CN109232744A - 改进型齐考诺肽 - Google Patents
改进型齐考诺肽 Download PDFInfo
- Publication number
- CN109232744A CN109232744A CN201811086050.0A CN201811086050A CN109232744A CN 109232744 A CN109232744 A CN 109232744A CN 201811086050 A CN201811086050 A CN 201811086050A CN 109232744 A CN109232744 A CN 109232744A
- Authority
- CN
- China
- Prior art keywords
- mviia
- ziconotide
- polypeptide
- amino acid
- administration
- 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.)
- Granted
Links
- BPKIMPVREBSLAJ-QTBYCLKRSA-N ziconotide Chemical compound C([C@H]1C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]2C(=O)N[C@@H]3C(=O)N[C@H](C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@H](C(N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CSSC2)C(N)=O)=O)CSSC[C@H](NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)CNC(=O)[C@H](CCCCN)NC(=O)CNC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CSSC3)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(N1)=O)CCSC)[C@@H](C)O)C1=CC=C(O)C=C1 BPKIMPVREBSLAJ-QTBYCLKRSA-N 0.000 title claims abstract description 53
- 229960002811 ziconotide Drugs 0.000 title claims abstract description 29
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 84
- 238000002360 preparation method Methods 0.000 claims abstract description 23
- 210000003462 vein Anatomy 0.000 claims abstract description 18
- 210000003928 nasal cavity Anatomy 0.000 claims abstract description 14
- 210000000683 abdominal cavity Anatomy 0.000 claims abstract description 6
- 229920001184 polypeptide Polymers 0.000 claims description 65
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 65
- 150000001413 amino acids Chemical class 0.000 claims description 22
- 239000003814 drug Substances 0.000 claims description 22
- MCYTYTUNNNZWOK-LCLOTLQISA-N penetratin Chemical compound C([C@H](NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CCCNC(N)=N)[C@@H](C)CC)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(N)=O)C1=CC=CC=C1 MCYTYTUNNNZWOK-LCLOTLQISA-N 0.000 claims description 14
- 108010043655 penetratin Proteins 0.000 claims description 14
- 101710149951 Protein Tat Proteins 0.000 claims description 10
- 239000008194 pharmaceutical composition Substances 0.000 claims description 10
- 210000004899 c-terminal region Anatomy 0.000 claims description 8
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 239000000816 peptidomimetic Substances 0.000 claims description 6
- 230000015572 biosynthetic process Effects 0.000 claims description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 4
- 238000003786 synthesis reaction Methods 0.000 claims description 4
- 239000004471 Glycine Substances 0.000 claims description 3
- 239000002253 acid Substances 0.000 claims description 3
- 238000003780 insertion Methods 0.000 claims description 3
- 230000037431 insertion Effects 0.000 claims description 3
- 230000035772 mutation Effects 0.000 claims description 3
- 101800004761 Magainin-2 Proteins 0.000 claims description 2
- 108010088535 Pep-1 peptide Proteins 0.000 claims description 2
- -1 S413- PV Proteins 0.000 claims description 2
- 210000001015 abdomen Anatomy 0.000 claims description 2
- 108010025307 buforin II Proteins 0.000 claims description 2
- 239000002552 dosage form Substances 0.000 claims description 2
- MGIUUAHJVPPFEV-ABXDCCGRSA-N magainin ii Chemical compound C([C@H](NC(=O)[C@H](CCCCN)NC(=O)CNC(=O)[C@@H](NC(=O)CN)[C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O)C1=CC=CC=C1 MGIUUAHJVPPFEV-ABXDCCGRSA-N 0.000 claims description 2
- 230000000202 analgesic effect Effects 0.000 abstract description 30
- 230000000694 effects Effects 0.000 abstract description 23
- 230000008499 blood brain barrier function Effects 0.000 abstract description 14
- 210000001218 blood-brain barrier Anatomy 0.000 abstract description 14
- 230000000857 drug effect Effects 0.000 abstract description 14
- 238000001727 in vivo Methods 0.000 abstract description 7
- 208000015181 infectious disease Diseases 0.000 abstract description 4
- 238000001990 intravenous administration Methods 0.000 abstract description 3
- 230000009471 action Effects 0.000 abstract description 2
- 210000000170 cell membrane Anatomy 0.000 abstract description 2
- 230000007547 defect Effects 0.000 abstract description 2
- 230000004927 fusion Effects 0.000 abstract 1
- 150000002333 glycines Chemical class 0.000 abstract 1
- 238000009776 industrial production Methods 0.000 abstract 1
- 238000007912 intraperitoneal administration Methods 0.000 abstract 1
- 230000000149 penetrating effect Effects 0.000 abstract 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 38
- 238000002474 experimental method Methods 0.000 description 27
- 208000002193 Pain Diseases 0.000 description 20
- 230000036407 pain Effects 0.000 description 18
- 235000001014 amino acid Nutrition 0.000 description 17
- 239000000243 solution Substances 0.000 description 16
- 238000000034 method Methods 0.000 description 15
- 229960000583 acetic acid Drugs 0.000 description 13
- 210000004556 brain Anatomy 0.000 description 13
- 108091006146 Channels Proteins 0.000 description 11
- 210000004027 cell Anatomy 0.000 description 11
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 11
- 239000002504 physiological saline solution Substances 0.000 description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
- 229940079593 drug Drugs 0.000 description 10
- 201000010099 disease Diseases 0.000 description 9
- 239000011780 sodium chloride Substances 0.000 description 9
- 206010003246 arthritis Diseases 0.000 description 8
- 239000000825 pharmaceutical preparation Substances 0.000 description 8
- 206010044565 Tremor Diseases 0.000 description 7
- 238000005096 rolling process Methods 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 230000001684 chronic effect Effects 0.000 description 6
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 6
- 238000002347 injection Methods 0.000 description 6
- 239000007924 injection Substances 0.000 description 6
- 150000003839 salts Chemical class 0.000 description 6
- RCQRKPUXJAGEEC-ZLUOBGJFSA-N Ala-Cys-Cys Chemical compound C[C@H](N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CS)C(O)=O RCQRKPUXJAGEEC-ZLUOBGJFSA-N 0.000 description 5
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 5
- PRXCTTWKGJAPMT-ZLUOBGJFSA-N Cys-Ala-Ser Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O PRXCTTWKGJAPMT-ZLUOBGJFSA-N 0.000 description 5
- IZUNQDRIAOLWCN-YUMQZZPRSA-N Cys-Leu-Gly Chemical compound CC(C)C[C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](CS)N IZUNQDRIAOLWCN-YUMQZZPRSA-N 0.000 description 5
- YIFUFYZELCMPJP-YUMQZZPRSA-N Gly-Leu-Cys Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(O)=O YIFUFYZELCMPJP-YUMQZZPRSA-N 0.000 description 5
- PNUCWVAGVNLUMW-CIUDSAMLSA-N Leu-Cys-Ser Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(=O)N[C@@H](CO)C(O)=O PNUCWVAGVNLUMW-CIUDSAMLSA-N 0.000 description 5
- OXRLYTYUXAQTHP-YUMQZZPRSA-N Leu-Gly-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](C)C(O)=O OXRLYTYUXAQTHP-YUMQZZPRSA-N 0.000 description 5
- JVTYXRRFZCEPPK-RHYQMDGZSA-N Leu-Met-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CCSC)NC(=O)[C@H](CC(C)C)N)O JVTYXRRFZCEPPK-RHYQMDGZSA-N 0.000 description 5
- DJDSEDOKJTZBAR-ZDLURKLDSA-N Thr-Gly-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O DJDSEDOKJTZBAR-ZDLURKLDSA-N 0.000 description 5
- 108010086434 alanyl-seryl-glycine Proteins 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 229910001424 calcium ion Inorganic materials 0.000 description 5
- 230000002401 inhibitory effect Effects 0.000 description 5
- 238000007913 intrathecal administration Methods 0.000 description 5
- 239000007791 liquid phase Substances 0.000 description 5
- 230000036515 potency Effects 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 208000011580 syndromic disease Diseases 0.000 description 5
- 108010051109 Cell-Penetrating Peptides Proteins 0.000 description 4
- 102000020313 Cell-Penetrating Peptides Human genes 0.000 description 4
- 206010019233 Headaches Diseases 0.000 description 4
- 208000007514 Herpes zoster Diseases 0.000 description 4
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 4
- 206010028980 Neoplasm Diseases 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 231100000869 headache Toxicity 0.000 description 4
- 238000010253 intravenous injection Methods 0.000 description 4
- 238000001543 one-way ANOVA Methods 0.000 description 4
- JMANVNJQNLATNU-UHFFFAOYSA-N oxalonitrile Chemical compound N#CC#N JMANVNJQNLATNU-UHFFFAOYSA-N 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 230000009885 systemic effect Effects 0.000 description 4
- 208000030507 AIDS Diseases 0.000 description 3
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 3
- 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 3
- 208000001640 Fibromyalgia Diseases 0.000 description 3
- 208000016604 Lyme disease Diseases 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 239000011575 calcium Substances 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 238000012512 characterization method Methods 0.000 description 3
- 108050003126 conotoxin Proteins 0.000 description 3
- 108010016616 cysteinylglycine Proteins 0.000 description 3
- 201000001981 dermatomyositis Diseases 0.000 description 3
- 206010012601 diabetes mellitus Diseases 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 229960003180 glutathione Drugs 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 208000004296 neuralgia Diseases 0.000 description 3
- 229940124636 opioid drug Drugs 0.000 description 3
- 230000001590 oxidative effect Effects 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000011347 resin Substances 0.000 description 3
- 229920005989 resin Polymers 0.000 description 3
- 238000004007 reversed phase HPLC Methods 0.000 description 3
- 206010039073 rheumatoid arthritis Diseases 0.000 description 3
- 238000010532 solid phase synthesis reaction Methods 0.000 description 3
- 238000010998 test method Methods 0.000 description 3
- RAVVEEJGALCVIN-AGVBWZICSA-N (2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-5-amino-2-[[(2s)-2-[[(2s)-2-[[(2s)-6-amino-2-[[(2s)-6-amino-2-[[(2s)-2-[[2-[[(2s)-2-amino-3-(4-hydroxyphenyl)propanoyl]amino]acetyl]amino]-5-(diaminomethylideneamino)pentanoyl]amino]hexanoyl]amino]hexanoyl]amino]-5-(diamino Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCN=C(N)N)NC(=O)CNC(=O)[C@@H](N)CC1=CC=C(O)C=C1 RAVVEEJGALCVIN-AGVBWZICSA-N 0.000 description 2
- 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 2
- 208000004998 Abdominal Pain Diseases 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 108090000312 Calcium Channels Proteins 0.000 description 2
- 102000003922 Calcium Channels Human genes 0.000 description 2
- 108010024636 Glutathione Proteins 0.000 description 2
- 239000007995 HEPES buffer Substances 0.000 description 2
- 241000713772 Human immunodeficiency virus 1 Species 0.000 description 2
- 108700000788 Human immunodeficiency virus 1 tat peptide (47-57) Proteins 0.000 description 2
- 208000004575 Infectious Arthritis Diseases 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- 208000004404 Intractable Pain Diseases 0.000 description 2
- 102000004129 N-Type Calcium Channels Human genes 0.000 description 2
- 108090000699 N-Type Calcium Channels Proteins 0.000 description 2
- 208000031845 Pernicious anaemia Diseases 0.000 description 2
- 208000007048 Polymyalgia Rheumatica Diseases 0.000 description 2
- 206010036376 Postherpetic Neuralgia Diseases 0.000 description 2
- 229920005654 Sephadex Polymers 0.000 description 2
- 239000012507 Sephadex™ Substances 0.000 description 2
- SLUWOCTZVGMURC-BFHQHQDPSA-N Thr-Gly-Ala Chemical compound C[C@@H](O)[C@H](N)C(=O)NCC(=O)N[C@@H](C)C(O)=O SLUWOCTZVGMURC-BFHQHQDPSA-N 0.000 description 2
- 108091023040 Transcription factor Proteins 0.000 description 2
- 102000040945 Transcription factor Human genes 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 230000036592 analgesia Effects 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 210000005013 brain tissue Anatomy 0.000 description 2
- AIYUHDOJVYHVIT-UHFFFAOYSA-M caesium chloride Chemical compound [Cl-].[Cs+] AIYUHDOJVYHVIT-UHFFFAOYSA-M 0.000 description 2
- HUCVOHYBFXVBRW-UHFFFAOYSA-M caesium hydroxide Chemical compound [OH-].[Cs+] HUCVOHYBFXVBRW-UHFFFAOYSA-M 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000002983 circular dichroism Methods 0.000 description 2
- 238000001142 circular dichroism spectrum Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 2
- 235000018417 cysteine Nutrition 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 238000007405 data analysis Methods 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 231100000673 dose–response relationship Toxicity 0.000 description 2
- 230000007831 electrophysiology Effects 0.000 description 2
- 238000002001 electrophysiology Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 230000000799 fusogenic effect Effects 0.000 description 2
- 108010000434 glycyl-alanyl-leucine Proteins 0.000 description 2
- XKUKSGPZAADMRA-UHFFFAOYSA-N glycyl-glycyl-glycine Chemical compound NCC(=O)NCC(=O)NCC(O)=O XKUKSGPZAADMRA-UHFFFAOYSA-N 0.000 description 2
- 210000003128 head Anatomy 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 229910052742 iron Inorganic materials 0.000 description 2
- 208000002551 irritable bowel syndrome Diseases 0.000 description 2
- 206010025135 lupus erythematosus Diseases 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- BQJCRHHNABKAKU-KBQPJGBKSA-N morphine Chemical compound O([C@H]1[C@H](C=C[C@H]23)O)C4=C5[C@@]12CCN(C)[C@@H]3CC5=CC=C4O BQJCRHHNABKAKU-KBQPJGBKSA-N 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- 201000001119 neuropathy Diseases 0.000 description 2
- 230000007823 neuropathy Effects 0.000 description 2
- 210000000929 nociceptor Anatomy 0.000 description 2
- 108091008700 nociceptors Proteins 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 201000008482 osteoarthritis Diseases 0.000 description 2
- 208000033808 peripheral neuropathy Diseases 0.000 description 2
- 231100000614 poison Toxicity 0.000 description 2
- 239000002574 poison Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 239000012266 salt solution Substances 0.000 description 2
- 208000007056 sickle cell anemia Diseases 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 208000006379 syphilis Diseases 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 238000010361 transduction Methods 0.000 description 2
- 230000026683 transduction Effects 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 1
- XUDUTRMKKYUAKI-UHFFFAOYSA-N 3-[1-(1-phenylethyl)piperidin-4-yl]-1h-benzimidazol-2-one Chemical compound C1CC(N2C(NC3=CC=CC=C32)=O)CCN1C(C)C1=CC=CC=C1 XUDUTRMKKYUAKI-UHFFFAOYSA-N 0.000 description 1
- 241000238876 Acari Species 0.000 description 1
- 208000026872 Addison Disease Diseases 0.000 description 1
- 208000003130 Alcoholic Neuropathy Diseases 0.000 description 1
- 206010053555 Arthritis bacterial Diseases 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 208000037157 Azotemia Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 206010005098 Blastomycosis Diseases 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 206010006811 Bursitis Diseases 0.000 description 1
- 208000010693 Charcot-Marie-Tooth Disease Diseases 0.000 description 1
- 208000006561 Cluster Headache Diseases 0.000 description 1
- 206010009900 Colitis ulcerative Diseases 0.000 description 1
- 241000237942 Conidae Species 0.000 description 1
- 206010012335 Dependence Diseases 0.000 description 1
- 201000004624 Dermatitis Diseases 0.000 description 1
- 208000032131 Diabetic Neuropathies Diseases 0.000 description 1
- 101000761020 Dinoponera quadriceps Poneritoxin Proteins 0.000 description 1
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 208000009386 Experimental Arthritis Diseases 0.000 description 1
- 208000024412 Friedreich ataxia Diseases 0.000 description 1
- 206010017533 Fungal infection Diseases 0.000 description 1
- 208000007882 Gastritis Diseases 0.000 description 1
- 208000005577 Gastroenteritis Diseases 0.000 description 1
- 108010053070 Glutathione Disulfide Proteins 0.000 description 1
- VSVZIEVNUYDAFR-YUMQZZPRSA-N Gly-Ala-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)CN VSVZIEVNUYDAFR-YUMQZZPRSA-N 0.000 description 1
- UQJNXZSSGQIPIQ-FBCQKBJTSA-N Gly-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)CN UQJNXZSSGQIPIQ-FBCQKBJTSA-N 0.000 description 1
- JQFILXICXLDTRR-FBCQKBJTSA-N Gly-Thr-Gly Chemical compound NCC(=O)N[C@@H]([C@H](O)C)C(=O)NCC(O)=O JQFILXICXLDTRR-FBCQKBJTSA-N 0.000 description 1
- 208000003807 Graves Disease Diseases 0.000 description 1
- 208000015023 Graves' disease Diseases 0.000 description 1
- 208000035895 Guillain-Barré syndrome Diseases 0.000 description 1
- 208000031886 HIV Infections Diseases 0.000 description 1
- 208000037357 HIV infectious disease Diseases 0.000 description 1
- 239000012981 Hank's balanced salt solution Substances 0.000 description 1
- 208000001204 Hashimoto Disease Diseases 0.000 description 1
- 208000030836 Hashimoto thyroiditis Diseases 0.000 description 1
- 206010019909 Hernia Diseases 0.000 description 1
- 201000001916 Hypochondriasis Diseases 0.000 description 1
- 208000022559 Inflammatory bowel disease Diseases 0.000 description 1
- 206010065390 Inflammatory pain Diseases 0.000 description 1
- 208000006877 Insect Bites and Stings Diseases 0.000 description 1
- 208000005615 Interstitial Cystitis Diseases 0.000 description 1
- 208000003456 Juvenile Arthritis Diseases 0.000 description 1
- 208000000913 Kidney Calculi Diseases 0.000 description 1
- 101710128836 Large T antigen Proteins 0.000 description 1
- 206010024229 Leprosy Diseases 0.000 description 1
- 206010072720 Medication overuse headache Diseases 0.000 description 1
- 206010049567 Miller Fisher syndrome Diseases 0.000 description 1
- 208000016285 Movement disease Diseases 0.000 description 1
- 206010049206 Muscle abscess Diseases 0.000 description 1
- 208000029578 Muscle disease Diseases 0.000 description 1
- 208000000112 Myalgia Diseases 0.000 description 1
- 208000031888 Mycoses Diseases 0.000 description 1
- 201000002481 Myositis Diseases 0.000 description 1
- MBBZMMPHUWSWHV-BDVNFPICSA-N N-methylglucamine Chemical compound CNC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO MBBZMMPHUWSWHV-BDVNFPICSA-N 0.000 description 1
- 206010029148 Nephrolithiasis Diseases 0.000 description 1
- 206010033645 Pancreatitis Diseases 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- 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 1
- 208000004983 Phantom Limb Diseases 0.000 description 1
- 206010056238 Phantom pain Diseases 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 208000004550 Postoperative Pain Diseases 0.000 description 1
- 201000001263 Psoriatic Arthritis Diseases 0.000 description 1
- 208000036824 Psoriatic arthropathy Diseases 0.000 description 1
- 208000001647 Renal Insufficiency Diseases 0.000 description 1
- 206010039020 Rhabdomyolysis Diseases 0.000 description 1
- 206010039207 Rocky Mountain Spotted Fever Diseases 0.000 description 1
- 206010040070 Septic Shock Diseases 0.000 description 1
- 208000021386 Sjogren Syndrome Diseases 0.000 description 1
- 206010044248 Toxic shock syndrome Diseases 0.000 description 1
- 231100000650 Toxic shock syndrome Toxicity 0.000 description 1
- 206010044608 Trichiniasis Diseases 0.000 description 1
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 1
- 208000037386 Typhoid Diseases 0.000 description 1
- 208000025865 Ulcer Diseases 0.000 description 1
- 201000006704 Ulcerative Colitis Diseases 0.000 description 1
- 208000001407 Vascular Headaches Diseases 0.000 description 1
- 206010052428 Wound Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 210000003489 abdominal muscle Anatomy 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 101150063416 add gene Proteins 0.000 description 1
- 238000009098 adjuvant therapy Methods 0.000 description 1
- 208000020701 alcoholic polyneuropathy Diseases 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 229940035676 analgesics Drugs 0.000 description 1
- 238000000540 analysis of variance Methods 0.000 description 1
- 239000000730 antalgic agent Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 206010003230 arteritis Diseases 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 206010064097 avian influenza Diseases 0.000 description 1
- 208000025255 bacterial arthritis Diseases 0.000 description 1
- PWHCIQQGOQTFAE-UHFFFAOYSA-L barium chloride dihydrate Chemical compound O.O.[Cl-].[Cl-].[Ba+2] PWHCIQQGOQTFAE-UHFFFAOYSA-L 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 208000002352 blister Diseases 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 230000000747 cardiac effect Effects 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000002490 cerebral effect Effects 0.000 description 1
- 201000001352 cholecystitis Diseases 0.000 description 1
- 208000025302 chronic primary adrenal insufficiency Diseases 0.000 description 1
- 210000000078 claw Anatomy 0.000 description 1
- 208000018912 cluster headache syndrome Diseases 0.000 description 1
- FDJOLVPMNUYSCM-WZHZPDAFSA-L cobalt(3+);[(2r,3s,4r,5s)-5-(5,6-dimethylbenzimidazol-1-yl)-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl] [(2r)-1-[3-[(1r,2r,3r,4z,7s,9z,12s,13s,14z,17s,18s,19r)-2,13,18-tris(2-amino-2-oxoethyl)-7,12,17-tris(3-amino-3-oxopropyl)-3,5,8,8,13,15,18,19-octamethyl-2 Chemical compound [Co+3].N#[C-].N([C@@H]([C@]1(C)[N-]\C([C@H]([C@@]1(CC(N)=O)C)CCC(N)=O)=C(\C)/C1=N/C([C@H]([C@@]1(CC(N)=O)C)CCC(N)=O)=C\C1=N\C([C@H](C1(C)C)CCC(N)=O)=C/1C)[C@@H]2CC(N)=O)=C\1[C@]2(C)CCC(=O)NC[C@@H](C)OP([O-])(=O)O[C@H]1[C@@H](O)[C@@H](N2C3=CC(C)=C(C)C=C3N=C2)O[C@@H]1CO FDJOLVPMNUYSCM-WZHZPDAFSA-L 0.000 description 1
- 206010009887 colitis Diseases 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 230000008602 contraction Effects 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- 238000013480 data collection Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000010511 deprotection reaction Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000000378 dietary effect Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 206010013023 diphtheria Diseases 0.000 description 1
- 239000002270 dispersing agent Substances 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 208000007784 diverticulitis Diseases 0.000 description 1
- 201000006549 dyspepsia Diseases 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000004945 emulsification Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 1
- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 description 1
- 210000003414 extremity Anatomy 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 208000021302 gastroesophageal reflux disease Diseases 0.000 description 1
- 230000030279 gene silencing Effects 0.000 description 1
- 239000003193 general anesthetic agent Substances 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000006481 glucose medium Substances 0.000 description 1
- YPZRWBKMTBYPTK-BJDJZHNGSA-N glutathione disulfide Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@H](C(=O)NCC(O)=O)CSSC[C@@H](C(=O)NCC(O)=O)NC(=O)CC[C@H](N)C(O)=O YPZRWBKMTBYPTK-BJDJZHNGSA-N 0.000 description 1
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 1
- 108010089804 glycyl-threonine Proteins 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 230000003054 hormonal effect Effects 0.000 description 1
- 208000033519 human immunodeficiency virus infectious disease Diseases 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 206010022000 influenza Diseases 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 238000001361 intraarterial administration Methods 0.000 description 1
- 238000000185 intracerebroventricular administration Methods 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 230000000302 ischemic effect Effects 0.000 description 1
- 230000009191 jumping Effects 0.000 description 1
- 206010023332 keratitis Diseases 0.000 description 1
- 201000006370 kidney failure Diseases 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 239000003589 local anesthetic agent Substances 0.000 description 1
- 230000033001 locomotion Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 210000003141 lower extremity Anatomy 0.000 description 1
- 239000007937 lozenge Substances 0.000 description 1
- DHRRIBDTHFBPNG-UHFFFAOYSA-L magnesium dichloride hexahydrate Chemical compound O.O.O.O.O.O.[Mg+2].[Cl-].[Cl-] DHRRIBDTHFBPNG-UHFFFAOYSA-L 0.000 description 1
- 201000004792 malaria Diseases 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 125000001160 methoxycarbonyl group Chemical group [H]C([H])([H])OC(*)=O 0.000 description 1
- 210000004925 microvascular endothelial cell Anatomy 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 208000005871 monkeypox Diseases 0.000 description 1
- 229960005181 morphine Drugs 0.000 description 1
- 201000006417 multiple sclerosis Diseases 0.000 description 1
- 230000004118 muscle contraction Effects 0.000 description 1
- 206010028417 myasthenia gravis Diseases 0.000 description 1
- SYSQUGFVNFXIIT-UHFFFAOYSA-N n-[4-(1,3-benzoxazol-2-yl)phenyl]-4-nitrobenzenesulfonamide Chemical class C1=CC([N+](=O)[O-])=CC=C1S(=O)(=O)NC1=CC=C(C=2OC3=CC=CC=C3N=2)C=C1 SYSQUGFVNFXIIT-UHFFFAOYSA-N 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 201000009240 nasopharyngitis Diseases 0.000 description 1
- 208000021722 neuropathic pain Diseases 0.000 description 1
- 239000000041 non-steroidal anti-inflammatory agent Substances 0.000 description 1
- 229940021182 non-steroidal anti-inflammatory drug Drugs 0.000 description 1
- 210000001331 nose Anatomy 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 229940127240 opiate Drugs 0.000 description 1
- 230000033116 oxidation-reduction process Effects 0.000 description 1
- 238000012858 packaging process Methods 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 208000005987 polymyositis Diseases 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000001020 rhythmical effect Effects 0.000 description 1
- YGSDEFSMJLZEOE-UHFFFAOYSA-M salicylate Chemical compound OC1=CC=CC=C1C([O-])=O YGSDEFSMJLZEOE-UHFFFAOYSA-M 0.000 description 1
- 229960001860 salicylate Drugs 0.000 description 1
- 201000000306 sarcoidosis Diseases 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 201000001223 septic arthritis Diseases 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 210000000278 spinal cord Anatomy 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 229920002994 synthetic fiber Polymers 0.000 description 1
- 208000020408 systemic-onset juvenile idiopathic arthritis Diseases 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- HNKJADCVZUBCPG-UHFFFAOYSA-N thioanisole Chemical compound CSC1=CC=CC=C1 HNKJADCVZUBCPG-UHFFFAOYSA-N 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 231100000167 toxic agent Toxicity 0.000 description 1
- 230000002110 toxicologic effect Effects 0.000 description 1
- 231100000027 toxicology Toxicity 0.000 description 1
- 238000002627 tracheal intubation Methods 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 208000003982 trichinellosis Diseases 0.000 description 1
- 201000007588 trichinosis Diseases 0.000 description 1
- 206010044652 trigeminal neuralgia Diseases 0.000 description 1
- 201000008827 tuberculosis Diseases 0.000 description 1
- 238000007492 two-way ANOVA Methods 0.000 description 1
- 201000008297 typhoid fever Diseases 0.000 description 1
- 231100000397 ulcer Toxicity 0.000 description 1
- 208000009852 uremia Diseases 0.000 description 1
- 208000019206 urinary tract infection Diseases 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 210000002845 virion Anatomy 0.000 description 1
- 102000038650 voltage-gated calcium channel activity Human genes 0.000 description 1
- 108091023044 voltage-gated calcium channel activity Proteins 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 108091058538 ω-conotoxin MVIIA Proteins 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Pain & Pain Management (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biochemistry (AREA)
- Rheumatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明为一种改进型齐考诺肽。本发明以齐考诺肽的C端与细胞膜穿透肽的N端通过两个甘氨酸进行连接,获得的融合肽克服了齐考诺肽不能通过血脑屏障,不能肌内注射,外科手术风险高、感染风险高等不足。本发明的改进型齐考诺肽适用于静脉、腹腔或鼻腔给药方式,操作方便,临床风险小,通过静脉、腹腔或鼻腔施用,其在体内的药效作用时间长,镇痛效果优良,副作用小,适用于大规模的临床应用。本发明的改进型齐考诺肽制备简单,制备工艺和制备过程中质量可控,适用于大规模产业化生产。
Description
技术领域
本发明属于多肽药物技术领域,特别涉及一种齐考诺肽的融合多肽。
背景技术
齐考诺肽(商品名PrialtTM,Elan Pharmaceuticals)是美国食药局(FDA)2004年批准的首例芋螺毒素类药物。其靶向作用位点是N型电压门控钙离子通道,是蛛网膜下腔(鞘内)复合镇痛的一线药物。齐考诺肽是太平洋食鱼螺——鸡心螺——体内毒液肽中亲水性多肽ω—MVIIA的人工合成物,是首个应用于临床的新型非吗啡类镇痛剂,其分子式为C102H172N36O32S7,结构式:
H-Cys-Lys-Gly-Lys-Gly-Ala-Lys-Cys-Ser-Arg-Leu-Met-Tyr-Asp-Cys-Cys-Thr-Gly-Ser-Cys-Arg-Ser-Gly-Lys-Cys-NH2。
齐考诺肽临床可以治疗带状疱疹后遗神经痛,幻肢痛,艾滋病相关神经病理性疼痛,难治性癌痛,手术后疼痛,不能耐受或拒绝其他治疗方法如全身性镇痛药、辅助治疗、缓解鞘内注射阿片类药物无效的疼痛等。与水杨酸盐,NSAIDs和局部麻醉剂主要通过外周神经/伤害感受器,阿片类药物和全身麻醉药主要作用于大脑水平以消除疼痛和意识不同,齐考诺肽的治疗作用机制是通过其结合N型钙通道受体的能力。N型钙离子通道受体位于脊髓后部表面Rexed′sI和II层内的主要伤害性A-δ和C-慢纤维疼痛纤维(伤害感受器)上,可缓解其他治疗手段包括鞘内注射吗啡无效的疼痛,且长时间使用该药物不会产生耐受性和成瘾性,应用指征为治疗与创伤、肿瘤和神经痛等相关的慢性疼痛,尤其在治疗对阿片类药物不敏感的难治性疼痛或对阿片类不能耐受的病人方面有独到的优势。然而,齐考诺肽不能穿过血脑屏障,目前仅使用鞘内插管给药途径输注,插管与输注泵埋藏于皮下,需要手术完成,临床使用不方便。目前仅用于对已有止痛药物抵抗型、慢性疼痛的长期、永久性治疗。这种给药方式极大限制了该药固有优点的临床应用。
血脑屏障(BBB)是存在于脑组织和血液之间的一个复杂细胞系统,能控制血脑两侧的物质转运,从而保证中枢神经织内环境的稳定血液中的有用物质通过微血管内皮细胞膜上各种各样的受体相互作用,并按机体的需要转运进入大脑,从而发挥作用;而一些有毒有害物质则被该屏障屏蔽于脑组织外,以防对大脑产生损害。血脑屏障(BBB)这种特殊的保护作用,导致了大部分药物难以进入大脑,这个中枢神经类疾病的治疗和给药造成困扰。
细胞穿透肽(cell penetrating peptides,CPP)是一类能够通过生物膜进入细胞的短肽(一般少于35个氨基酸残基)。其发现源于1988年,有学者发现HIV-1的反式激活蛋白Tat能跨膜转导至胞内,接着又有人发现果蝇转录蛋白也具有类似特性。此后,其他许多CPP陆续被发现,CPP在相对分子质量、氨基酸组成、氨基酸排列序列上表现出多样性,所含氨基酸数量和种类不同,极性和带电荷量亦不同,但是它们具有一些共同的特点,比如:在较低的浓度条件下,可以穿过细胞膜进入细胞并且不会对膜造成明显破坏和损伤;本身具有穿透膜能力,并且还能介导各种物质包括小分子、核酸、蛋白多肽以及纳米粒子等入胞;高效、低毒。近年来,研究发现,采用细胞穿透肽与药物分子连接,可以达到穿过血脑屏障效果,这给中枢神经给药带来了新的方向。
目前,现有技术中存在利用细胞穿透肽辅助芋螺毒素穿过血脑屏障的技术方法,例如,将齐考诺肽包装于病毒颗粒中,并在病毒颗粒表面连接TAT多肽,该病毒颗粒可以递送齐考诺肽通过血脑屏障,但是,这样的方法制备的工艺复杂,病毒的包装过程,无法进行很好的质量控制,难以在产业上大规模应用。又如,将TAT肽与芋螺毒素的N端通过两个GG作为连接单元,制备获得融合多肽,该融合多肽可以通过血脑屏障,适用于静脉注射给药,但是,N端连接的融合多肽通过静脉注射给药,在体内的镇痛效果和存在时间都无法达到临床应用的要求,无法进行大规模的推广。因此,获得一种能够通过血脑屏障,克服鞘内插管给药的缺点,且能在临床上大规模使用的改进型齐考诺肽是目前急需解决的问题。
发明内容
本发明目的之一是提供一种改进型齐考诺肽。本发明人通过长期研究发现,通过齐考诺肽的C端与细胞膜穿透肽的N端连接,获得的改进型齐考诺肽的融合肽,可以克服现有技术的缺陷,适用于静脉、腹腔或鼻内给药,且在体内的镇痛效果良好和药效时间较长,能够在临床上大规模使用。
具体的,实现上述目的的技术方案如下所示。
一种融合多肽,由齐考诺肽与细胞膜穿透肽组成。优选的,所述融合多肽由齐考诺肽通过C端与细胞膜穿透肽连接组成,或者齐考诺肽的C端通过连接子与细胞膜穿透肽的N端连接,优选的,所述连接子为两个甘氨酸(GG)。
进一步,齐考诺肽的氨基酸为CKGKGAKCSRLMYDCCTGSCRSGKC(如SEQ ID NO.1所示),或者,融合多肽中齐考诺肽也可以是CKGKGAKCSRLMYDCCTGSCRSGKC(如SEQ ID NO.1所示)通过少于10个、少于8个,少于6个,少于4个,2个或1个的氨基酸进行缺失、突变或插入的氨基酸的变体。
进一步,所述细胞膜穿透肽可以是Penetratin、TAT肽、Pep-1肽、S413-PV、Magainin2或Buforin 2。
其中,TAT肽来源HIV-1的反式激活蛋白Tat,其能跨膜转导至胞内。TAT肽的氨基酸为YGRKKRRQRRR(如SEQ ID NO.2所示),或者,融合多肽中TAT肽也可以是YGRKKRRQRRR(如SEQ ID NO.2所示)通过少于10个、少于8个,少于6个,少于4个,2个或1个的氨基酸进行缺失、突变或插入的氨基酸的变体,或其拟肽。
优选的,前述多肽或融合多肽或改进型齐考诺肽的氨基酸序列为CKGKGAKCSRLMYDCCTGSCRSGKCGGYGRKKRRQRRR(如SEQ ID NO.3所示)或者,通过少于10个、少于8个,少于6个,少于4个,2个或1个的氨基酸进行缺失、突变或插入的氨基酸的变体,或其拟肽。
所述拟肽,指具有与由天然氨基酸组成的肽基本上相同的结构和/或功能特征的合成化学化合物。拟肽可以完全地包含氨基酸的合成的非天然类似物,或是部分天然肽氨基酸和部分非天然氨基酸类似物的嵌合分子。拟肽还可以掺入任何数量的天然氨基酸保守替换位点,只要此种替换基本上不改变模拟物的结构和/或抑制活性或结合活性。多肽模拟成分可以含有非天然结构组分的任何组合,所述非天然结构组分一般来自3个结构组:a)非天然酰胺键(“肽键”)连接的残基连接基团;b)代替天然存在的氨基酸残基的非天然残基;或c)诱导二级结构模拟,即诱导或稳定二级结构例如β转角、γ转角、β折叠、α螺旋构象等的残基。
本发明的第二个目的是提供一种药物组合物或制剂,优选为药物制剂,进一步,所述药物组合物或制剂/药物制剂包含本发明的多肽和/或可接受载体。
所述药物组合物或制剂/药物制剂可以包含以下所示的任意剂量的单位剂量形式(即,用于单次施用的剂量)提供药物组合物。可以利用常规的混合、溶解、制粒、制备糖锭剂、磨细、乳化、包囊、包埋或冻干方法制备。可以使用一种或多种便于将活性剂加工为可药用制剂的生理学上可接受的载体、稀释剂、赋形剂或辅料,以常规方式配制所述药物组合物或制剂/药物制剂。适合的制剂取决于所选择的给药途径。
施用方式可以是胃肠外、静脉内、口服、皮下、动脉内、颅内、鞘内、腹膜内、局部、鼻内或肌内施用。优选静脉内施用或腹腔注射。用于肠胃外施用的所述药物组合物或制剂/药物制剂优选是无菌和基本上等渗的。对于注射,可将活性剂配制在水溶液,优选生理上相容的缓冲液例如汉克氏溶液、林格溶液或生理盐水或乙酸盐缓冲液中(以减少注射部位的不适)。该溶液可以含有配制剂,诸如助悬剂、稳定剂和/或分散剂。
本发明的第三个目的是提供一种改进型齐考诺肽的用途。所述用途为:用于制备药物,优选的,用于制备镇痛药物,优选的,所述镇痛药物作用于钙通道。
进一步,所述药物可以用于治疗疼痛、疼痛相关的疾病,例如,可导致慢性疼痛的疾病包括糖尿病、关节炎(例如,骨关节炎、类风湿关节炎和青少年慢性关节炎)、癌症或化疗的毒性作用、纤维肌痛、带状疱疹、肠易激综合征、血管问题或镰状细胞病。
与偶发的一般疼痛相关的疾病包括风湿性多肌痛、臆想病、抑郁症、糖尿病、恶性贫血、镰状细胞病和梅毒。与神经性疼痛相关的疾病包括神经痛(例如,三叉神经痛、不典型面痛以及由带状疱疹或疱疹引起的带状疱疹神经痛)、外周的神经病、Charcot-Marie-Tooth病、弗里德赖希共济失调、糖尿病(例如,糖尿病性神经病变)、饮食缺陷(尤其维生素B-12)、过度的酒精使用(酒精性神经病变)、尿毒症(来自肾衰竭)、癌、艾滋病、肝炎、科罗拉多蜱传热、白喉、格-巴二氏综合征、没有发展为艾滋病的HIV感染、麻风病、莱姆病、多发性结节性动脉炎、类风湿关节炎、结节病、舍格伦综合征、梅毒、系统性红斑狼疮,以及暴露于有毒化合物。
与炎性疼痛相关的疾病包括:(A)关节炎疾病,例如类风湿关节炎;青少年慢性关节炎;系统性红斑狼疮(SLE);痛风性关节炎;硬皮病;骨关节炎;银屑病关节炎;强直性脊柱炎;莱特尔氏综合征(反应性关节炎);成年斯蒂尔病;来自病毒感染的关节炎;来自细菌传染的关节炎,例如,淋病性关节炎和非淋病性细菌性关节炎(脓毒性关节炎);三级莱姆病;结核性关节炎;以及来自真菌感染的关节炎,诸如酵母病;(B)自身免疫疾病,例如格-巴二氏综合征、桥本甲状腺炎、恶性贫血、艾迪生病、I型糖尿病、系统性红斑狼疮、皮肌炎、舍格伦综合征、红斑狼疮、多发性硬化、重症肌无力、莱特尔氏综合征和格雷夫斯病。(C)结缔组织病,例如脊椎关节炎、皮肌炎和纤维肌痛;(D)损伤引起的炎症;(E)感染,例如结核病或间质性角膜炎;以及(G)关节炎症,例如滑囊炎或肌腱炎。头疼的类型包括肌肉的/肌源性头痛、血管性头痛、牵引性或炎症性头痛、丛集性头痛、激素性头痛、反跳性头痛或慢性鼻窦炎头痛。
躯体疼痛可与以下相关:过度的肌肉收缩、反复运动疾病、诸如多肌炎的肌肉疾病、皮肌炎、狼疮、纤维肌痛、风湿性多肌痛,以及横纹肌溶解、肌痛、感染诸如肌肉脓肿、旋毛虫病、流行性感冒、莱姆病、疟疾、落矶山斑疹热、禽流感、普通感冒、社会获得性肺炎、脑膜炎、猴痘、严重急性呼吸综合征、中毒性休克综合征、旋毛虫病、伤寒,以及上呼吸道感染。内脏痛可与诸如以下的疾病相关:肠易激综合征、慢性功能性腹痛(CFAP)、功能性便秘、功能性消化不良、非心脏胸痛(NCCP)和慢性腹痛、慢性胃肠炎,例如胃炎、炎性肠病,例如克罗恩病、溃疡性结肠炎、微观结肠炎、憩室炎和肠胃炎;间质性膀胱炎;肠局部缺血;胆囊炎;阑尾炎;胃食管反流;溃疡、肾结石、尿路感染、胰腺炎和疝。
本发明的第四个目的是提供一种改进型齐考诺肽的制备方法,优选的,可以采用化学合成的方式制备改进型齐考诺肽。进一步优选的,改进型齐考诺肽采用固相合成法或重组表达法进行制备,进一步,采用F-moc全自动固相合成法制备本发明的多肽。
与现有技术相比,本发明有益效果为:通过将齐考诺肽的C端与细胞膜穿透肽连接获得一种改进型齐考诺肽,克服了齐考诺肽不能通过血脑屏障,不能肌内注射,主要通过脑室和椎管给药所带来的外科手术风险高、感染风险高等不足。本发明的改进型齐考诺肽能够通过血脑屏障,适用于静脉、腹腔和鼻腔给药方式,操作方便,临床风险小,通过静脉、腹腔或鼻腔施用,其在体内的药效作用时间长,镇痛效果优良,而且本发明的改进型齐考诺肽副作用小,适用于大规模的临床应用。另外,本发明的改进型齐考诺肽制备简单,制备工艺和制备过程中质量可控,适用于大规模产业化生产。
附图说明
图1:MVIIA和MVIIA-a,b,c,d一步氧化折叠HPLC分析图谱;
图2:MVIIA和MVIIA-a,b,c,d的圆二色谱图,多肽的终浓度是35μmol/L,分别溶解在磷酸盐缓冲液(10mM,pH=7.2);
图3:MVIIA和MVIIA-a,b,c,d对CaV2.2通道电流的抑制作用,MVIIA的剂量效应曲线如图3A所示,MVIIA变体的剂量效应曲线如图3B-3E所示。半抑制浓度和坡度值的数据均显示在图上,数据以平均值±标准误表示,每组5只小鼠。如F图所示,在10μM L-MVIIA(蓝色)和2μM MVIIA(红色)时,由从-80mv到10mv的电压步长所激发的全细胞钙通道电流痕迹的叠加;G图是MVIIA及其变体的半抑制浓度的汇总表格;
图4:MVIIA和MVIIA-c热板疼痛比较结果,侧脑室给药MVIIA(图4A),尾静脉给药MVIIA(图4B)和MVIIA-c(图4C)后的体内镇痛效果。镇痛效果以反应潜伏时间表示。数据以平均值±标准误表示,每组6-8只小鼠。*p<0.05,**p<0.01和***p<0.001表示与生理盐水组进行比较(数据分析采用重复多因素方差分析和邓肯多重范围检验法);
图5:MVIIA-a,b,d热板疼痛实验结果,图5A-图5C为尾静脉给MVIIA-a,b,d多肽后的体内镇痛效果。镇痛效果以百分比表示最大可能的影响(%MPE)。数据以平均值±标准误表示,每组8-10只小鼠。*p<0.05,**p<0.01和***p<0.001表示与生理盐水组进行比较;
图6:醋酸扭体实验中MVIIA和MVIIA-a,b,c,d的镇痛效果,腹腔注射1%醋酸后的5到20分钟内,记录小鼠的翻滚次数;如图A所示,侧脑室给药30分钟后,腹腔注射1%的醋酸的作用比较;如图B所示,尾静脉给药30分钟后,腹腔注射1%的醋酸的作用比较;#,与生理盐水组(saline)组比较;*,与MVIIA组比较;&,与MVIIA-C组比较;*,#,&,p<0.05;***,###,&&&,p<0.001。数据以平均值±标准误表示,每组9-11只小鼠;
图7:MVIIA和MVIIA-a,b,c,d对小鼠震颤时间的影响,侧脑室给予6μL的多肽(0.9nmol/kg)和生理盐水。给药后30分钟和120分钟,记录5分钟内小鼠的累计震颤时间。数据以平均值±标准误(n=12)表示;
图8:MVIIA的质谱图;
图9:MVIIA-a的质谱图;
图10:MVIIA-b的质谱图;
图11:MVIIA-c的质谱图;
图12:MVIIA-d的质谱图;
图13:MVIIA和不同剂量MVIIA-c鼻腔给药后的镇痛能力;
图14:MVIIA-a,b,d鼻腔给药时在热板疼痛实验中的镇痛能力。
具体实施方式
为了克服现有技术中齐考诺肽的不足,本发明人通过长期研究发现,通过齐考诺肽的C端与细胞膜穿透肽的N端连接,获得的改进型齐考诺肽的融合肽,适用于静脉或腹腔给药。为了进一步研究不同类型的改进型齐考诺肽的镇痛效果,本发明设计合成了多种不同类型和结构的融合多肽,包括,不使用连接子,通过齐考诺肽的C端与细胞膜穿透肽的N端直接连接的融合多肽;使用一个或多个甘氨酸作为连接子构建的融合多肽。进一步,对上述不同类型的融合多肽进行结构表征,细胞实验、体内实验以及副作用验证实验,用以说明不同类型的的改进型齐考诺肽的效果
为了更好的理解本发明的技术方案,下面结合实施例进行详细说明。
实施例1不同类型改进型齐考诺肽的制备
制备4种不同类型的改进型齐考诺肽,分别命名为受保护的多肽MVIIA-a、MVIIA-b、MVIIA-c、MVIIA-d。同时制备齐考诺肽,命名为MVIIA,作为对照。本实验是采用F-moc全自动固相合成法,具体步骤如下:
多肽的合成:使用433A自动合成器(ABI,Foster City,CA)的模型在树脂上组装受保护的多肽及其衍生物。室温下,肽树脂在悬浮液中孵育2.5小时,脱保护基。悬浮液体系是由10毫升TFA、0.75克苯酚、0.25毫升的1,2-乙二硫醇、0.5毫升的苯甲硫醚和0.5毫升的水组成的。(笏甲氧羰基(Fmoc),一种常见的烷氧羰基类氨基保护基)。通过过滤,从多肽去保护基混合物中分离树脂。粗多肽在150ml预冷的乙醚溶液中沉淀,并以10%冰醋酸作为洗脱剂在葡聚糖凝胶G-25柱中进行层析纯化。随后,含有多肽的组分被汇集并冻干,并使用高效液相色谱法测得粗多肽的纯度为80%左右。
多肽折叠:MVIIA包含六个半胱氨酸残基,维持其三个二硫键结构,在氧化条件下的折叠可产生多种异构体。在经过氧化还原系统、缓冲液、盐、浓度和温度的筛选以后,选取了两个MVIIA的高效折叠条件:(a)0.5M NH4Ac缓冲液(pH 7.9),其中包含1mM GSH,0.1mMGSSG,1mM EDTA,和0.2mg/mL MVIIA;(b)0.5M NH4Ac缓冲液,其中包含1mM cysteine,1mMEDTA,和0.2mg/mL MVIIA。4℃时,直线型多肽MVIIA在a条件下48-72小时和在b条件24-48h下被折叠。
多肽纯化和表征:MVIIA被氧化后,先用醋酸对反应混合物进行酸化(pH<4.5)处理,随后过滤。滤液直接上样至Zorbax 21.2×250mm的C18液相色谱柱,其中使用的是制备高效液相色谱泵(Waters 2000series,Milford,MA)。C18柱先使用缓冲液A(0.1%TFA的水溶液)预清洗柱,随后采用10-40%缓冲液B(0.1%TFA的乙氰溶液),以8mL/min的速度进行40分钟线性梯度洗脱。得到的馏分是含90%的MVIIA的浓缩液,随后我们采用装上9.4×250mm Zorbax C18液相色谱柱的半制备的反相高效液相色谱进行进一步纯化。最后,我们在葡聚糖凝胶G-25层析柱中以20%乙酸溶液为洗脱液,将终产物从TFA盐溶液中转换至乙酸盐溶液中。多肽的纯度是由分析型的反相高效液相色谱进行评估的,评估时是在1ml每分钟的流速下,使用Zorbax C18液相色谱柱(4.6×250mm),以8-40%缓冲液B(0.1%TFA的乙氰溶液)进行25分钟线性梯度洗脱。最终,我们得到的终产物-多肽的纯度为98%。
实施例2:不同类型改进型齐考诺肽化学特性和结构表征
1.MVIIA及其变体的化学特性
4℃时,缓冲液处理线性多肽24-48小时,随后使用高效液相色谱进行分析,发现线性多肽的折叠导致了一个主要的高峰和几个小峰的出现。缓冲液体系包括1mM谷胱甘肽,0.1mM氧化型谷胱甘肽,1mM EDTA,和0.2mg/mL线性多肽,溶液的pH为7.9。主要的产物经过提纯,并通过分析反相高效液相色谱进行评估,同时多肽的纯度被确定为大于98%。用Ultraflex III TOF/TOF质谱仪进行确定(Bruker)。制备获得的多肽序列如表1所示,其一步氧化折叠HPLC分析图谱如图1所示。
表1:制备获得的多肽序列
2.圆二色谱
多肽在PBS(10mM,pH=7.2)溶液中溶解,终浓度为35μM。室温下,检测190nm到260nm波长范围内的圆二色谱,使用的是Chirascan Plus spectropolarimeter(AppliedPhotophysics Ltd.,Leatherhead,Surrey,UK)仪器。设置的检测指数如下所示:stepresolution 1.0nm;speed 20nm/min,和cell path length of 1.0mm。
如图2所示,MVIIA在195nm-205nm处呈现了明显的β折叠结构。我们发现TAT变体有相似的随机线圈结构,并在200nm左右,有明显的减弱波段出现。这些结果表明,MVIIA和TAT间的连接序列的长度扩大时,多肽的二级结构并没有发生改变。当连接序列扩增时,TAT变体的摩尔椭圆度随之加深,表明MVIIA和TAT间连接序列的扩增有助于形成随机线圈结构。采用质谱(采用Voyager MALDI-TOF光谱仪)的方法鉴定产物多肽准确的分子量,如表2所示,MVIIA和MVIIA-a,b,c,d的质谱图如图8-图12所示。二硫键的桥接模式是由于部分减少半胱氨酸耦合和氨基酸沉默的方法进行分配的。合成的多肽与MVIIA标准品的高效液相色谱图和圆二色谱图结果是一致的。
表2.MVIIA及其变体的分子量
样品 | 理论MW | 实测m/z | 理论值与实测的差值 |
MⅦA | 2645.54 | 2639.0198 | 6.5202 |
MⅦA-a | 4186.0784 | 4180.0108 | 6.0676 |
MⅦA-b | 4243.0978 | 4237.0300 | 6.0678 |
MⅦA-c | 4299.1353 | 4292.0362 | 7.0991 |
MⅦA-d | 4356.1568 | 4351.0842 | 5.0726 |
实施例3:不同类型改进型齐考诺肽的电生理学实验
为了进一步研究不同类型改进型齐考诺肽电生理学效果和对钙离子(CaV2.2)通道的抑制作用,进行如下实验:
HEK293T细胞(能表达SV40大T抗原)培养在含10%胎牛血清,1%的青霉素、链霉素的DMEM高糖培养基(Gibco)中。培养箱环境为37℃,5%CO2。Dr.Diane Lipscombe提供了大鼠CaV2.2通道的α1B拼接变体e37a,辅助亚单位α2δ1和β3质粒(Addgene plasmid#26569,#26575,#26574)。随后三种质粒(3μg)、0.4μg增强型绿色荧光蛋白基因和脂质体一起瞬转染至HEK293T细胞中。转染24小时后,细胞接种在玻璃载玻片上,在培养箱(37℃,5%CO2)培养至少6小时,随后进行电生理记录。
本研究是按照之前已发表的研究文献中细胞电压钳位记录的方法进行记录的(F.Wang et al.,2016)。简单来说,记录电极有大约3MΩ的电阻,被内部溶液所充满。内部溶液包含135mM CsCl,10mM NaCl,10mM HEPES,和5mM EGTA,并用CsOH调节溶液pH为7.2。细胞外记录溶液包含135mM N-Methyl-D-glucamine,10mM BaCl2.2H2O,2mM MgCl2.6H2O和10mM HEPES,溶液最终pH为7.4。室温下(~22℃),采用MultiClamp 700B放大器(MolecularDevices,Sunnyvale,CA)和Clampex 10.3/Digidata1440A数据采集系统和数模转换器记录采集的电流。膜电流在2kHz过滤后,在10kHz取样。所有的数据采用数据分析系统clampfit10.3进行分析(Molecular Devices),且以平均值±标准误表示。阻断N型Ca离子电流的毒素的剂量-效应曲线用GraphPad Prism (GraphPad Software,San Diego,CA)软件绘制的,将电流幅度抑制曲线作为药物浓度的函数,使用希尔方程拟合。
MVIIA及其变体MVIIA-a,b,c,d的主要氨基酸序列及其电生理活性如表3所示。
表3.MVIIA及其变体的主要氨基酸序列及其电生理活性
MVIIA及其变体对钙离子(CaV2.2)通道的抑制作用
众所周知,MVIIA是一种选择性CaV2.2通道阻滞剂。浓度为2μM MVIIA能90%以上的阻断CaV2.2通路。(F.Wang.2016,and other articles)在本研究中,我们在293T细胞中记录了CaV2.2通道(α1B,α2δ1andβ3)的Ca2+峰值电流(ICa)。所有的电流被100ms从-80mv到10mv的电压步长所激发。1μM浓度的MVIIA、MVIIA-a、MVIIA-b、MVIIA-c和MVIIA-d处理可以降低Ca2+峰值电流,其降低值分别为98.24±0.708%,89.45±0.752%,91.70±1.477%,98.81±0.427%和84.26±3.127%。我们发现,MVIIA-c与MVIIA有相似的阻断CaV2.2通道的能力。L-MVIIA阻断CaV2.2通道的能力显著降低,且在10μM的浓度下仅能降低23.28±3.347%的Ca2+峰值电流。MVIIA的浓度和抑制CaV2.2通道的响应关系的半抑制浓度是0.0436μM,与TAT变体相比,几乎大了5-10倍。TAT变体(MVIIA-a,MVIIA-b,MVIIA-c andMVIIA-d)的半抑制浓度分别为0.413,0.379,0.237和0.345μM,如图3所示。这些结果表明,MVIIA-a,MVIIA-b,MVIIA-c和MVIIA-d对CaV2.2通道的具有一定的抑制效果,且MVIIA和TAT变体间连接序列的长度能够影响CaV2.2通道的结合能力。
实施例4:不同类型改进型齐考诺肽的体内镇痛效果实验
1.热板疼痛实验
1.1实验方法
本实验中,一共九组小鼠,每组小鼠6-8只,分别采用侧脑室给药MVIIA(0.11,0.33or 1.00nmol/kg),尾静脉给药MVIIA和MVIIA-a,MVIIA-b,MVIIA-c和MVIIA-d(0.33,1.00or 3.00μmol/kg)。两种途径给药时,生理盐水组均作为空白对照组。动物被放在一个温度恒定为55±0.5℃的电烫斗上,延迟时间是记录从将小鼠放置在电烫斗的表面到第一次舔后腿的爪子或者第一次跳起来的时间作为疼痛指数的阈值(Eddy and Leimbach,1953)。以60s的时间为界限,若超过60s则将小鼠取出,避免小鼠组织受到损伤。在给药前,延迟时间被提前测量作为基线值;随后,记录给药MVIIA、MVIIA-c和Saline(侧脑室给药或者尾静脉给药)后的0.5、1、2、3、4、6、8、10和12h时的延迟时间。与延迟基线时间相比,延迟时间少于5s或者多于20s的小鼠均被认为是不敏感的和超敏感的小鼠,随后被剔除。镇痛效果以潜伏期时间表示。
1.2镇痛能力比较
如图4所示,侧脑室给药MVIIA(0.11,0.33和1.00nmol/kg)1小时后,MVIIA的药效达到最高值;到4小时时,MVIIA的药效基本消失(图4A)。然而,尾静脉注射时,多剂量的MVIIA均未产生药效(图4B)。MVIIA-c是MVIIA的TAT变体中有最强抑制CaV2.2通道电流作用的变体。如图4C所示,MVIIA-c在给药3小时时展现出最强的药效,且其的最强药效持续4小时左右,药效持续时间为12小时。
如图5所示,尾静脉注射不同剂量的MVIIA-a,b,d(0.11umol/kg,0.33umol/kg和1.00μmol/kg)1小时后,均呈现出镇痛效果,并在给药2-3小时时展现出最强的药效,药效持续4小时左右,随时间逐步降低,在给药12小时后给药组与盐水组仍有显著差别,药效持续时间为12小时。
2.醋酸扭体实验(Koster et al.,1959)
2.1实验方法
三种剂量的MVIIA-a,b,c,d多肽组(0.6,1.8和5.4nmol/kg,图中低,中,高剂量),盐水对照组(saline),三种剂量的阳性参考药物组MVIIA(0.11,0.33和1.00nmol/kg,图中低,中,高剂量)处理动物。扭体实验时,在腹腔注射1%醋酸前30分钟分别给药MVIIA(侧脑室)or MVIIA-a,b,c,d(侧脑室),随后测量MVIIA和MVIIA-MVIIA-a,b,c,d的体内镇痛效果。为了检测MVIIA and MVIIA-a,b,c,d通过血脑屏障的能力,在腹腔注射1%醋酸前3小时,通过尾静脉给药的方式分别给药MVIIA和MVIIA-a,b,c,d。生理盐水组均作为空白对照组(侧脑室给药或者尾静脉给药)。记录醋酸注射后5分钟到20分钟内小鼠的翻滚次数(Galeottiet al.,2008)。扭转运动的次数是以腹部肌肉的收缩并伴随着后肢的拉伸和身体的伸长为特点进行记录的。
2.2镇痛能力比较
在醋酸扭体实验中,三种剂量的MVIIA-a,b,c,d多肽组(0.6,1.8和5.4nmol/kg,图6中低,中,高剂量),盐水对照组(saline),三种剂量的阳性参考药物组MVIIA(0.11,0.33和1.00nmol/kg,图6中低,中,高剂量)处理动物,对比各组在三种不同剂量,静脉给药和侧脑室给药条件下的翻滚次数。发现MVIIA-a,b,c,d多肽组与阳性参考药物组MVIIA均减少醋酸诱导的翻滚次数,且呈剂量依赖性。在侧脑室给药条件下,MVIIA,MVIIA-a,b,c,d分别使得小鼠的翻滚次数降低为(相对于盐水组):MVIIA8.97%,53.37%,76.88%;MVIIA-A,2.94%,13.36%,48.35%;MVIIA-B,10.82%,42.79%,77.60%;MVIIA-C,14.75%,39.53%,81.77%;MVIIA-D,12.08%,23.95%,56.54%。在静脉给药条件下,阳性参考药物MVIIA无降低小鼠的翻滚次数的作用,MVIIA-a,b,c,d分别使小鼠的翻滚次数降低为(相对于盐水组):MVIIA-a,10.47%,27.82%,30.03%;MVIIA-b,17.08%,45.94%,51.79%;MVIIA-c,19.81%,49.30%,62.95%;MVIIA-d,6.33%,35.86%,47.57%,如图6所示。
结论:从上述的实验结果可以看出,相比于MVIIA,MVIIA-a,b,c,d多肽能在静脉注射的情形,起到镇痛的效果且呈剂量依赖性,特别是,在中,高剂量的情况下,MVIIA-a,b,c,d多肽通过静脉注射,能够达到良好的镇痛效果,满足临床使用需求。进一步,相比于MVIIA,通过静脉注射给药MVIIA-a,b,c,d药效长达12小时,在体内有良好的缓释效果。
前述镇痛实验采用单因素方差分析(one-way ANOVA),重复测量的多因素方差分析(two-way ANOVA with repeated measures),组间用邓肯或纽曼一柯尔测验法的方法进行分析。所有的数据均用平均值±标准差或标准误或95%的置信区间。当p值的差异小于0.05时,认为数据具有统计学意义。
实施例5:不同类型改进型齐考诺肽的副作用实验
为了进一步研究不同类型改进型齐考诺肽在体内的副作用,进行如下实验:
1.实验方法
震颤时间被认为是齐考诺肽一类典型的副作用。震颤时间是记录一段时间内小鼠四肢、头部和躯干有节奏的震动的总时间。小鼠随机分组:MVIIA(0.9nmol/kg)组,MVIIA-a,b,c,d(0.9nmol/kg)组和正常对照组(6μL,侧脑室给药;n=12,雌雄各半)。给药30分钟和120分钟后,用数码相机录制5分钟内小鼠的动态视频,并由一位对实验不了解的人统计每只小鼠5分钟内的累计震颤时间。
毒理学实验采用单因素方差分析(one-way ANOVA)和纽曼一柯尔测验法的方法进行分析。所有的数据均用平均值±标准差或标准误或95%的置信区间。当p值的差异小于0.05时,认为数据具有统计学意义。
2.1副作用比较
如图7所示,给药30分钟时,MVIIA造成了更明显的震颤症状和更长的震颤时间;给药120分钟时,各组多肽与MVIIA相比,造成的震颤症状和更长的震颤时间没有明显差异。从上述结果中可以看出,MVIIA于MVIIA-a,b,c,d多肽在副作用上无明显差异,甚至于,在给药开始阶段,MVIIA-a,b,c,d的副作用还要低于MVIIA,可见,本申请的MVIIA-a,b,c,d多肽毒副作用较小。
实施例6:MVII-A脑室给药与MVIIA-a,b,c,d滴鼻镇痛实验比较
1.1热板疼痛实验方法
热板疼痛实验方法如前所述。小鼠本实验中,一共九组小鼠,每组小鼠10只,脑室内给予MVIIA(1.00nmol/kg,5ul/10g)作为阳性对照组(实验中发现MVIIA鼻内给药无效),鼻腔分别给予生理盐水(saline,2ul/10g),MVIIA-C(3.3,6.6or 9.9nmol/kg,2ul/10g)。生理盐水组作为空白对照组。记录脑室内给药MVIIA、鼻腔给药MVIIA-c和Saline后的0.5、1、2、3、4、6、8、10h时的延迟时间。与延迟基线时间相比,延迟时间少于5s或者多于20s的小鼠均被认为是不敏感的和超敏感的小鼠,随后被踢除。
镇痛效果是以百分比表示最大可能的影响(%MPE),最后使用下面的方程进行计算:%MPE=(T1-T0)×100/(T2-T0)
其中,T0和T1分别表示给药前后的延迟时间,T2是每次测试的界限时间。
1.2实验结果
MVIIA和不同剂量MVIIA-c鼻腔给药后的镇痛能力如图13所示。图13展示了MVIIA脑室和MVIIA-c鼻腔给药时在热板疼痛实验中的镇痛效果。脑室给药MVIIA(1.00nmol/kg)后,药效持续4小时。MVIIA-C(3.3,6.6,9.9nmol/kg)鼻腔给药后迅速起效,高剂量的MVIIA-C药效持续时间长,在8小时仍与生理盐水组有显著差异,在给药10小时后药效消失。*p<0.05,**p<0.01和***p<0.001表示与生理盐水组进行比较。
1.3MVIIA-a,b,d滴鼻镇痛实验
如图14所示,MVIIA-a,b,d鼻腔给药时在热板疼痛实验中的镇痛效果。与MVIIA-C类似,MVIIA-a,b,d(9.9nmol/kg)鼻腔给药后迅速起效,MVII-b在8小时仍与生理盐水组有显著差异,在给药10小时后药效消失。*p<0.05,***p<0.001表示与生理盐水组进行比较。
上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
序列表
<110> 北京大学深圳研究生院
<120> 改进型齐考诺肽
<141> 2018-09-11
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 25
<212> PRT
<213> Artificial Sequence
<400> 1
Cys Leu Gly Leu Gly Ala Leu Cys Ser Ala Leu Met Thr Ala Cys Cys
1 5 10 15
Thr Gly Ser Cys Ala Ser Gly Leu Cys
20 25
<210> 2
<211> 11
<212> PRT
<213> Artificial Sequence
<400> 2
Thr Gly Ala Leu Leu Ala Ala Gly Ala Ala Ala
1 5 10
<210> 3
<211> 38
<212> PRT
<213> Artificial Sequence
<400> 3
Cys Leu Gly Leu Gly Ala Leu Cys Ser Ala Leu Met Thr Ala Cys Cys
1 5 10 15
Thr Gly Ser Cys Ala Ser Gly Leu Cys Gly Gly Thr Gly Ala Leu Leu
20 25 30
Ala Ala Gly Ala Ala Ala
35
<210> 4
<211> 36
<212> PRT
<213> Artificial Sequence
<400> 4
Cys Leu Gly Leu Gly Ala Leu Cys Ser Ala Leu Met Thr Ala Cys Cys
1 5 10 15
Thr Gly Ser Cys Ala Ser Gly Leu Cys Thr Gly Ala Leu Leu Ala Ala
20 25 30
Gly Ala Ala Ala
35
<210> 5
<211> 37
<212> PRT
<213> Artificial Sequence
<400> 5
Cys Leu Gly Leu Gly Ala Leu Cys Ser Ala Leu Met Thr Ala Cys Cys
1 5 10 15
Thr Gly Ser Cys Ala Ser Gly Leu Cys Gly Thr Gly Ala Leu Leu Ala
20 25 30
Ala Gly Ala Ala Ala
35
<210> 6
<211> 39
<212> PRT
<213> Artificial Sequence
<400> 6
Cys Leu Gly Leu Gly Ala Leu Cys Ser Ala Leu Met Thr Ala Cys Cys
1 5 10 15
Thr Gly Ser Cys Ala Ser Gly Leu Cys Gly Gly Gly Thr Gly Ala Leu
20 25 30
Leu Ala Ala Gly Ala Ala Ala
35
Claims (10)
1.一种多肽,其特征在于,所述多肽包含齐考诺肽与细胞膜穿透肽。
2.权利要求1所述的多肽,其特征在于,所述多肽由齐考诺肽通过C端与细胞膜穿透肽连接组成;优选的,所述齐考诺肽的C端通过连接子与细胞膜穿透肽的N端连接。
3.权利要求2任意一项所述的多肽,其特征在于,所述连接子为两个甘氨酸。
4.权利要求1-3任意一项所述的多肽,其特征在于,所述齐考诺肽的氨基酸如SEQ IDNO.1所示,或所述齐考诺肽为SEQ ID NO.1所示氨基酸少于10个的缺失、突变或插入的氨基酸的变体。
5.权利要求1-3任意一项所述多肽,其特征在于,所述细胞膜穿透肽选自:Penetratin、TAT肽、Pep-1肽、S413-PV、Magainin 2或Buforin 2;优选的,所述TAT肽的氨基酸如SEQ IDNO.2所示,或所述TAT肽为SEQ ID NO.2所示氨基酸少于10个的缺失、突变或插入的氨基酸的变体,或其拟肽。
6.权利要求1-3任意一项所述的多肽,其特征在,所述多肽的氨基酸序列如SEQ IDNO.3所示。
7.一种药物组合物,其特征在于,所述药物组合物包含权利要求1-6的多肽和可接受载体。
8.权利要求7所述的药物组合物,其特征在于,所述药物组合物用于静脉、腹腔或鼻腔给药,药物组合物剂型为静脉、腹腔或鼻腔给药剂型。
9.一种制剂,其特征在于,所述制剂包含权利要求1-6的多肽,优选的,制剂为静脉、腹腔或鼻腔给药制剂。
10.一种权利要求1-6所述多肽的制备方法,其特征在于,所述多肽采用合成的方式制备而成。
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201811086050.0A CN109232744B (zh) | 2018-09-18 | 2018-09-18 | 改进型齐考诺肽 |
PCT/CN2018/124256 WO2020056989A1 (zh) | 2018-09-18 | 2018-12-27 | 改进型齐考诺肽 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201811086050.0A CN109232744B (zh) | 2018-09-18 | 2018-09-18 | 改进型齐考诺肽 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN109232744A true CN109232744A (zh) | 2019-01-18 |
CN109232744B CN109232744B (zh) | 2020-02-28 |
Family
ID=65059662
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201811086050.0A Active CN109232744B (zh) | 2018-09-18 | 2018-09-18 | 改进型齐考诺肽 |
Country Status (2)
Country | Link |
---|---|
CN (1) | CN109232744B (zh) |
WO (1) | WO2020056989A1 (zh) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2020056987A1 (zh) * | 2018-09-18 | 2020-03-26 | 深圳瑞健生物科技有限公司 | 一种可通过血脑屏障的多肽 |
WO2020056985A1 (zh) * | 2018-09-18 | 2020-03-26 | 深圳瑞健生物科技有限公司 | 一种齐考诺肽和tat肽的融合多肽 |
WO2020056991A1 (zh) * | 2018-09-18 | 2020-03-26 | 深圳瑞健生物科技有限公司 | 一种适用于静脉、腹腔或鼻腔给药的多肽 |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20020193285A1 (en) * | 2001-03-02 | 2002-12-19 | Hesson David P. | Neuroprotectants formulations and methods |
US11053295B2 (en) * | 2015-08-21 | 2021-07-06 | Iucf-Hyu (Industry-University Cooperation Foundation Hanyang University) | Peptides having effects of preventing or treating central nervous system diseases and pharmaceutical compositions for preventing or treating central nervous system diseases containing same as active ingredient |
CN108992661B (zh) * | 2018-09-18 | 2019-09-20 | 北京大学深圳研究生院 | 一种多肽在制备静脉、腹腔或鼻腔给药药物中的应用 |
-
2018
- 2018-09-18 CN CN201811086050.0A patent/CN109232744B/zh active Active
- 2018-12-27 WO PCT/CN2018/124256 patent/WO2020056989A1/zh active Application Filing
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2020056987A1 (zh) * | 2018-09-18 | 2020-03-26 | 深圳瑞健生物科技有限公司 | 一种可通过血脑屏障的多肽 |
WO2020056985A1 (zh) * | 2018-09-18 | 2020-03-26 | 深圳瑞健生物科技有限公司 | 一种齐考诺肽和tat肽的融合多肽 |
WO2020056991A1 (zh) * | 2018-09-18 | 2020-03-26 | 深圳瑞健生物科技有限公司 | 一种适用于静脉、腹腔或鼻腔给药的多肽 |
Also Published As
Publication number | Publication date |
---|---|
CN109232744B (zh) | 2020-02-28 |
WO2020056989A1 (zh) | 2020-03-26 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN108992661B (zh) | 一种多肽在制备静脉、腹腔或鼻腔给药药物中的应用 | |
CN109106942B (zh) | 一种可通过血脑屏障的多肽在制备药物中的应用 | |
CN109265558A (zh) | 改进型齐考诺肽在制备药物中的应用 | |
CN109265557A (zh) | 一种齐考诺肽和tat肽的融合多肽在制备药物中的应用 | |
RU2676713C2 (ru) | Терапевтические пептиды | |
BR112016004889B1 (pt) | Derivados de 1,2,4-oxadiazol como imunomoduladores | |
CN109232744A (zh) | 改进型齐考诺肽 | |
JP7252674B2 (ja) | 新規なオリゴペプチド及びこれを有効成分として含む癌の予防又は治療用薬学的組成物 | |
CN109265556A (zh) | 一种适用于静脉、腹腔或鼻腔给药的多肽 | |
CN109232745A (zh) | 一种可通过血脑屏障的多肽 | |
CN109232746A (zh) | 一种齐考诺肽和tat肽的融合多肽 | |
JPH07505371A (ja) | 免疫調節活性を有する新規親油性オリゴペプチド | |
CN107592865B (zh) | 中和流感病毒的拟肽化合物 | |
CN107459557B (zh) | 左旋维c-2-氧乙酰-grpak,其合成,活性和应用 | |
WO2024037263A1 (zh) | 一种体内低毒性的抑制金葡菌毒素产生的合成肽及其应用 | |
LV10109B (en) | New oligopeptides selectively inhibiting haemopoietic stem cells, pharmaceutical composition on their base and method for preparing thereof | |
CN118103387A (zh) | 肽 | |
EA039991B1 (ru) | Соединения-пептидомиметики, нейтрализующие вирус гриппа | |
JPWO2014148489A1 (ja) | 環状ペプチド | |
JPH05331195A (ja) | 新規なペプチド |
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 | ||
TA01 | Transfer of patent application right | ||
TA01 | Transfer of patent application right |
Effective date of registration: 20191017 Address after: 518000 Guangdong city of Shenzhen province Qianhai Shenzhen Hong Kong cooperation zone before Bay Road No. 1 building 201 room A (located in Shenzhen Qianhai business secretary Co. Ltd.) Applicant after: Shenzhen Ruijian Biotechnology Co., Ltd. Address before: 518000 no.2199 Lishui Road, Beida Park, Xili University City, Nanshan District, Shenzhen City, Guangdong Province Applicant before: Shenzhen Graduate School of Peking University |
|
GR01 | Patent grant | ||
GR01 | Patent grant |