CN108553639B - 一种壳聚糖/胰岛素纳米缓释经皮制剂及其制备方法 - Google Patents
一种壳聚糖/胰岛素纳米缓释经皮制剂及其制备方法 Download PDFInfo
- Publication number
- CN108553639B CN108553639B CN201810377863.9A CN201810377863A CN108553639B CN 108553639 B CN108553639 B CN 108553639B CN 201810377863 A CN201810377863 A CN 201810377863A CN 108553639 B CN108553639 B CN 108553639B
- Authority
- CN
- China
- Prior art keywords
- insulin
- chitosan
- preparation
- nano
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 title claims abstract description 129
- 102000004877 Insulin Human genes 0.000 title claims abstract description 64
- 108090001061 Insulin Proteins 0.000 title claims abstract description 64
- 229940125396 insulin Drugs 0.000 title claims abstract description 64
- 229920001661 Chitosan Polymers 0.000 title claims abstract description 48
- 238000002360 preparation method Methods 0.000 title claims abstract description 30
- 238000013268 sustained release Methods 0.000 title claims abstract description 11
- 239000012730 sustained-release form Substances 0.000 title claims abstract description 11
- 239000003814 drug Substances 0.000 claims abstract description 35
- 229940079593 drug Drugs 0.000 claims abstract description 28
- 239000003961 penetration enhancing agent Substances 0.000 claims abstract description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 5
- 239000010410 layer Substances 0.000 claims description 20
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 18
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 12
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 claims description 12
- 239000000463 material Substances 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- NIXOWILDQLNWCW-UHFFFAOYSA-N Acrylic acid Chemical compound OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 claims description 5
- 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 claims description 5
- 229930195725 Mannitol Natural products 0.000 claims description 5
- 239000004820 Pressure-sensitive adhesive Substances 0.000 claims description 5
- 229920002125 Sokalan® Polymers 0.000 claims description 5
- 229960001631 carbomer Drugs 0.000 claims description 5
- 239000000594 mannitol Substances 0.000 claims description 5
- 235000010355 mannitol Nutrition 0.000 claims description 5
- 239000011159 matrix material Substances 0.000 claims description 5
- 235000019832 sodium triphosphate Nutrition 0.000 claims description 5
- PQUXFUBNSYCQAL-UHFFFAOYSA-N 1-(2,3-difluorophenyl)ethanone Chemical compound CC(=O)C1=CC=CC(F)=C1F PQUXFUBNSYCQAL-UHFFFAOYSA-N 0.000 claims description 4
- 229920003063 hydroxymethyl cellulose Polymers 0.000 claims description 4
- 229940031574 hydroxymethyl cellulose Drugs 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 229940047670 sodium acrylate Drugs 0.000 claims description 4
- 238000003756 stirring Methods 0.000 claims description 4
- 238000005303 weighing Methods 0.000 claims description 4
- 239000011248 coating agent Substances 0.000 claims description 3
- 238000000576 coating method Methods 0.000 claims description 3
- 239000011241 protective layer Substances 0.000 claims description 3
- 238000007789 sealing Methods 0.000 claims description 3
- AYEKOFBPNLCAJY-UHFFFAOYSA-O thiamine pyrophosphate Chemical compound CC1=C(CCOP(O)(=O)OP(O)(O)=O)SC=[N+]1CC1=CN=C(C)N=C1N AYEKOFBPNLCAJY-UHFFFAOYSA-O 0.000 claims 1
- 239000008280 blood Substances 0.000 abstract description 12
- 210000004369 blood Anatomy 0.000 abstract description 12
- 239000002105 nanoparticle Substances 0.000 abstract description 8
- 230000000694 effects Effects 0.000 abstract description 6
- 238000000034 method Methods 0.000 abstract description 6
- 230000002218 hypoglycaemic effect Effects 0.000 abstract description 4
- 238000010521 absorption reaction Methods 0.000 abstract description 3
- 229920005615 natural polymer Polymers 0.000 abstract description 2
- 102000004190 Enzymes Human genes 0.000 abstract 1
- 108090000790 Enzymes Proteins 0.000 abstract 1
- 230000003321 amplification Effects 0.000 abstract 1
- 229940088597 hormone Drugs 0.000 abstract 1
- 239000005556 hormone Substances 0.000 abstract 1
- 238000004519 manufacturing process Methods 0.000 abstract 1
- 238000003199 nucleic acid amplification method Methods 0.000 abstract 1
- 239000004014 plasticizer Substances 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 21
- 238000002347 injection Methods 0.000 description 11
- 239000007924 injection Substances 0.000 description 11
- 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 8
- 206010012601 diabetes mellitus Diseases 0.000 description 8
- 239000008103 glucose Substances 0.000 description 8
- 239000002245 particle Substances 0.000 description 8
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 241000699670 Mus sp. Species 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 239000002994 raw material Substances 0.000 description 4
- ZSJLQEPLLKMAKR-GKHCUFPYSA-N streptozocin Chemical compound O=NN(C)C(=O)N[C@H]1[C@@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O ZSJLQEPLLKMAKR-GKHCUFPYSA-N 0.000 description 4
- 239000004094 surface-active agent Substances 0.000 description 4
- AXTGDCSMTYGJND-UHFFFAOYSA-N 1-dodecylazepan-2-one Chemical compound CCCCCCCCCCCCN1CCCCCC1=O AXTGDCSMTYGJND-UHFFFAOYSA-N 0.000 description 3
- 208000013016 Hypoglycemia Diseases 0.000 description 3
- 208000002193 Pain Diseases 0.000 description 3
- 150000001298 alcohols Chemical class 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 230000001186 cumulative effect Effects 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 230000036407 pain Effects 0.000 description 3
- 238000005070 sampling Methods 0.000 description 3
- 210000003491 skin Anatomy 0.000 description 3
- 238000013271 transdermal drug delivery Methods 0.000 description 3
- 239000007836 KH2PO4 Substances 0.000 description 2
- 238000012449 Kunming mouse Methods 0.000 description 2
- ZSJLQEPLLKMAKR-UHFFFAOYSA-N Streptozotocin Natural products O=NN(C)C(=O)NC1C(O)OC(CO)C(O)C1O ZSJLQEPLLKMAKR-UHFFFAOYSA-N 0.000 description 2
- 230000003187 abdominal effect Effects 0.000 description 2
- 210000000577 adipose tissue Anatomy 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 235000014113 dietary fatty acids Nutrition 0.000 description 2
- 239000002552 dosage form Substances 0.000 description 2
- 238000012377 drug delivery Methods 0.000 description 2
- 230000000857 drug effect Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 239000000194 fatty acid Substances 0.000 description 2
- 229930195729 fatty acid Natural products 0.000 description 2
- 150000004665 fatty acids Chemical class 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 210000001035 gastrointestinal tract Anatomy 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 239000003223 protective agent Substances 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 229960001052 streptozocin Drugs 0.000 description 2
- 238000010254 subcutaneous injection Methods 0.000 description 2
- 239000007929 subcutaneous injection Substances 0.000 description 2
- 150000003462 sulfoxides Chemical class 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- UNXRWKVEANCORM-UHFFFAOYSA-I triphosphate(5-) Chemical compound [O-]P([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O UNXRWKVEANCORM-UHFFFAOYSA-I 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- 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 1
- 241000220479 Acacia Species 0.000 description 1
- 241001143500 Aceraceae Species 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
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 229920000858 Cyclodextrin Polymers 0.000 description 1
- 206010015150 Erythema Diseases 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 206010016654 Fibrosis Diseases 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 101000976075 Homo sapiens Insulin Proteins 0.000 description 1
- 206010060708 Induration Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 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
- 235000010643 Leucaena leucocephala Nutrition 0.000 description 1
- 206010024604 Lipoatrophy Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 208000003251 Pruritus Diseases 0.000 description 1
- 108010026951 Short-Acting Insulin Proteins 0.000 description 1
- 229940123958 Short-acting insulin Drugs 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 239000012790 adhesive layer Substances 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 229940072056 alginate Drugs 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000000181 anti-adherent effect Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-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
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 1
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 1
- 201000011529 cardiovascular cancer Diseases 0.000 description 1
- 239000012876 carrier material Substances 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 229940097362 cyclodextrins Drugs 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000013118 diabetic mouse model Methods 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000004761 fibrosis Effects 0.000 description 1
- 238000010579 first pass effect Methods 0.000 description 1
- 239000004088 foaming agent Substances 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
- 238000007429 general method Methods 0.000 description 1
- 238000007446 glucose tolerance test Methods 0.000 description 1
- 230000002641 glycemic effect Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- PBGKTOXHQIOBKM-FHFVDXKLSA-N insulin (human) Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@H]1CSSC[C@H]2C(=O)N[C@H](C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3C=CC(O)=CC=3)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3NC=NC=3)NC(=O)[C@H](CO)NC(=O)CNC1=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O)=O)CSSC[C@@H](C(N2)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](NC(=O)CN)[C@@H](C)CC)[C@@H](C)CC)[C@@H](C)O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC=1C=CC=CC=1)C(C)C)C1=CN=CN1 PBGKTOXHQIOBKM-FHFVDXKLSA-N 0.000 description 1
- 230000003914 insulin secretion Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 230000007794 irritation Effects 0.000 description 1
- 230000007803 itching Effects 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 229940040145 liniment Drugs 0.000 description 1
- 239000000865 liniment Substances 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 230000006996 mental state Effects 0.000 description 1
- 229920000609 methyl cellulose Polymers 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- 235000010981 methylcellulose Nutrition 0.000 description 1
- 239000004530 micro-emulsion Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000002674 ointment Substances 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- -1 patch Substances 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 238000000053 physical method Methods 0.000 description 1
- 229920001495 poly(sodium acrylate) polymer Polymers 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 229940024999 proteolytic enzymes for treatment of wounds and ulcers Drugs 0.000 description 1
- 150000004040 pyrrolidinones Chemical class 0.000 description 1
- 230000000384 rearing effect Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- NNMHYFLPFNGQFZ-UHFFFAOYSA-M sodium polyacrylate Chemical compound [Na+].[O-]C(=O)C=C NNMHYFLPFNGQFZ-UHFFFAOYSA-M 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 210000000434 stratum corneum Anatomy 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 150000003505 terpenes Chemical class 0.000 description 1
- 235000007586 terpenes Nutrition 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
- 239000002023 wood Substances 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
- A61K38/28—Insulins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/70—Web, sheet or filament bases ; Films; Fibres of the matrix type containing drug
- A61K9/7023—Transdermal patches and similar drug-containing composite devices, e.g. cataplasms
-
- 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
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Public Health (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Diabetes (AREA)
- Epidemiology (AREA)
- Endocrinology (AREA)
- Obesity (AREA)
- Gastroenterology & Hepatology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Hematology (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Emergency Medicine (AREA)
- Inorganic Chemistry (AREA)
- Dermatology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicinal Preparation (AREA)
Abstract
本发明涉及一种壳聚糖/胰岛素纳米缓释经皮制剂及其制备方法。本发明以天然高分子载体‑‑壳聚糖包载胰岛素，制备出一种纳米降血糖制剂。胰岛素是体内唯一能降低血糖的激素，载药层由胰岛素和壳聚糖、传递体、促渗剂和增塑剂组成，通过水溶性较好的壳聚糖将难溶于水的胰岛素包载形成纳米溶液从而制得制剂。本发明实现无痛、缓释、便捷的给药，纳米粒载体可保护胰岛素避免生物体酶的破坏,提高生物利用度,具有缓释作用，同时降低副作用，具有经皮吸收率快、安全稳定、缓释给药以及无痛便捷等特点。本发明的壳聚糖/胰岛素纳米经皮制剂制备工艺简单，生产成本低，工艺易于放大。
Description
技术领域
本发明属于药物制剂技术领域，具体涉及一种壳聚糖/胰岛素纳米经皮制剂及其制备方法。
背景技术
糖尿病是危害人类健康的常见病，发病率约为 3 %～ 5 %，由其引起的死亡率仅次于心血管病和肿瘤。目前治疗糖尿病的药物中，胰岛素仍然占据大部分比例，是治疗糖尿病的最后一道防线。如何合理使用胰岛素能够帮助病人控制血糖，是研究人员必须考虑的事情。由于胰岛素为蛋白质多肽类药物，能被胃肠道的蛋白水解酶降解且不被吸收，所以口服无效。因此，目前临床上使用的剂型是注射剂。为了使血糖得到控制，患者需要至少每天注射一次。但长期频繁注射胰岛素会给病人身心带来相当大的压力和痛苦，同时注射部位会出现皮肤红肿 、发痒 、硬结及感染，皮下脂肪萎缩或纤维化增生等严重的副作用。
由此，我们应用先进的纳米缓释技术，使用自然界中不可多得天然高分子载体材料:壳聚糖作为载体制备出一种胰岛素纳米制剂。壳聚糖具有良好的生物可降解性 、生物相容性、低毒性以及打开上皮细胞间紧密连接，促进大分子药物通过组织上皮的转运作用。从而研究出一种更加长效稳定的胰岛素纳米缓释系统，不仅可以使患者减少注射次数，还可以避免较大的机体血糖波动给患者带来的不适，与短效胰岛素合理配合使用，会使糖尿病治疗更为便捷有效。
经皮给药系统(transdermal drug delivery system, TDDS)可实现无创伤性给药，具有超越一般给药方法的独特优点：直接作用于靶部位发挥药效；避免肝脏的首过效应和胃肠道的干扰；血药浓度稳定减少给药次数等。而且患者可以自主用药，特别适合于婴儿、老人及不宜口服给药的患者，提高患者的用药依从性；发现副作用时，可随时中断给药等。但是当药物分子量较大，并且易形成聚集体而难以渗透皮肤表面的致密角质层时，我们需要对药物进行促渗处理。经皮给药的促渗方法有药剂学方法包括囊泡、脂质体、微乳和纳米粒；化学方法包括亚枫类、脂肪酸类、表面活性剂类、脂类和醇；物理方法离子导入、微针、起声波、电致孔和驻极体。本发明结合了药剂学和化学两种方法，以胰岛素为主要药物活性成分制备出一种葡萄糖浓度响应的胰岛素纳米缓释系统。
发明内容
本发明目的在于，克服现有胰岛素注射剂带来的副作用，解决胰岛素注射生理痛苦、瞬时低血糖、长期注射形成脂肪垫、价格昂贵、注射频繁等问题。本发明利用纳米医药技术提高胰岛素的经皮渗透作用，增加缓释释药，降低副作用，避免低血糖，具有药效期长以及经济便捷的优点。
本发明技术问题是提供一种胰岛素缓释系统制剂：葡萄糖浓度响应的壳聚糖/胰岛素缓释系统。每片制剂中至少含25U的胰岛素，纳米平均粒径达到80~200nm，纳米凝胶越小，凝胶的经皮吸收性越好。
所述壳聚糖/胰岛素纳米缓释经皮制剂，其由背衬层、载药层和防粘层组成，其特征在于：载药层组分包括壳聚糖包载的胰岛素、压敏胶、促渗剂、基质、亲水性辅料和冻干保护剂；载药层以凝胶形式被封闭于背衬层和防粘层之间。
所述的胰岛素包括动物胰岛素、人胰岛素和胰岛素类似物。
所述亲水性辅料为海藻酸盐、阿拉伯胶、淀粉、明胶、甲基纤维素、羧甲基纤维素、卡波姆、聚丙烯酸钠中的两种或两种以上，冻干保护剂为甘露醇、乳糖、葡萄糖中的两种或两种以上。
其中载药层的组分以重量分数计包括：壳聚糖10-30%、胰岛素3.5-17%、压敏胶5.2-7%、促渗剂16-20%、基质0.17-2%、亲水性辅料7-10%、冻干保护剂35-40%。
促渗剂为吡咯烷酮类、亚砜类、Azone及其类似物、氨基酸及其酯、萜烯类、脂肪酸及其酯、环糊精类、胺类、表面活性剂类、醇类和磷脂类中一种或几种；
优选为Azone及其类似物、表面活性剂、醇类、亚砜类中的一种或几种组合，其中各组分在载药层中的含量以重量分数计为：Azone及其类似物0-20%，表面活性剂0-20%，醇类0-40%。
本发明的第二个技术问题是提供所述胰岛素纳米经皮制剂的制备方法，包括以下步骤：
A：将胰岛素原料药溶解于有机溶剂中，配制成浓度1 mmol/mL的原料药溶液，备用；
同时将壳聚糖溶于醋酸中，配制成1-10mg/mL的原料药溶液，并向壳聚糖/醋酸溶液中加入三聚磷酸钠TPP，制成壳聚糖纳米溶液，备用；
B：在25℃室温搅拌条件下，向壳聚糖纳米溶液中缓缓加入胰岛素原料药溶液，充分搅拌混合1h后制得壳聚糖/胰岛素纳米溶液，其平均粒径为112.6 nm；
C：将B步骤得到的胰岛素纳米溶液与亲水性辅料溶液、促渗剂、基质、压敏胶混匀，其中壳聚糖/胰岛素纳米溶液与亲水性辅料的水溶液的体积比为1/5-1/20，即得载药层；
D：载药层均匀涂布在背衬层上，盖上保护层，监测合格，抽真空密封，即得所述纳米缓释经皮制剂。
本发明的优点在于：
1、本发明制备的壳聚糖/胰岛素纳米经皮制剂大大提高了药物的溶解度。
2、本发明制备的壳聚糖/胰岛素纳米经皮制剂中的壳聚糖/胰岛素纳米具有很大的比表面积，具有很高的载药量，提高药物缓释释放。
3、本发明制备的壳聚糖/胰岛素纳米经皮制剂具有缓释的特性，能够减少患者的用药次数，也可避免注射给患者带来的生理和心理痛苦。
4、胰岛素纳米制剂能够避免由于皮下注射而引起的脂肪垫结现象而影响药物的吸收，同时有效避免皮下注射有可能导致的瞬时低血糖。
本发明技术内容制备的经皮给药纳米制剂可用于经皮的软膏剂、贴剂、涂剂、喷雾剂、气雾剂、泡沫剂、微型海绵剂以及微针贴剂等剂型。
附图说明
图1是壳聚糖/胰岛素纳米的粒径和电势；
图2是壳聚糖/胰岛素纳米的粒径的优化；
图3是对壳聚糖/胰岛素纳米的原子力测试；
图4是壳聚糖/胰岛素纳米的药物释放；
图5是氧浓度响应的壳聚糖/胰岛素纳米药物的释放；
图6是壳聚糖/胰岛素载药层的累积渗经率；
图7是本发明的壳聚糖/胰岛素纳米经皮制剂对糖尿病的治疗效果。
具体实施方式
根据下述实施例，可以更好地理解本发明，下面结合具体实施方式对本发明所述的技术方案作进一步的说明，但是本发明不仅限于此。
实施例1：
A：称取甘油0.1ml，羟甲基纤维素200mg，甘露醇1g，丙二醇0.4g，卡波姆5mg，丙烯酸钠150mg，。称取胰岛素5.5538mg溶于1ml甲醇中，形成含药的有机溶液。取11.1076 mg壳聚糖溶于1mL 1wt%醋酸中，并向壳聚糖醋酸溶液中加入 2mg 三聚磷酸钠（TPP）制成壳聚糖纳米溶液。
B：将步骤A中称取的甘油、羟甲基纤维素、甘露醇、丙二醇、卡波姆、丙烯酸钠溶于5ml水中，同时取100μL胰岛素甲醇溶液和250μL壳聚糖纳米溶液混合均匀后，逐滴滴加到亲水性辅料中，在室温条件下搅拌1h，再加入促渗剂、基质和压敏胶混匀，即得载药层；
D：将载药层均匀涂布在背衬层上，盖上保护层，检验合格，抽真空密封，即得。
实施例2
取胰岛素甲醇溶液500μL和壳聚糖纳米水溶液750μL混合均匀后，使用马尔文粒径仪测定纳米溶液的粒径，如图1所示。同时取用不同分子量的壳聚糖对胰岛素进行包载，并测定其粒径，如图2所示，可知，壳聚糖分子量越小，壳聚糖/胰岛素纳米颗粒粒径越小。并对粒径较好的壳聚糖/胰岛素纳米颗粒拍摄原子力图像，如图3所示。
实施例3
将1ml载药层置于截留分子量为10000的透析袋中，同时将透析袋置于pH为7.4的30 ml 的PBS溶液（NaCl,137 mM;KCl,2.7 mM，Na2HPO4,10 mM; KH2PO4, 2 mM; pH值7.4）中，向缓冲溶液中加入葡萄糖，以达到人体较高的血糖浓度，再将PBS缓冲溶液置于37℃恒温的摇床中，转速233min/r，在不同的时间点取样500 μl，同时再补加500 μl的较高血糖浓度的PBS。采用紫外分光光度法检测波长276nm，计算累积量，如图4所示。
实施例4
将1ml载药层置于截留分子量为10000的透析袋中，同时将透析袋置于pH为7.4的30 ml的PBS（NaCl,137 mM;KCl,2.7 mM，Na2HPO4,10 mM; KH2PO4, 2 mM; pH值7.4）中，再将PBS缓冲溶液置于37℃恒温的摇床中，转速233r/min，在不同的时间点取样1ml，同时再补加1ml的空白PBS。采用紫外分光光度法检测波长276nm，计算累积量，与在高葡萄糖浓度下的药物释放相对比，如图5所示，表明本发明制备的壳聚糖/胰岛素纳米缓释经皮给药制剂具有缓释的特性。
实施例4
制备12张完整的小鼠腹部皮肤，采用扩散池实验，根据公式计算渗透量
(Q，μg.cm2)
式中，Cn为第n个取样点测得的药物浓度；Ci为第i个取样点测得的药物浓度；A为有效接触面积(cm2)；V为接收池体积(ml)；V0为取样体积(ml)。
以单位面积累计渗透量(Q)为纵坐标，时间(t)为横坐标，绘制经皮吸收实验，如图6所示。
实施例5
胰岛素纳米凝胶经皮制剂对糖尿病模型小鼠的治疗：
建模及分组：体重18-20g的昆明小鼠、链脲佐菌素（streptozotocin STZ）50～150mg／kg，正常饲养7天后，尾静脉注射STZ如果连续两天血糖值超过11.1mmol／Ｌ或者葡萄糖耐量试验120min时的血糖值仍未恢复到注射前水平则认为糖尿病小鼠造模成功，30只小鼠成功建立其模型。将糖尿病小鼠分为3组，实验组为经皮给药组，胰岛素注射组，设阳性对照组。
称量实验小鼠的体重，按40mg/kg剂量进行经皮治疗，实验小鼠固定在木板上，除去腹部的毛后，紧贴壳聚糖/胰岛素纳米经皮制剂，面积为3 cm2，将小鼠放回笼子正常饲养。
血糖指标检测：将各组给药治疗0、2、4、6、10、24h后从尾静脉取血，在测定血糖浓度，考察血糖浓度变化情况，如图7所示。
实施例6
经皮制剂的刺激性观察：
实验小鼠经壳聚糖/胰岛素纳米经皮制剂治疗后，无明显的精神状态不正常现象，连续观察2天后，未发现经皮部位皮肤出现红肿和发炎等现象。
Claims (1)
1.一种壳聚糖/胰岛素纳米缓释经皮制剂的制备方法，其特征在于：具体制备步骤包括：
A：称取甘油0.1ml，羟甲基纤维素200mg，甘露醇1g，丙二醇0.4g，卡波姆5mg，丙烯酸钠150mg；称取胰岛素5.5538mg溶于1ml甲醇中，形成含药的有机溶液,取11.1076 mg壳聚糖溶于1mL 1wt%醋酸中，并向壳聚糖醋酸溶液中加入 2mg 三聚磷酸钠TPP制成壳聚糖纳米溶液；
B：将步骤A中称取的甘油、羟甲基纤维素、甘露醇、丙二醇、卡波姆、丙烯酸钠溶于5ml水中，同时取100μL胰岛素甲醇溶液和250μL壳聚糖纳米溶液混合均匀后，逐滴滴加到亲水性辅料中，在室温条件下搅拌1h，再加入促渗剂、基质和压敏胶混匀，即得载药层；
C：将载药层均匀涂布在背衬层上，盖上保护层，检验合格，抽真空密封，即得。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810377863.9A CN108553639B (zh) | 2018-04-25 | 2018-04-25 | 一种壳聚糖/胰岛素纳米缓释经皮制剂及其制备方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810377863.9A CN108553639B (zh) | 2018-04-25 | 2018-04-25 | 一种壳聚糖/胰岛素纳米缓释经皮制剂及其制备方法 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108553639A CN108553639A (zh) | 2018-09-21 |
| CN108553639B true CN108553639B (zh) | 2022-04-19 |
Family
ID=63536405
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810377863.9A Expired - Fee Related CN108553639B (zh) | 2018-04-25 | 2018-04-25 | 一种壳聚糖/胰岛素纳米缓释经皮制剂及其制备方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108553639B (zh) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113893333B (zh) * | 2021-10-18 | 2024-04-09 | 中国药科大学 | 一种胰岛素缓释口腔贴片及其制备方法、应用 |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6068853A (en) * | 1994-04-13 | 2000-05-30 | Novartis Corporation | Temporally controlled drug delivery systems |
| CN1438881A (zh) * | 2000-06-27 | 2003-08-27 | 株式会社Mitech | 胰岛素控释制剂及其方法 |
| WO2007113665A2 (en) * | 2006-04-05 | 2007-10-11 | Transgene Biotek Limited | Polymerized solid lipid nanoparticles for oral or mucosal delivery of therapeutic proteins and peptides |
| US8137697B1 (en) * | 2004-10-05 | 2012-03-20 | Gp Medical, Inc. | Nanoparticles for protein/peptide delivery and delivery means thereof |
| CN103705494A (zh) * | 2013-12-26 | 2014-04-09 | 湖北医药学院 | 一种胰岛素纳米透皮贴剂及其制备方法 |
| CN105963304A (zh) * | 2016-07-05 | 2016-09-28 | 福州大学 | 一种二甲双胍与熊果酸及其衍生物的药物组合物及其在制备抗肿瘤药物中的应用 |
-
2018
- 2018-04-25 CN CN201810377863.9A patent/CN108553639B/zh not_active Expired - Fee Related
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6068853A (en) * | 1994-04-13 | 2000-05-30 | Novartis Corporation | Temporally controlled drug delivery systems |
| CN1438881A (zh) * | 2000-06-27 | 2003-08-27 | 株式会社Mitech | 胰岛素控释制剂及其方法 |
| US8137697B1 (en) * | 2004-10-05 | 2012-03-20 | Gp Medical, Inc. | Nanoparticles for protein/peptide delivery and delivery means thereof |
| WO2007113665A2 (en) * | 2006-04-05 | 2007-10-11 | Transgene Biotek Limited | Polymerized solid lipid nanoparticles for oral or mucosal delivery of therapeutic proteins and peptides |
| CN103705494A (zh) * | 2013-12-26 | 2014-04-09 | 湖北医药学院 | 一种胰岛素纳米透皮贴剂及其制备方法 |
| CN105963304A (zh) * | 2016-07-05 | 2016-09-28 | 福州大学 | 一种二甲双胍与熊果酸及其衍生物的药物组合物及其在制备抗肿瘤药物中的应用 |
Non-Patent Citations (4)
| Title |
|---|
| Blocking Central Galanin Receptors Attenuates Insulin Sensitivity in Myocytes of Diabetic Trained Rats;Le Bu等;《Journal of Neuroscience Research》;20130507;第971-977页 * |
| Transdermal patches of chitosan nanoparticles for insulin delivery;LINGHESWAR SADHASIVAM等;《International Journal of Pharmacy and Pharmaceutical Sciences》;20150131;第7卷(第5期);摘要，第85页Synthesis of insulin-chitosan nanoparticles和Transdermal patches部分，第87页左栏正文倒数第1段 * |
| 胰岛素样生长因子结合蛋白-1启动子的克隆与序列分析;吴志香等;《生物技术通讯》;20051230;第16卷(第6期);第624-626页 * |
| 驻极体联合季铵化壳聚糖纳米粒促进胰岛素经皮转运及其机制的研究;苑旺;《万方学位论文》;20151102;全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108553639A (zh) | 2018-09-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108434125B (zh) | 一种介孔二氧化硅-胰岛素纳米缓释经皮贴剂的制备方法 | |
| JP5538897B2 (ja) | 体表適用製剤、並びに、体表適用製剤保持シート | |
| Yang et al. | Polymeric microneedle‐mediated sustained release systems: Design strategies and promising applications for drug delivery | |
| CN103705494A (zh) | 一种胰岛素纳米透皮贴剂及其制备方法 | |
| EP3572119B1 (en) | Preparation of a portable transdermal administration patch apparatus | |
| CN112022919A (zh) | 经皮吸收的苍艾油传递体凝胶及其制备方法 | |
| WO2020103832A1 (zh) | 一种制备柔性脂质体的方法 | |
| CN108553639B (zh) | 一种壳聚糖/胰岛素纳米缓释经皮制剂及其制备方法 | |
| Al-Tabakha et al. | Recent advances and future prospects of non-invasive insulin delivery systems | |
| CN108272778A (zh) | 一种熊果酸/胰岛素纳米缓释经皮给药制剂及其制备方法 | |
| Jose et al. | Recent Progress of Transdermal Drug Delivery Systems for Biomedical Applications | |
| KR20170000747A (ko) | 트러블 개선 및 피부 재생을 위한 용해성 미세바늘 패치 | |
| Elçioğlu et al. | Nanoparticle insulin drug delivery—applications and new aspects | |
| CN113893333A (zh) | 一种胰岛素缓释口腔贴片及其制备方法、应用 | |
| Jyothika et al. | Types of transdermal drug delivery systems: a literature report of the past decade | |
| CN110115710B (zh) | 一种用于治疗哮喘的透皮吸收制剂 | |
| CN109528693B (zh) | 一种雷帕霉素巴布剂及其制备方法 | |
| CN112741897A (zh) | 一种胰高血糖素样肽-1受体激动剂的微阵列递药系统的组合物 | |
| CN109289041B (zh) | 一种维生素d-胰岛素纳米缓释经皮制剂及其制备方法 | |
| Kumar et al. | A Review on Transdermal Drug Delivery Patches | |
| CN108273042A (zh) | 一种人参皂苷-胰岛素纳米凝胶及其制备方法与应用 | |
| Krisnamurti et al. | Glucose-responsive pectin insulin patch for diabetes mellitus: A review | |
| CN116173188B (zh) | 口服glp-1类似物固体脂质纳米粒及其制备方法和应用 | |
| Sindhu et al. | Development of and advances in insulin delivery | |
| CN115317439A (zh) | 一种克立硼罗微针贴剂及其制备方法 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB03 | Change of inventor or designer information |
Inventor after: Zou Junjie Inventor after: Shao Jingwei Inventor after: Lin Juanfang Inventor before: Shao Jingwei Inventor before: Zou Junjie Inventor before: Lin Juanfang |
|
| CB03 | Change of inventor or designer information | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20220419 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |