeptide which demonstrates NOS activity, for example a fragment of a NOS.
- a coding sequence which codes for a functional variant of a NOS may be, for example a fragment of a full length NOS coding sequence.
- a fragment may be of any length, so long as the polypeptide for which it codes has NOS activity.
- a functional variant of a NOS typically comprises a sequence substantially similar to that of the naturally occurring form of the relevant NOS sequence.
- a functional variant of a NOS will generally have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the relevant NOS sequence, calculated over the full length of those sequences
- the coding sequence may be modified by nucleotide substitutions or deletions For example up to 1, 2 or 3 to 10, 25, 50, 75 or 100 substitutions or deletions oi combinations thereof may be made to produce a functional variant of a NOS
- a polynucleotide encoding a NOS may alternatively or additionally be modified by one or more insertions and/or deletions and/or by an extension at either or both ends
- the modified polynucleotide generally encodes for a polypeptide which has NOS activity Degenerate substitutions may be made and/or substitutions may be made which would result in a conservative ammo acid substitution when the modified sequence is translated, for example as shown in the Table below Ammo acids in the same block in the second column and preferably m the same line m the third column may be substituted for each other
- Sequence identity may be calculated as follows
- the UWGCG Package provides the BESTFIT program which can be used to calculate identity (for example used on its default settings) (Devereux et al (1984) Nucleic Acids Research 12, p387-
- the PILEUP and BLAST algonthms can be used to calculate identity or line up sequences (typically on their default settings), for example as descnbed in Altschul S F (1993) J Mol Evol 36 290-300. Altschul, S, F et al (1990) J Mol Biol 215 403-10 Software for performing BLAST analyses is publicly available through the National Centre for Biotechnology Information (http://ww .ncbi.nlm.nih.gov ⁇ (ii) Vectors
- Both types of constmct of the invention can be incorporated into a recombinant replicable vector.
- the vector may be used to replicate the construct in a compatible host cell.
- a vector may also provide for expression of the NOS coding sequence when the vector is harboured by an appropriate host cell.
- the vectors may be for example, plasmid, virus or phage vectors provided with an origin of replication and optionally a regulator of the ecdysone-responsive promoter or promoter used in a tetracycline-responsive construct.
- the vectors may contain one or more selectable marker genes, for example an ampicillin resistance gene for selection in bacterial cells or a G418 or a zeocin resistance gene for selection in mammalian cells.
- selectable marker genes for example an ampicillin resistance gene for selection in bacterial cells or a G418 or a zeocin resistance gene for selection in mammalian cells.
- Vectors of the invention may also comprise so-called "suicide genes", for example the herpes simplex virus thymidine kinase gene (HSV-tk).
- HSK-tk gene may be induced in the presence of a small molecule such as ganciclovir, resulting in the elimination of a cell harbouring the HSV-tk gene.
- a cell which harbours a vector of the invention which does not comprises the HSK-tk gene may be provided with the HSV-tk gene by integration of the HSV gene into the host cell genome or by transfection or trans formation of the cell with a further vector comprising the HSV-tk gene.
- Vectors of the invention which incorporate an ecdysone-inducible construct, may be introduced into a suitable host cell by any appropriate transformation or transfection technique.
- the host cell will permit the expression of the NOS coding sequence.
- the cells may be chosen to be compatible with the said vector and may be for example bacterial, yeast, insect or mammalian cells.
- NOS gene expression to be induced in the presence of ecdysone or an analog thereof a cell harbouring an ecdysone inducible construct must preferably also be capable of expressing a functional ecdysone receptor.
- the wild type Drosophila functional ecdysone receptor is a heterodimer of the ecdysone receptor (EcR) and the product of the ultraspiracle gene (USP)
- EcR ecdysone receptor
- USP ultraspiracle gene
- cells of the invention may be capable of expressmg EcR and USP
- EcR * s natural heterodimenc partner USP with its mammlian homologue retmoid X receptor gives a heterodimer which can give more potent induction of an ecdysone responsive promoter
- cells of the invention may be capable of expressing EcR and RXR
- Functional vanants of EcR and USP/RXR are polypeptides which can heteiodimense with their partner and can, when heterodimensed, allow ecdysone- responsive dime ⁇ sation to occui
- functional vanants may
- EcR293 cells Invitrogen, San Diego, CA, Catalogue No R650-07.
- EcR293 is a denvative of the human fetal kidney cell lme HEK293 (ECACC accession number 85/20602))
- EcR293 cells are particularly suitable as they stably transformed with the vector pVgRXR That vector is capable of expressing a functional vanant of EcR, VgEcR, and RXR m mammalian cells and thus allows the expression of a functional ecdysone receptoi
- Other suitable cell lines include EcR-CHO and EcR- 3T3 (Invitrogen, San Diego, CA, Cat Nos R660-07 and R680-07 respectively)
- Vectoi s of the invention which incorporate an tetracyclme-mducible construct, may be introduced into a suitable host cell by any appropnate transformation or transfection technique
- the host cell will permit the expression of the NOS coding sequence in the presence of tetracyclme or an analog thereof
- the cells may be chosen to be compatible with the said vector and may be for example bacterial, yeast, insect or mammalian cells
- NOS gene expression to be regulated such that expression does not occur m the absence of tetracyclme
- a cell harbouring a tetracyclme-mducible construct must preferably also be capable of expressmg the tetracycline repressor protein (tetR) or a functional variant thereof.
- tetR tetracycline repressor protein
- a functional variant of tetR is a polypeptide which is similar to tetR and which remains capable of binding, as a homodimer, the Tet0 2 site or a functional variant thereof and tetracycline or an analog thereof.
- the binding affinity of a functional variant sequence of tetR for the Tet0 2 site or a functional variant thereof or tetracycline or an analog thereof may be substantially the same as the binding affinity of the tetR polypeptide for the Tet0 2 site or a functional variant thereof or tetracycline or an analog thereof.
- a functional variant sequence may have a binding affinity which may be greater or less than that of the tetR polypetide.
- the TetR gene encodes a repressor protein of 207 amino acids with a calculated molecular weight of 23 kDa (Hillen and Berens, 1994. supra).
- a functional variant of tetR typically comprises an amino acid sequence substantially similar to that of the tetR sequence.
- a functional variant of a tetR will generally have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%. at least 98%) or at least 99%) sequence identity to tetR, calculated over the full length of those sequences. The calculation of sequence identities is described above.
- a functional variant of the tetR sequence may be a modified version of that sequence obtained by, for example, amino acid substitution or deletion. Up to 1, up to 10, up to 20, up to 50, up to 75, up to 100 or more amino acid substitutions or deletions or combinations thereof may be made to the tetR sequence to produce a functional variant of that sequence. Substitutions are preferably made which result in a conservative amino acid substitution, for example as shown in the Table above.
- Preferred cells for use in the invention are human cells. Particularly preferred cells are T-REx cells (Invitrogen, San Diego, CA; Catalogue Nos: R710-07, R712-
- T-Rex cells are particularly suitable as they stably transformed with the plasmid pcDNA6/TR which generates high level expression of the tetR polypeptide.
- any cell line can be used which expresses tetR or a functional variant thereof.
- Cells of the invention may be encapsulated to give microcapsules.
- microcapsules of the invention will be suitable for administration to a human or animal.
- microcapsules which are suitable for administration to a human or animal are biocompatible, immunoisolatory and mechanically stable.
- Biocompatible microcapsules do not elicit a host response sufficient to result in rejection of the microcapsules or to render the microcapsules non-functioning.
- host responses includes all responses by a host to heterologous material, including for example immune responses such as cytotoxic and systemic responses, fibrotic responses, foreign body responses and other types of inflammatory responses induced by heterologous material.
- Biocompatibility may be determined by a combination of factors, including for example, the microcapsule material, surface texture and shape.
- Materials used to form the microcapsules are thus typically selected for their ability to be accepted by a host harbouring the microcapsules. Suitable materials should generally be stable under in vivo physiological conditions and stresses. Typically, suitable materials will be stable in vivo over extended periods of time, for example up to 2 months, up to six months, up to 1 year or up to 2 years.
- Suitable materials should also preferably be free from or have a content of leachable pyrogenic substances or other harmful, irritating or immunogenic substances. If the material to be used to form microcapsules is not free of those substances, it may be purified to remove the unwanted substances.
- Prefen-ed materials include reversibly and irreversibly gellable substances (for example, those that form hydrogels) and water-insoluble thermoplastic polymers.
- Suitable hydrogel forming materials include alginates.
- Alginate is a linear copolymer composed of two monomeric units, D-mannuronic acid and L-guluronic acid.
- Preferted alginates are those low in or free from toxic substances, pyrogens and immunogenic materials such as proteins and complex carbohydrates, for example highly purified high-G alginates. Alginates may be induced to form gels in the presence of certain divalent cations, for example Ca 2 ".
- Ca 2 ⁇ alginate as a microcapsule material
- chelating compounds such as phosphate, citrate and lactate or anti-gelling cations such as Mg 2"1" or Na 2 ".
- alginate gels can be stabilized by replacing Ca 2+ with other divalent cations having a higher affinity for alginate.
- the affinity series for various divalent caitons is , in order of decreasing affinity for alginate: Pb 2 " > Cu 2+ > Cd 2 > > Ba 2+ > Sr 2 " " > Ca 2+
- Alginate forms strong complexes with polycations such as chitosan or polypeptides, or synthetic polymers such as poiyethylenimin ⁇ . These complexes do not dissolve in the presence of Ca 2+ chelators or anti-gelling cations and may therefore optionally be used both to stabilize the gel and to reduce the porosity. Additionally, covalent crosslinking may be used to stabilize alginate gels. Covalent crosslinking may be carried out by techniques such as direct crosslinking of the carboxyl groups and covalent grafting of alginate with synthetic polymers. This gives gels with improved stability and mechanical strength. However, these techniques involve reactive chemicals and thus may be difficult to apply to the encapsulation of living cells.
- Suitable thermoplastics are typically those which are mildly hydrophobic, for example those having a solubility parameter of from 8 to 15 (joules/m 3 ) 54 or more preferably from 6 to 14 (joules/m 3 ) 5 [Solubility parameter as defined in Polymer Handbook 3 rd Ed., John Wiley and Sons, NY] .
- Suitable polymers are selected to have a solubility parameter low enough so that they are soluble in organic solvents, but high enough so that they may partition to from a membrane. Such polymers should be free from labile nucleophilic moieties and be resistant to oxidants and enzymes even, for example, in the absence of stabilizing agents.
- thermoplastic polymers examples include polyacylonitrile vinyl chloride (PAN/PVC), polyacrylonitrile (PAN), polymethylmethacrylate (PMMA), polyvinyldifuoride (PVDF), polyethylene oxide, polyolefins (eg. polyisobutylene or polypropylene), polysulfones or cellulose derivatives (eg. cellulose acetate or cellulose butyrate).
- the microcapsule forming material may also comprise one or more substance which reduces or eliminates local host inflammatory responses to the microcapsules and/or stimulates the formation of a suitable environment for the microcapsules.
- an antibody to a mediator of the immune response could be included.
- Suitable antibodies include antibodies to the lymphokines, tumour necrosis factor
- TNF tumor necrosis factor
- IFN interferon
- An anti-inflammatory steroid may be included in the microcapsule forming material.
- a substance which stimulates angiogenesis ingrowth of capillary beds can be included. This may be desirable where the isolated cells or tissues are required to be in close contact with the host bloodstream for proper functioning. Cells which are encapsulated may themselves be capable of generating any of the substances mentioned in this paragraph.
- the texture of the exterior of the microcapsules is typically formed so after implantation it provides an optimal interface with the tissues of the host.
- the appropriate texture will in part be determined by the site of implantation. For example, if the microcapsules are to be introduced into the intraperitoneal cavity of the host, its surface should be smooth. A smooth surface may discourage excessive overgrowth by fibroblasts. The overgrowth of fibroblasts may be disadvantageous as it may result in the deposition of a poorly permeable basement membrane around microcapsules and consequent isolation of microcapsules from the host. However, if the microcapsules are to be embedded in the soft tissues of the host the surface may be moderately rough or stippled. A factor to be taken into account is whether it is desired for host cells to attach to the surface of the microcapsules or alternatively if such contact is to be avoided.
- Microcapsules of any shape may be formed. However, certain microcapsule shapes may elicit fibrotic responses and therefore those shapes are typically avoided.
- microcapsules preferably should not comprises structures having interlayers such as brush surfaces or folds.
- opposing microcapsule surfaces or edges either from the same or adjacent microcapsules should be at least 2mm apart, more preferably at least 5mm apart.
- Preferred shapes for microcapsules are thus, substantially spherical, substantially cylindrical, U-shaped cylinders and flat sheets or sandwiches.
- Substantially spherical microcapsules may be formed and offer the advantage that delivery of cells so encapsulated may be carried out by intraperitoneal injection. This may be advantageous if systemic delivery is required. Cylinders, by contrast, generally require surgical implantation, but as described below, may have advantages in terms of surface area to volume ratios.
- Microcapsules of the invention are typically immunoisolatory.
- Immunoisolatory microcapsules protect encapsulated cells from the host immune system.
- the encapsulating material may protect encapsulated cells by: (1) substantially preventing or minimising components of the host foreign body response mechanisms from entering a microcapsule; (2) providing a barrier which substantially prevents or minimises contact between the encapsulated cells and components of the host immune system; and (3) providing a barrier which substantially prevents or minimises contact between inflammatory, antigenic or other harmful substances that may be present in a microcapsule and components of the host foreign body response mechanisms.
- the microcapsule material will generally allow passage of substances up to a predetermined size, but prevent the passage of larger substances and therefore microcapsules are differentially permeable.
- the molecular weight cutoff (MWCO) selected for particular microcapsules may be determined in part by (1) the type and extent of immunological rejection anticipated after introduction of microcapsules to the host; and (2) the molecular size of the largest substance whose passage into and out of the microcapsules is required.
- the type and extent of immunological rejection encountered by microcapsules may depend in part on the type of genotype of the encapsulated cells and the genotype of the host.
- the encapsulated cells are allogenic to the host rejection may proceed largely through cell-mediated attack by the host's immune cells against the implanted cells.
- rejection through assembly of the host ' s cytolytic complement attack complex may predominate, as well as antibody interaction with complement
- the MWCO of the encapsulation material must therefore be sufficiently low to prevent access of the molecules required to carry out the above mentioned rejection mechanisms to the encapsulated cells, yet sufficiently high to allow passage of any therapeutic substance produced by the encapsulated cells.
- materials can be used which allow passage of molecules up to about the size of Clq ( ⁇ 400kd), the largest polypeptide required for the assembly of the cytolytic complement attack complex
- microcapsule materials which allow passage of molecules up to the size of immunoglobulin G
- microcapsules of the invention may have a low MWCO NO is a small molecule and thus microcapsules of the invention can have a low MWCO, which will in turn increase immunoisolation of the encapsulated cells Diffusion of small molecules appears unaffected by alginate gels, whereas passage of larger molecules may be restricted.
- poly-L-lysine An example of a suitable polycation is poly-L-lysine; the lower the molecular weight , of the poly-L-lysine, the less permeable the capsules Contact between components of the host response mechanisms and encapsulated cells and other inflammatory, antigenic or other harmful substances may be substantially prevented or minimised of the microcapsule material forms a physical barrier between those substances.
- the thickness of the barrier may vary, but typically it should be sufficiently thick to prevent direct contact between substances on either side of the barrier Thus the thickness of the barrier generally ranges from 5 to 200 ⁇ m, preferably from 10 to lOO ⁇ m and most preferably from 20 to 50 ⁇ m
- Microcapsules of the invention may harbour from 1 to at least 100 cells, at least 200 cells, at least 500 cells, at least 1000 cells or at least 1 x 10 4 cells, up to 1 x 10 5 cells or up to 1 x 10 7 cells.
- Factors which influence the number of cells that a microcapsule may harbour include: (1) microcapsule size and shape; (2) mitotic activity within the microcapsule; and (3) viscocity of the interior of the microcapsule.
- microcapsule size and shape the diffusion of nutrients into cells and of waste products out of cells is critical to the continued viability of the encapsulated cells. Since diffusion in and out of the microcapsule may be limited by the microcapsule surface area, the surface to volume ratio of microcapsules will be critical in determining how many cells may be maintained within the microcapsules.
- the oxygen requirement of cells must be determined for any cell type of choice.
- a typical example may be that for cells encapsulated in a cylindrical microcapsule of 900 ⁇ m outer diameter implanted in the intraperitoneal space (p0 2 ⁇ 45-50mmHg) the optimal total cell volume is in the range of up to 20%), preferably 1 to 15%) and most preferably about 5% of the microcapsule volume.
- the optimal cell volume may be from 30 to 65%) total microcapsule volume and preferably may be 35%.
- microcapsules that will be filled to confluency with cells will be no more than 250 ⁇ m in cross-section, such that cells in the interior will have less than 15 between them and an external surface, preferably less than 120 cells and more preferably less than 5 cells.
- Microcapsules are preferably substantially sphencal or substantially cylindrical They may be of any appropnate size, but the size will be mfluenced by a number of factors including some of those mentioned above For example, nutnent diffusion may limit microcapsule size
- Substantially sphencal microcapsules may be for example from 0 01 to 4 0 mm, preferably from 0 1 to 3 0 mm and more preferably from 0 2 to 2 0 mm m diameter
- Substantially cylmdncal microcapsules may be from 0 01 to 4 0 mm, preferably from 0 1 to 3 0 mm and more preferably from 0 2 to 2 0 mm m outer diameter
- Such cylmdncal microcapsules may be for example ft om 0 1 mm, 0 2 or 0 5 mm in length to for example 5 mm, 10 mm or 20 mm m length
- the above dimensions may be average dimensions for
- Microcapsules may be produced according to methods well known to those skilled in the art
- Alginate microcapsules may be formed, for example, by mixing an algmate solution with a cell suspension and then dnppmg the algmate-cell suspension into a solution containing Ca 2+ ions
- a poly-L-lysme layer may be added by to the microcapsules thus formed by incubation m a solution of poly-L-lysme
- the poly-L- lysme layer may increase mechanical stability and decrease permeability of the resulting macocapsules
- a final algmate layer may be added by a further incubation in an algmate solution
- an initial sodium algmate solution at a concentration of from 1 5 to 12%), more preferably from 2 to 4% is prepared by dissolving an appropnate amount of Na + -alg ⁇ nate m distilled water or buffer
- the suspension may be stirred for 6 hours by a magnetic stirrer or left overnight on a rotary shaker at room temperature
- the resulting suspension may be filter sterilized by use of a 0.22 ⁇ m membrane filter.
- Cells may then be mixed with the Na + -alginate solution. If the cells are added as a suspension in a buffer, the presence of phosphate, citrate, EDTA and divalent cations should be avoided.
- Microcapsules may formed by dripping the alginate-cell suspension (from a syringe with an inner diameter of for example from 0.22 to
- the size of the microcapsules may be controlled by the application of a coaxial airstream. For a given Na + -alginate solution (concentration, type and intrinsic viscosity) the size of the microcapsules depends only on the airflow for a given syringe diameter and microcapsules of diameters in the range of, for example, from 0.2mm to 3mm may be produced.
- the microcapsules may be left to harden in the CaCl, for from 5 to 30 minutes, depending on their diameter.
- the resulting microcapsules may be washed in saline or a suitable buffer and then suspended in a solution of poly-L-lysine.
- the poly-L-lysine used may be a molecular weight of for example from 24, 000 to 65, 000, preferably 27, 000 and the microcapsules may typically be suspended in a 0.05% (w/v) solution of poly-L- lysine.
- the microcapsules may be suspended in a Na -alginate solution (at a concentration of for example, 0.15% ).
- the microcapsules may further be suspended in a solution of sodium citrate
- microcapsules are generally given a further saline wash before use.
- Cylindrical microcapsules can be formed by loading thermoplastic polymer hollow fibres, such as polyethersulfone hollow fibers, with a cell suspension and sealing the ends of the hollow fibre.
- Newly-formed microcapsules may be maintained until use under sterile conditions in a non-pyrogenic, serum-free defined nutrient medium or balanced salt solution at about 37°C until implantation. Alternatively the microcapsules may be cryopreserved.
- Microcapsules of the invention may be used to treat conditions which are associated with deficient NO production
- Microcapsules may also be used for the manufacture of a medicament for use in the treatment of a condition associated with deficient NO production.
- Conditions associated with deficient NO production include those that may be a result of abnormal deficient NO production and also those that although not a result of abnormal deficient NO production as such, may be treated by raising NO levels
- the condition of a patient suffering from such an infection can be improved by administration of an inhibitor.
- a therapeutically effective amount of an inhibitor may be given to a human patient in need thereof
- Microcapsules of the invention may therefore be used to treat, for example, hyperlipidaemia. renal failure, hypertension, restenosis after angioplasty, atherscloerosis and its complications, complications of heart failure or schizophrenia
- microcapsules of the invention may be used as artificial macrophages in the treatment of cancers
- Microcapsules may be administered to a human or animal in need thereof and ecdysone or an analog thereof administered subsequently.
- ecdysone or an analog thereof may be administered at the same time as the microcapsules.
- the invention provides a product containing microcapsules of the invention and ecdysone or an analog thereof as a combined preparation for simultaneous, separate or sequential use in the treatment of a condition associated with deficient NO production
- microcapsules are implanted at a site in the host which (1) allows for appropriate delivery of NO produced by the microcapsules to the host; and (2) allows for passage of nutrients to the encapsulated cells Also the site may be chosen to allow for the recapture of the microcapsules
- Microcapsules may be administered in a variety of dosage forms. Thus, they can be administered orally, for example as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules.
- the inhibitors may also be administered parenterally, either subcutaneously, intravenously, intramuscularly, intrasternally, transdermally or by infusion techniques.
- the microcapsules may also be administered as suppositories. A physician will be able to determine the required route of administration for each particular patient.
- microcapsules for use in preventing or treating a condition associated with deficient NO production will depend upon factors such as the nature of the microcapsules, whether a pharmaceutical or veterinary use is intended, etc.
- Microcapsules are typically formulated for administration in the present invention with a pharmaceutically acceptable carrier or diluent.
- the pharmaceutical carrier or diluent may be, for example, an isotonic solution.
- solid oral forms may contain, together with the microcapsules, diluents, e.g. lactose, dextrose, saccharose, cellulose, corn starch or potato starch; lubricants, e.g. silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols; binding agents; e.g.
- Liquid dispersions for oral administration may be syrups, emulsions and suspensions.
- the syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.
- Suspensions may contain as a carrier, for example a natural gum, agar, sodium alginte, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol.
- the suspensions or solutions for intramuscular or intratumor injections may contain, together with the microcapsules, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, sesame oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.
- Solutions for intravenous administration or infusions may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.
- a therapeutically effective amount of microcapsules is administered to a patient.
- the dose of microcapsules may be determined according to various parameters, especially according to the microcapsules used; the age. weight and condition of the patient to be treated; the route of administration; the required regimen; and the condition to be treated. Again, a physician will be able to determine the required route of administration and dosage for any particular patient.
- a typical dose is from about lmg/kg to lOmg/kg of body weight, according to the activity of the microcapsules. the age, weight and conditions of the subject to be treated, the type and seventy of the degeneration of the microcapsules and the frequency and route of administration.
- Ecdysone or an analog thereof or tetracycline or an analog thereof may be administered simultaneously, separately or sequentially with microcapsules of the invention.
- Ecdysone or an analog thereof or tetracycline may be administered and formulated for administration, for example as described above for microcapsules of the invention.
- the amount of ecdysone or tetracycline required and the frequency of admininstration will depend on similar considerations as apply to microcapsule dosages. It may be necessary to give several doses of ecdysone or tetracycline over a period of time or alternatively, it may only be necessary to administer a single dose at the beginning of the treatment.
- Ecdysone or an analog thereof or tetracycline or an analog thereof can itself be microencapsulated, for example by a method for microencapsulation as described above. Such microcapsules could be administered along with microcapsules of the invention.
- the invention provides a product containing microcapsules of the invention and microcapsules harbouring ecdysone or an analog thereof or tetracycline or an analog thereof as a combined preparation for simultaneous, separate or sequential use in the treatment of a condition associated with deficient NO production.
- Ecdysone or tetracycline may also be incorporated into the material used to encapsulate cells of the invention.
- Encapsulation of ecdysone or an analog thereof or tetracycline or an analog thereof or incorporation of ecdysone or tetracycline into the encapsulation material may abrogate the need for more than one administration of ecdysone or an analog thereof or tetracycline or an analog thereof.
- the cells were then transferred to three or four T-25 flasks (or equivalent) and placed in a humidified, 37 °C, 5% C0 2 incubator. It typically took 3-4 days for cell cultures to reach 80-90% confluency. Media were changed 2 times a week and cells subcultured at a ratio of 1:3 to 1:4 when they reached 80-90% confluency. Cells were subcultured when approaching confluency to avoid the accumulation of floating and dead cells. Cells were frozen for storage in 95%> FBS and 5%> DMSO. Induction using Muristerone A
- Cells could be maintained in non selection medium for 2-3 weeks without losing inducibility following muristerone A treatment.
- Non-selection medium was used by choice, although selection medium worked equally well.
- Cells were seeded at 1-2 x 10 5 /ml concentration for 12 well or 96 well plates. Semi-confluent or confluent plates or flasks were used for induction.
- Figure 1(a) shows overnight induction in 12 well plates (3-30 hours) following the addition of different doses of muristerone A. Nitrate concentrations are measured using the Griess Reaction (see below).
- Poly(A) + mRNA was isolated using a micro-fastrack mRNA purification kit (invitrogen), separated by electrophoresis and transferred onto Hybond N membrane
- Example 1 Generation of NOS transfected cell lines under the control of an ecdysone-responsive or a tetracycline-responsive promoter
- Three plasmids were generated, each of which expressed one of the human NOS isoforms under the control of an ecdysone-responsive promoter
- An additional plasmid was constructed placing the iNOS cDNA under the control of a tetracycline- regulated promoter.
- 4164bp of the human iNOS cDNA (GenBank accession number: X73029, Coding sequence 226-3687) was cut from its original vector (Bluescript KS) using the restriction endonucleases, Kpnl and Spel and cloned into pIND (Invitrogen, San Diego, CA, Catalogue No: V705-20) which had been cut with Kpnl and ⁇ b ⁇ l.
- the resulting plasmid was sequenced to confirm that the cDNA had inserted in the correct orientation
- the wild type human eNOS (GenBank accession number: M95296, Coding sequence 21-3632) was cut from its original vector (Bluescript KS) using the restriction endonucleases.
- Hindill and Notl and cloned into pLND Invitrogen, San Diego, CA; Catalogue No: V705-20
- the resulting plasmid was sequenced to confirm that the cD ⁇ A had inserted in the correct orientation.
- the human i ⁇ OS cD ⁇ A (GenBank accession number: X73029, Coding sequence 226-3687) was cut from its original vector (Bluescript KS) using the restriction endonucleases, Kpnl and Spel and cloned into pcD ⁇ A4/TO (Invitrogen, San Diego, CA) which had been cut with Kpnl and Xbal. The resulting plasmid was sequenced to confirm that the cDNA had inserted in the correct orientation.
- the pIND plasmid contains 5 modified EcREs called E/GREs which bind a modified functional ecdysone receptor. That modified functional ecdysone receptor can be expressed by another plasmid, pVgRXR (Invitrogen, San Diego, CA;
- pVgRXR constitutively expresses a heterodimeric receptor comprising a modified ecdysone receptor (VgEcR) and RXR.
- VgEcR modified ecdysone receptor
- a cell transformed with pVgRXR and one of the three plasmids described above (I, II or III) will express NOS in the presence of ecdysone or an analog thereof.
- the functional ecdysone receptor binds to the E/GREs and transcription of the NOS cDNA is initiated.
- the plasmid pIND-hiNOS-f was used to transfect a human fetal kidney cell line, EcR293 (Invitrogen, San Diego, CA; Catalogue No: R650-07), which is stably transformed with pVgRXR. Transfections were carried out using Superfect reagent (Qiagen) and transfectants were isolated following double selection on G418 (400 ⁇ g/ml) and zeocin (250 ⁇ g/ml) for 14 days.
- cells were isolated which constitutively expressed the subunits of a functional ecdysone receptor, RXR/VgEcR and the human iNOS cDNA under the control of an ecdysone-inducible promoter.
- the T-Rex system (Invitrogen, San Diego, CA; Catalogue No: Kl 020-01) is a tetracycline-regulated mammalian expression system that uses regulatory elements from the E. coli Tn70-encoded tetracycline resistance operon.
- the pcDNA4/TO plasmid allows expression of a gene of interest under te control of the strong human cytomegalovirus immediate-early (CMV) promoter and two tetracycline operator 2 (Tet0 2 ) sites.
- CMV cytomegalovirus immediate-early
- Tet0 2 tetracycline operator 2
- a cell transformed with pcDNA6/TR and the plasmid described above (IV) will express NOS in the presence of tetracycline or an analog thereof.
- tetracycline binds to tetR which undergoes a conformational change such that it dissociates from the TetO, sites.
- Expression of the iNOS gene is then induced, driven by the CMV promoter.
- the plasmid pTet-hiNOS-f was used to transfect the cell line T-REx-293 ((Invitrogen, San Diego, CA, Catalogue No: R710-07).
- the T-REx-293 cell line is a human embryonic kidney 293 cell line which has been transfected with the pcDNA6/TR plasmid and thus generates high level expression of the tetracycline repressor protein (TetR).
- Transfections were carried out using conditions as described above for the muristerone A inducible constructs. Transfectants were isolated following selection on zeocin (200 ⁇ g/ml) for the human iNOS cDNA expressing plasmid and blasticidin (5 ⁇ g/ml) for the tetR expressing plasmid.
- NOS detect system (Stratagene NOS detect kit, Cat. No.204500). The kit measures the conversion of [ 14 C] arginine to [ 14 C] citrulline, and is specific for NOS.
- transfectants were plated out on 12 well Falcon tissue culture plates at a cell density of 1 x 10 5 /ml and nitrite was measured by the Griess reaction.
- Muristerone A (Invitrogen) was added at a final concentration of 1 OO ⁇ M to specific wells, and after induction for 24hr, lOO ⁇ l of culture supernatant was used to measure nitrite concentration using the Griess reagent. The results are reported as the average of assays run on triplicate wells. Well-to-well variation was less than 10%.
- transfectants clone 11
- clone 11 One of the transfectants, clone 11, was selected for further study.
- Cells were grown with varying concentrations of muristerone A, and at different time intervals, supernatants were taken and the Griess reaction was used to measure the nitrite concentration. The results are reported as the average of assays run in triplicate. Well-to-well variation was less than 10%. See Figure 2a.
- Muristerone A-dependent expression of the human iNOS gene (i) Muristerone A-dependent expression of the human iNOS gene.
- Northern blot analysis was carried out with 2 ⁇ g of poly A+ RNA isolated from cells which had been treated with muristerone A for 24 hr.
- a human iNOS cDNA probe was used to detect the presence of a 4kb band in mRNA extracted from cells treated with either 1 ⁇ M or 1 O ⁇ M muristerone A. Human ⁇ -actin mRNA was used as a loading control.
- clone 1 A panel of 4 tetracycline-regulated iNOS expressing cell lines were isolated, designated clone 1, clone 2. clone 5 and clone 22.
- the iNOS transfectants were unable to express mRNA as the two Tet operator sites (Tet0 2 ) are occupied by the repressor protein effectively blocking transcription.
- Tet0 2 Tet operator sites
- tetracycline is added to the culture medium, it binds to the TetR protein and changes its conformation. The altered conformation of the repressor is unable to bind the operator sites, and consequently iNOS can be expressed.
- Nitrite concentrations were determined using the Griess reaction (see figure 3).
- EcR293 clone 11 cells were used for encapsulation studies. Prior to encapsulation, EcR293 clone 11 cells were maintained in Dulbecco's media supplemented with 10%o fetal bovine serum (FBS) and penicillin (100 units/ml) and streptomycin (100 ⁇ g/ml) in the absence of both G418 and zeocin. The cells were encapsulated in alginate-poly-L-ly sine- alginate microcapsules as follows:
- Cells were harvested with trypsin then resuspended in a 1.8% sodium alginate solution (Sigma, catalogue no. A2033, medium viscosity, filter sterilised through a 0.25 ⁇ m membrane) in PBS at between 0.5-5 x 10 7 cells/ml.
- the suspension was placed in a syringe and held 20 to 40cm above a beaker containing an excess (100ml) of stirred ice cold calcium chloride solution (0.2M, pH7, filter sterilized).
- the cell suspension was extruded through a 21 G needle and droplets made to fall into the solution of sterile ice cold calcium chloride.
- alginate gelation Upon contact with the calcium chloride buffer, alginate gelation is immediate and the cells are encapsulated in an alginate complex.
- the microcapsule size was controlled by the extrusion pressure and the needle gauge.
- a typical hypodermic needle produced beads of from 0.5 to 2mm in diameter. Other shapes could be obtained by using a mould the wall of which was permeable to calcium ions.
- Example 4 In vitro culture of the encapsulated cells
- EcR293 clone 1 1 cells were encapsulated with immunoisolating microcapsules fabricated from alginate -poly-L-lysine-alginate, the cells survive for at least 3 days. Moreover, the encapsulated cells could be induced to produce nitric oxide at a controlled level upon stimulation with muristone A at a concentration of 10 ⁇ M..
- Example 5 In vivo injection and retrieval of the encapsulated cells
- mice Male CT-1 mice (3-4 weeks old, about 15 grams body weight), were intraperitoneally injected with 1ml of microcapsules from Example 3 with a 16G catheter. The inducer, lmg per mouse of muristerone A, was added at the same time.
- the cells were washed three times with ice-cold phosphate-buffered saline (PBS) and then supplemented with Dulbecco's media.
- the capsules were then tested for survival and inducibility by muristerone A.
- the test showed that the microencapsulated cells were still capable of induction by 1 O ⁇ M muristerone A and could produce nitric oxide accordingly.
- microencapsulated cell line could be used as an artificial endothelial cell or macrophage for human disease treatment.
- Example 6 Microencapsulated cells as an artificial macrophage for treating human ovarian and colon cancer
- a nude mouse/xenograf model was used to assess the ability of microencapsulated cells to kill tumor cells in vivo.
- the present studies utilized two human carcinoma cell lines that produce rapidly growing subcutaneous tumors in nude mice were used.
- DLD-1 is a human colon adenocarcinoma cell line
- SK- OV-3 is an ovarian carcinoma cell line. Both were obtained from ATCC.
- the cell lines were maintained as adherent monolayers in DMEM supplemented with 10% FBS. Cultures were maintained on plastic and incubated in 5% C0 2 -95% air at 37°C All cultures were free of Mycoplasma.
- tumor cells in the exponential phase were harvested by a 1 to 2 min treatment with 0.25% trypsin, 0.02%) EDTA solution (w/v). The flasks were tapped sharply to dislodge the cells, DMEM added, and the cell suspension pipetted to produce a single-cell suspension. The cells were washed, resuspended in Ca27Mg2 + -free PBS and diluted to the desired concentration. Cell viability was determined by trypan blue exclusion, and only single-cell suspensions of greater than 95% viability were used for in vivo experiments.
- a total volume of 300 ⁇ l of the cell suspension/mixture was injected (with a 21G needle) subcutaneously into the lateral flank proximal to the midline of the unanaesthetized animal. Each experiment used between 5 and 10 animals.
- the mean diameter of the tumors was determined by measuring tumor length (1) and width (w) by calipers.
- Tumors obtained from each of the experiments were either frozen in liquid nitrogen or subsequent analysis by northern or western blotting or for histological evaluation. The above procedure was carried out with both of the human carcinoma lines.
- Colon cancer DLD-1 cells (lxl 0 6 per mouse) were injected subcutaneously along with microencapsulated EcR293 clone 1 1 cells; 5x10 6 cells microencapsulated as described in Example 3 above.
- the microencapsulated cells were injected in the presence of either sesame oil alone or sesame oil and ponasterone A (lmg per mouse).
- the results are shown in Figure 6a and demonstrate that the microencapsulated cells plus ponasterone A treatment slowed the growth of DLD-1 cells considerably compared to the treatment of microencapsulated cells alone.
- the experiment was repeated with SK-OV-3 ovarian cancer cells.
- the results are shown in Figure 6b and demonstrate that the microencapsulated cells plus ponasterone A treatment completely inhibited the growth of SK-OV-3 cells.
- OV-3 cells showed considerable growth in the absence of ponasterone A.
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Cardiology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- General Chemical & Material Sciences (AREA)
- Heart & Thoracic Surgery (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Hospice & Palliative Care (AREA)
- Diabetes (AREA)
- Microbiology (AREA)
- Hematology (AREA)
- Obesity (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Vascular Medicine (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicinal Preparation (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Manufacturing Of Micro-Capsules (AREA)
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GBGB9918076.2A GB9918076D0 (en) | 1999-07-30 | 1999-07-30 | Microencapsulated nitric oxide synthase source |
GB9918076 | 1999-07-30 | ||
GB0016172A GB0016172D0 (en) | 2000-06-30 | 2000-06-30 | Microencapsulated nitric oxide synthase source |
GB0016172 | 2000-06-30 | ||
PCT/GB2000/002924 WO2001009342A1 (en) | 1999-07-30 | 2000-07-28 | Microencapsulated nitric oxide synthase source |
Publications (1)
Publication Number | Publication Date |
---|---|
EP1203084A1 true EP1203084A1 (de) | 2002-05-08 |
Family
ID=26244581
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP00948182A Withdrawn EP1203084A1 (de) | 1999-07-30 | 2000-07-28 | Stickstoffmonoxid-synthase-quelle in mikrokapseln |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP1203084A1 (de) |
JP (1) | JP2003506047A (de) |
AU (1) | AU6174500A (de) |
CA (1) | CA2380787A1 (de) |
WO (1) | WO2001009342A1 (de) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
PT105489B (pt) * | 2011-01-18 | 2014-05-13 | Ass For The Advancement Of Tissue Engineering And Cell Based Technologies & Therapies A4Tec | Dispositivo, método e sistema de fabrico de microcápsulas |
US9555007B2 (en) | 2013-03-14 | 2017-01-31 | Massachusetts Institute Of Technology | Multi-layer hydrogel capsules for encapsulation of cells and cell aggregates |
CN115487779B (zh) * | 2022-06-01 | 2023-07-28 | 同济大学 | 一种二价铁离子/纤维素纳米晶体/海藻酸钠复合材料的制备方法及对四环素的吸附应用 |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5800829A (en) * | 1991-04-25 | 1998-09-01 | Brown University Research Foundation | Methods for coextruding immunoisolatory implantable vehicles with a biocompatible jacket and a biocompatible matrix core |
US5658565A (en) * | 1994-06-24 | 1997-08-19 | University Of Pittsburgh Of The Commonwealth System Of Higher Education | Inducible nitric oxide synthase gene for treatment of disease |
US5972650A (en) * | 1997-06-26 | 1999-10-26 | Brigham And Women's Hospital | Tetracycline repressor regulated mammalian cell transcription and viral replication switch |
US6333318B1 (en) * | 1998-05-14 | 2001-12-25 | The Salk Institute For Biological Studies | Formulations useful for modulating expression of exogenous genes in mammalian systems, and products related thereto |
-
2000
- 2000-07-28 JP JP2001514134A patent/JP2003506047A/ja active Pending
- 2000-07-28 AU AU61745/00A patent/AU6174500A/en not_active Abandoned
- 2000-07-28 EP EP00948182A patent/EP1203084A1/de not_active Withdrawn
- 2000-07-28 CA CA002380787A patent/CA2380787A1/en not_active Abandoned
- 2000-07-28 WO PCT/GB2000/002924 patent/WO2001009342A1/en not_active Application Discontinuation
Non-Patent Citations (1)
Title |
---|
See references of WO0109342A1 * |
Also Published As
Publication number | Publication date |
---|---|
AU6174500A (en) | 2001-02-19 |
JP2003506047A (ja) | 2003-02-18 |
WO2001009342A1 (en) | 2001-02-08 |
CA2380787A1 (en) | 2001-02-08 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Sagot et al. | Polymer encapsulated cell lines genetically engineered to release ciliary neurotrophic factor can slow down progressive motor neuronopathy in the mouse | |
Nikkhah et al. | A microtransplantation approach for cell suspension grafting in the rat Parkinson model: a detailed account of the methodology | |
Al-Hendy et al. | Correction of the growth defect in dwarf mice with nonautologous microencapsulated myoblasts—an alternate approach to somatic gene therapy | |
JP2005239726A (ja) | カプセル化細胞の宿主への移植方法 | |
JPH08509215A (ja) | 移植治療のための神経由来胎児セルラインの使用 | |
Serguera et al. | Control of erythropoietin secretion by doxycycline or mifepristone in mice bearing polymer-encapsulated engineered cells | |
US20070059835A1 (en) | Ocular gene therapy using avalanche-mediated transfection | |
EP3424498B1 (de) | Nanokapsel mit monoklonalem antikörper für tumortherapie sowie herstellungsverfahren und verwendung davon | |
US6103226A (en) | Genetically engineered cells that produce produce L. Dopa | |
Kwak et al. | The role of inducible 70-kDa heat shock protein in cell cycle control, differentiation, and apoptotic cell death of the human myeloid leukemic HL-60 cells | |
IE83522B1 (en) | Gene therapy for proliferative disease | |
EP0289034B1 (de) | Transkaryotische Einpflanzung | |
JP4119952B2 (ja) | ウイルス粒子産生カプセル化細胞 | |
CN103638002B (zh) | 包囊的肝脏细胞组合物 | |
WO1993010234A1 (en) | Therapy of central nervous system by genetically modified cells | |
JP2004505647A (ja) | 条件的阻害によるトランスジーンの発現をインビボで調節する方法 | |
KR19990022414A (ko) | 통증 치료용 진통 화합물을 생성하는 세포주 | |
JPH0235091A (ja) | 哺乳動物細胞における発現の制御 | |
AU2006279395A1 (en) | Ocular gene therapy using avalanche-mediated transfection | |
EP1534832A2 (de) | Zusammensetzungen und verfahren zur gewebsspezifischen oder induzierbaren hemmung der genexpression | |
DE10108412A1 (de) | Pigmentepithelzelle des Auges, ihre Herstellung und Verwendung in der Therapie einer Augen- oder Nervenerkrankung | |
Basic et al. | Microencapsulation and transplantation of genetically engineered cells: a new approach to somatic gene therapy | |
WO2001009342A1 (en) | Microencapsulated nitric oxide synthase source | |
Efron et al. | A novel method of studying wound healing | |
AU724537B2 (en) | Method for gene transfer to the central nervous system |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
17P | Request for examination filed |
Effective date: 20020227 |
|
AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE |
|
AX | Request for extension of the european patent |
Free format text: AL;LT;LV;MK;RO;SI |
|
GRAP | Despatch of communication of intention to grant a patent |
Free format text: ORIGINAL CODE: EPIDOSNIGR1 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
|
18D | Application deemed to be withdrawn |
Effective date: 20060101 |