CN117659122A - Carbon monoxide release molecule-transmembrane peptide complex and preparation and application thereof - Google Patents
Carbon monoxide release molecule-transmembrane peptide complex and preparation and application thereof Download PDFInfo
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- CN117659122A CN117659122A CN202311191001.4A CN202311191001A CN117659122A CN 117659122 A CN117659122 A CN 117659122A CN 202311191001 A CN202311191001 A CN 202311191001A CN 117659122 A CN117659122 A CN 117659122A
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- corm
- corm401
- resin
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 25
- UGFAIRIUMAVXCW-UHFFFAOYSA-N Carbon monoxide Chemical compound [O+]#[C-] UGFAIRIUMAVXCW-UHFFFAOYSA-N 0.000 title claims abstract description 17
- 229910002091 carbon monoxide Inorganic materials 0.000 title claims abstract description 17
- 238000002360 preparation method Methods 0.000 title claims description 6
- 238000010668 complexation reaction Methods 0.000 title description 2
- 239000003814 drug Substances 0.000 claims abstract description 16
- 208000002177 Cataract Diseases 0.000 claims abstract description 10
- 108091010883 CORM-401 Proteins 0.000 claims abstract description 9
- 238000011282 treatment Methods 0.000 claims abstract description 8
- 238000000034 method Methods 0.000 claims abstract description 6
- 108010011110 polyarginine Proteins 0.000 claims abstract description 6
- 150000001413 amino acids Chemical class 0.000 claims description 7
- 238000006243 chemical reaction Methods 0.000 claims description 6
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 230000015572 biosynthetic process Effects 0.000 claims description 3
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 3
- 238000004949 mass spectrometry Methods 0.000 claims description 2
- 238000006467 substitution reaction Methods 0.000 claims description 2
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims 15
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims 12
- 239000011347 resin Substances 0.000 claims 11
- 229920005989 resin Polymers 0.000 claims 11
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims 9
- 239000003153 chemical reaction reagent Substances 0.000 claims 8
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims 6
- 238000005406 washing Methods 0.000 claims 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims 4
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims 4
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims 4
- 238000001514 detection method Methods 0.000 claims 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims 3
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims 3
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 claims 3
- 229920001184 polypeptide Polymers 0.000 claims 3
- 239000002904 solvent Substances 0.000 claims 3
- HNICLNKVURBTKV-NDEPHWFRSA-N (2s)-5-[[amino-[(2,2,4,6,7-pentamethyl-3h-1-benzofuran-5-yl)sulfonylamino]methylidene]amino]-2-(9h-fluoren-9-ylmethoxycarbonylamino)pentanoic acid Chemical compound C12=CC=CC=C2C2=CC=CC=C2C1COC(=O)N[C@H](C(O)=O)CCCN=C(N)NS(=O)(=O)C1=C(C)C(C)=C2OC(C)(C)CC2=C1C HNICLNKVURBTKV-NDEPHWFRSA-N 0.000 claims 2
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims 2
- JFLSOKIMYBSASW-UHFFFAOYSA-N 1-chloro-2-[chloro(diphenyl)methyl]benzene Chemical compound ClC1=CC=CC=C1C(Cl)(C=1C=CC=CC=1)C1=CC=CC=C1 JFLSOKIMYBSASW-UHFFFAOYSA-N 0.000 claims 2
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims 2
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 claims 2
- 238000012512 characterization method Methods 0.000 claims 2
- 238000009833 condensation Methods 0.000 claims 2
- 230000005494 condensation Effects 0.000 claims 2
- 239000012043 crude product Substances 0.000 claims 2
- 238000010511 deprotection reaction Methods 0.000 claims 2
- 238000004108 freeze drying Methods 0.000 claims 2
- 238000010438 heat treatment Methods 0.000 claims 2
- CMWYAOXYQATXSI-UHFFFAOYSA-N n,n-dimethylformamide;piperidine Chemical compound CN(C)C=O.C1CCNCC1 CMWYAOXYQATXSI-UHFFFAOYSA-N 0.000 claims 2
- 238000004806 packaging method and process Methods 0.000 claims 2
- 239000002245 particle Substances 0.000 claims 2
- 238000005086 pumping Methods 0.000 claims 2
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims 2
- 239000002994 raw material Substances 0.000 claims 2
- 238000003786 synthesis reaction Methods 0.000 claims 2
- GSNUFIFRDBKVIE-UHFFFAOYSA-N DMF Natural products CC1=CC=C(C)O1 GSNUFIFRDBKVIE-UHFFFAOYSA-N 0.000 claims 1
- 238000000861 blow drying Methods 0.000 claims 1
- 210000004899 c-terminal region Anatomy 0.000 claims 1
- 230000021523 carboxylation Effects 0.000 claims 1
- 238000006473 carboxylation reaction Methods 0.000 claims 1
- 238000003776 cleavage reaction Methods 0.000 claims 1
- 230000018044 dehydration Effects 0.000 claims 1
- 238000006297 dehydration reaction Methods 0.000 claims 1
- 229910001873 dinitrogen Inorganic materials 0.000 claims 1
- 238000004090 dissolution Methods 0.000 claims 1
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- 230000000887 hydrating effect Effects 0.000 claims 1
- 239000007788 liquid Substances 0.000 claims 1
- 239000006166 lysate Substances 0.000 claims 1
- 239000000203 mixture Substances 0.000 claims 1
- 229910052757 nitrogen Inorganic materials 0.000 claims 1
- 150000003053 piperidines Chemical class 0.000 claims 1
- NNFCIKHAZHQZJG-UHFFFAOYSA-N potassium cyanide Chemical compound [K+].N#[C-] NNFCIKHAZHQZJG-UHFFFAOYSA-N 0.000 claims 1
- 239000000843 powder Substances 0.000 claims 1
- 239000002244 precipitate Substances 0.000 claims 1
- 230000001376 precipitating effect Effects 0.000 claims 1
- 239000000047 product Substances 0.000 claims 1
- 238000011321 prophylaxis Methods 0.000 claims 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 claims 1
- 230000007017 scission Effects 0.000 claims 1
- 238000007789 sealing Methods 0.000 claims 1
- 238000010532 solid phase synthesis reaction Methods 0.000 claims 1
- 239000006228 supernatant Substances 0.000 claims 1
- 230000008961 swelling Effects 0.000 claims 1
- 238000001308 synthesis method Methods 0.000 claims 1
- 229940079593 drug Drugs 0.000 abstract description 13
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 abstract description 9
- 150000001875 compounds Chemical class 0.000 abstract description 5
- 210000004087 cornea Anatomy 0.000 abstract description 5
- 238000012377 drug delivery Methods 0.000 abstract description 5
- 230000000694 effects Effects 0.000 abstract description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 4
- 230000000149 penetrating effect Effects 0.000 abstract description 4
- 239000012528 membrane Substances 0.000 abstract description 3
- 210000003484 anatomy Anatomy 0.000 abstract description 2
- 230000036770 blood supply Effects 0.000 abstract description 2
- 238000002347 injection Methods 0.000 abstract description 2
- 239000007924 injection Substances 0.000 abstract description 2
- 238000001990 intravenous administration Methods 0.000 abstract description 2
- 230000035479 physiological effects, processes and functions Effects 0.000 abstract description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 13
- 210000004027 cell Anatomy 0.000 description 11
- 210000001542 lens epithelial cell Anatomy 0.000 description 11
- 230000006378 damage Effects 0.000 description 8
- 230000004888 barrier function Effects 0.000 description 7
- 208000027418 Wounds and injury Diseases 0.000 description 6
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 6
- 230000001965 increasing effect Effects 0.000 description 6
- 208000014674 injury Diseases 0.000 description 6
- 239000007800 oxidant agent Substances 0.000 description 6
- 230000036542 oxidative stress Effects 0.000 description 6
- 239000001301 oxygen Substances 0.000 description 6
- 229910052760 oxygen Inorganic materials 0.000 description 6
- 229910052751 metal Inorganic materials 0.000 description 5
- 239000002184 metal Substances 0.000 description 5
- 230000035515 penetration Effects 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 210000001742 aqueous humor Anatomy 0.000 description 4
- 230000001590 oxidative effect Effects 0.000 description 4
- 230000001988 toxicity Effects 0.000 description 4
- 231100000419 toxicity Toxicity 0.000 description 4
- 230000009471 action Effects 0.000 description 3
- 239000003963 antioxidant agent Substances 0.000 description 3
- 230000003078 antioxidant effect Effects 0.000 description 3
- JYHHJVKGDCZCCL-UHFFFAOYSA-J carbon monoxide;dichlororuthenium Chemical compound [O+]#[C-].[O+]#[C-].[O+]#[C-].[O+]#[C-].[O+]#[C-].[O+]#[C-].Cl[Ru]Cl.Cl[Ru]Cl JYHHJVKGDCZCCL-UHFFFAOYSA-J 0.000 description 3
- 230000003833 cell viability Effects 0.000 description 3
- 230000003013 cytotoxicity Effects 0.000 description 3
- 231100000135 cytotoxicity Toxicity 0.000 description 3
- 239000011572 manganese Substances 0.000 description 3
- 230000002441 reversible effect Effects 0.000 description 3
- 229910052707 ruthenium Inorganic materials 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 239000011550 stock solution Substances 0.000 description 3
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 108010033276 Peptide Fragments Proteins 0.000 description 2
- 102000007079 Peptide Fragments Human genes 0.000 description 2
- KJTLSVCANCCWHF-UHFFFAOYSA-N Ruthenium Chemical compound [Ru] KJTLSVCANCCWHF-UHFFFAOYSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- UORVGPXVDQYIDP-UHFFFAOYSA-N borane Chemical compound B UORVGPXVDQYIDP-UHFFFAOYSA-N 0.000 description 2
- 125000002091 cationic group Chemical group 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 210000002919 epithelial cell Anatomy 0.000 description 2
- 230000002349 favourable effect Effects 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 238000001356 surgical procedure Methods 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 239000004475 Arginine Substances 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 1
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- PWHULOQIROXLJO-UHFFFAOYSA-N Manganese Chemical compound [Mn] PWHULOQIROXLJO-UHFFFAOYSA-N 0.000 description 1
- 102000015728 Mucins Human genes 0.000 description 1
- 108010063954 Mucins Proteins 0.000 description 1
- 102000019197 Superoxide Dismutase Human genes 0.000 description 1
- 108010012715 Superoxide dismutase Proteins 0.000 description 1
- 239000003732 agents acting on the eye Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 229910000085 borane Inorganic materials 0.000 description 1
- 150000001728 carbonyl compounds Chemical class 0.000 description 1
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 210000003683 corneal stroma Anatomy 0.000 description 1
- 230000007402 cytotoxic response Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000009510 drug design Methods 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 210000003560 epithelium corneal Anatomy 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 208000030533 eye disease Diseases 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 230000007760 free radical scavenging Effects 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 231100000171 higher toxicity Toxicity 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 210000002490 intestinal epithelial cell Anatomy 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
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- 229910052748 manganese Inorganic materials 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
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- 230000002503 metabolic effect Effects 0.000 description 1
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- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229940051875 mucins Drugs 0.000 description 1
- 229940023490 ophthalmic product Drugs 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000007903 penetration ability Effects 0.000 description 1
- 230000002688 persistence Effects 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
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- 230000035790 physiological processes and functions Effects 0.000 description 1
- 230000035806 respiratory chain Effects 0.000 description 1
- 238000009531 respiratory rate measurement Methods 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
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- 230000004083 survival effect Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000011200 topical administration Methods 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
-
- 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
- A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
-
- 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
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0048—Eye, e.g. artificial tears
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
- A61P27/12—Ophthalmic agents for cataracts
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Ophthalmology & Optometry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Epidemiology (AREA)
- Molecular Biology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Biochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The present invention relates to a carbon monoxide releasing molecule-transmembrane peptide complex CORM401-R9 (CRs), a method of preparing the same and use thereof, in which the adult lens does not have any blood supply, and oral and intravenous drugs are hardly accumulated therein, so that the mode of administration of the lens is limited to local administration, which is the simplest and least invasive route of delivering the drug to the lens, or intraocular injection. Due to the unique physiology and anatomy of the eye, drug delivery through the cornea is limited, and thus the bioavailability of CORM-401 administered alone is low, less than 5%. The oligoarginine R9 is a hydrophilic peptide containing 9 positively charged arginine residues and has stronger biological membrane penetrating capacity. The compound breaks through the technical problems of the prior art that the drug delivery disorder and the treatment effect for age-related cataract are limited, and the sequence of the compound is (CORM-401) -Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg; the molecular weight of CORM401-R9 is 1709kDa. The invention can be used for preparing the medicine for preventing and treating age-related cataract.
Description
Technical Field
The invention relates to an ophthalmic drug, in particular to a carbon monoxide release molecule-transmembrane peptide complex CORM401-R9 (CRs) and a preparation method and application thereof.
Background
Age-related cataract (ARC) is a disease in which the transparency of the lens decreases with age to affect vision, and is the first blinding eye disease in the world, and the etiology and pathogenesis of the disease are not completely known. Oxidative stress mediated apoptosis of Lens Epithelial Cells (LECs) is closely related to the development of ARC, hydrogen peroxide (H2O 2) being the predominant oxidative species in the lens and aqueous humor of age-related cataracts patients, and significantly higher than normal H2O2 concentrations are also detected in the aqueous humor of patients.
The only viable method of treating cataracts is currently surgery. Over 20 ten thousand cataract patients worldwide receive lens removal and intraocular lens implantation treatments each year, but surgery has many limitations and potential complications, with a significant economic burden. Based on the proposed mechanism of ARC formation, attempts have been made to slow down the development of cataracts using antioxidants and the like. To date, compounds with antioxidant and free radical scavenging activity have shown great potential in relevant experimental studies.
CORM-401 is a newly discovered carbon monoxide releasing molecule, and has become the focus of research in recent years, playing an important role in reducing oxidative stress damage. However, since the adult lens does not have any blood supply, oral and intravenous drugs are difficult to accumulate there, and thus, the mode of lens administration is limited to topical administration, which is the simplest and least invasive route of drug delivery to the lens, or intraocular injection. Due to the unique physiology and anatomy of the eye, drug delivery through the cornea is limited, and thus the bioavailability of CORM-401 administered alone is low, less than 5%. The oligoarginine R9 is a hydrophilic peptide containing 9 positively charged arginine residues and has stronger biological membrane penetrating capacity.
The reason for selecting CORM-401: although many CORMs are currently known that contain different kinds of metal centers or organic molecules, most CORMs lack some of the necessary properties for clinically useful drugs. The ideal CORMs should meet a number of properties 1, effective therapeutic effect and low toxicity; 2. proper absorption, distribution, metabolism, and excretion properties; 3. good water solubility and stability; 4. good biocompatibility. CORM-1 is only dissolved in an organic solvent, and CO is slowly released under a cold light source, and the half life is less than 1min and is eliminated; CORM-2 (soluble in organic solvents) and CORM-3 (water-soluble) are metal carbonyls, ruthenium (Ru) being their metal centers; CORM-A1 is a water-soluble borane carbonate that spontaneously releases CO in solution in a pH dependent manner in solution. These above release only one molar amount of CO per mole of CORMs, despite their different half-lives. CORMs of metal carbonyl compounds have more favorable pharmacological actions than CORMs-A1, but CORMs-2 must release CO under the stimulation of physicochemical conditions and are not suitable for the actual environment of the organism, and the metal ruthenium center of CORMs-2 is combined with glycine to obtain CORMs-3, so that the CORMs-3 has better water solubility, but ruthenium-based CORMs have higher toxicity than CO gas and CORMs-401. In terms of toxicity, manganese is relatively low in toxicity, is one of trace elements necessary for the body, and can constitute enzymes or coenzymes (such as Mn-SOD) having important physiological functions in organisms. CORM-401 has greatly increased water solubility compared with the previous CORM, and the introduced-CH 2CO2H group makes CORM-401 soluble in Phosphate Buffer (PBS), so that the biocompatibility is improved, and the CO generating capacity is three times that of the commercial CORM-3. CORM-401 is a CO-releasing molecule sensitive to oxidizing agents, and can accelerate CO release under the action of the oxidizing agents, so that the generation of ROS induced by H2O2 can be reduced. In terms of stability, CORM-2 and CORM-3 release CO very quickly (< 5 minutes), while CORM-401 releases CO continuously (> 50 minutes). Regarding the storage stability of the CORM stock solution, only the storage of CORM-401 remained stable for 7 days. The effect of CORMs on the respiratory chain was studied using high resolution respiratory measurements and extracellular flux techniques, with interference of CORM-2 and CORM-3 on oxygen measurements occurring because rapid consumption of oxygen was detected in the medium even in the absence of cells, whereas CORM-401 did not interfere with oxygen measurements, yielding the most reliable CO-specific results for typical CO targeting modulation. In terms of therapeutic effects, it has been found that CORM-401 is superior to other CO-RMs in protecting intestinal epithelial cells from high concentrations of exogenous H2O 2-induced oxidative stress and apoptosis under H2O 2-induced oxidative stress conditions. The ability of CORM-401 to release multiple CO molecules is therapeutically important because administration of lower doses of the compound is sufficient to achieve the delivery of pharmacologically relevant amounts of CO to the tissue, inducing more intracellular CO accumulation. And the interaction with biologically relevant oxidants (such as H2O 2) increases the CO released by CORM-401, with the rate of CO release from CORM-401 increasing with increasing oxidant concentration. In the stock solution stored, CORM-401 releases CO in a reversible manner, which also explains why it has stability. Under the action of an oxidant such as H2O2, the H2O2 can coordinate with 16-electron Mn (CO) 3 (S2 CNR 2) after the first CO is lost by CORM-401 (Mn (CO) 4 (S2 CNR 2)), and oxidize the CO rapidly, so that the CO is prevented from being subjected to reverse reaction, and the rest 3 COs can be released immediately, thereby realizing the targeted modulation of the CO.
The reason for selecting R9: depending on physicochemical properties, transmembrane peptides (CPPs) can be divided into three major classes: cationic, amphiphilic, and hydrophobic. Cationic transmembrane peptides are the most important subject of current research, and generally contain more than 5 positively charged amino acids, and the more basic amino acids they contain, the more penetrating they are. Positively charged agents can interact with negatively charged ocular components (e.g., epithelial cell membranes and external mucins) to increase the persistence of the drug on the ocular surface and then mediate drug absorption. Arginine residues are emphasized as "magic residues" and have been shown to internalize effectively and penetrate the plasma membrane most effectively. The transduction capacity of the oligoarginine peptide increases with the number of consecutive arginine residues and the concentration of the peptide, and the cytotoxicity of the peptide fragment is proportionally more pronounced with increasing length of the peptide fragment, the minimum number of arginine residues required for the internalization of the oligoarginine being 9, which represents the best result between cellular internalization efficacy, low cytotoxicity and low production costs. In addition, R9 is also a typical hydrophilic peptide that contains multiple positively charged arginine residues, which allows it to hydrogen bond with water molecules and dissolve well in aqueous solutions such as PBS.
In view of the structural formula of the complex (CORM-401) -R9, the CO-releasing site of CORM-401 and the arginine residue of R9 providing penetration ability are distributed at both poles of the molecule, and their binding does not interfere with the CO-releasing ability of the respective CORM-401 and the cell penetrating ability of R9. Secondly, they are covalently bound, which, in addition to allowing R9 to more stably carry CORM-401 across the cornea to the target site, is simpler in structure, less in molecular weight, and simpler and more cost-effective to prepare. CORM-401 and R9 are both soluble in PBS, and the complex is also soluble in PBS solution, with good biocompatibility, however, water-soluble drugs are hindered by lipophilic epithelial and endothelial cells during penetration of the cornea into the aqueous humor contact lens. Their combination is also "1+1 > 2" in effect, R9 being favourable for penetration of the corneal barrier but not having a targeting function itself, whereas CORM-401 has an oxidative response release mechanism which releases CO in a reversible manner in the stock solution stored, and which rapidly releases large amounts of CO in response to an oxidising agent, in particular H2O 2.
Disclosure of Invention
The invention aims to solve the technical problems of drug delivery disorder and limited treatment effect on age-related cataract drugs in the prior art, and provides a carbon monoxide release molecule-transmembrane peptide complex CORM401-R9 (CRs) with good antioxidant effect, and a preparation method and application thereof.
To this end, the invention provides a carbon monoxide releasing molecule-transmembrane peptide complex CORM401-R9 (CRs), which has the sequence (CORM-401) -Arg-Arg-Arg-Arg-Arg; the molecular weight of CORM401-R9 (CRS) is 1709kDa.
The invention has the following beneficial effects:
CORM401-R9 (CRS) provided by the invention can be safely and controllably released to increase the CO content in lens epithelial cells, can obviously reduce the accumulation of ROS in the cells, enhance the activity of antioxidant enzyme and reduce oxidative stress damage; meanwhile, the challenges of the anterior ocular segment administration barrier, including static barrier (corneal epithelium, corneal stroma and blood-aqueous humor barrier), dynamic barrier (conjunctival blood flow, lymphatic flow and tear drainage) and metabolic barrier, can be significantly overcome, and the anterior ocular segment administration barrier penetrates through the cornea to enter aqueous humor and lens epithelial cells and is used as a candidate drug for local ocular administration.
Drawings
FIG. 1 evaluates CORM-401's ability to bind to different oligoarginines (R5-R12) for lens epithelial cell penetration and toxicity. The uptake and cytotoxic response of cells to the oligoarginine was quantified by a fluorescence microplate reader using a living cell assay method. Wherein FIG. 1A shows that the penetration of arginine lens epithelial cells increases with increasing number of arginine residues and drug concentration, and FIG. 1B shows that at a concentration of 15. Mu.M CORM 401-oligoarginine complex treatment for 3 hours, the survival rate of lens epithelial cells decreases with increasing number of arginine residues, combining the performance of cell penetration and toxicity, and selecting R9 for drug design.
FIG. 2 is a reaction scheme for the preparation of carbon monoxide releasing molecule-transmembrane peptide complexes CORM401-R9 (CRs) of the present invention;
fig. 3A, 3B, and 3C are a freeze-dried product, a mass spectrometry and an HPLC analysis of carbon monoxide releasing molecule-transmembrane peptide complexes CORM401-R9 (CRs), respectively.
FIGS. 4A, 4B and 4C show the CCK8 results of CORM401-R9 (CRs), respectively, for carbon monoxide releasing molecule-transmembrane peptide complexes of the present invention. Wherein FIG. 4A shows that CORM401 and complex CORM401-R9 (CRs) alone treated lens epithelial cells have high cell viability for 3 hours, and the drug has no significant cytotoxicity; FIGS. 4B and 4C show cell viability of CORM401 and CORM401-R9 (CRs) lens epithelial cells treated for 3 hours and 24 hours, respectively, under 500 μMH2O2 injury treatment, and the drug significantly reduced H2O2 injury to the cells.
FIGS. 5A and 5B are respectively graphs showing the results of an active oxygen assay of CORM401-R9 (CRs) for the carbon monoxide release molecule-transmembrane peptide complex of the present invention; FIG. 5A shows the fluorescence intensity of active oxygen of CORM401 and of CORM401-R9 (CRs) complexes of the complex measured by a fluorescence enzyme-labeled instrument 1 hour after 600 μMH2O2 injury stimulus; FIG. 5B is a graph showing fluorescence intensity observed by a fluorescence microscope under the same stimulus. The medicine can obviously reduce the level of active oxygen in cells under H2O2 treatment, thereby reducing the oxidative stress state of the cells.
FIG. 6 shows the results of an experiment for detecting the H2O2 level of the carbon monoxide releasing molecule-penetrating peptide complex CORM401-R9 (CRs) in the present invention; CORM401 and the complex CORM401-R9 (CRs) treated lens epithelial cell H2O2 levels measured by the enzyme-labeled instrument at 600 μMH2O2 injury stimulus for 2 hours. The drug can obviously reduce the level of H2O2 in cells under H2O2 treatment, wherein the compound CORM401-R9 (CRs) in the invention can reduce the level of H2O2 to a normal level.
FIG. 7 shows the cell viability of lens epithelial cells treated with CORM401-R9 (CRs) at different concentrations for 3 hours of 600 μM 2O2 injury in accordance with the present invention, with a range of concentrations of CORM401-R9 (CRs) significantly reducing H2O2 injury to the cells, wherein the 15 μM-25 μM concentration provides better protection and no statistical differences between groups.
The carbon monoxide release molecule-transmembrane peptide complex CORM401-R9 (CRs) prepared in the invention can be used for preventing and treating age-related cataract.
However, the foregoing description is only illustrative of the embodiments of the present invention and is not intended to limit the scope of the invention, so that the substitution of equivalent elements or equivalent variations and modifications within the scope of the invention are intended to fall within the scope of the claims.
Claims (3)
1. A carbon monoxide release molecule-transmembrane peptide complex CORM401-R9 (CRs) is characterized in that the carbon monoxide release molecule-transmembrane peptide complex is CORM-401 (C) 8 H 8 MnNO 6 S 2 ) Oligo-arginine R9 (C) 54 H 110 N 36 O 10 ) Is prepared by dehydration and carboxylation; the complex sequence is (CORM-401) -Arg-Arg-Arg-Arg-Arg-Arg and has a molecular weight of 1709kDa.
2. The method for preparing carbon monoxide releasing molecule-penetrating peptide complex CORM401-R9 (CRs) as defined in claim 1, wherein the reaction formula is shown in figure 2 of the accompanying drawings, and the synthesis method is Fmoc solid-phase synthesis method, and comprises the following steps:
synthetic raw materials and related reagents:
1) Fmoc-Arg (pbf) -OH, CORM-401 as a protected amino acid raw material
2) Condensation reagent
HBTU，DIEA
3) Solvents and solvents
DMF, DCM, methanol, acetonitrile
4) Resin
Chlorotrityl chloride resin with substitution degree of 1.1mmol/g
5) Deprotection reagent
Piperidine compounds
6) Detection reagent:
phenol reagent, pyridine reagent, ninhydrin reagent
7) Reagent for cutting
TFA, TIS, EDT, anhydrous diethyl ether
8) Nitrogen gas
9) Precision electronic balance
Instrument apparatus:
1) Twelve-channel semiautomatic polypeptide synthesizer
2) High performance liquid chromatograph
3. Freeze dryer
4) Centrifuge with a centrifugal separator
The synthesis process comprises the following steps:
swelling of resin
The chlorotrityl chloride resin was placed in a reaction tube, DMF (15 ml/g) was added, and shaking was performed for 60min.
Second, connect the first amino acid
The solvent was filtered off with suction through a sand core, 3-fold molar excess of Fmoc-Arg (pbf) -OH (C-terminal first amino acid) was added, followed by 10-fold molar excess of DIEA, and finally dissolved by adding DMF and shaking for 30min. Methanol seal head for 30min.
Three, deprotection
DMF was removed, 20% piperidine DMF solution (15 ml/g) was added for 5min, and 20% piperidine DMF solution (15 ml/g) was removed for 15min.
Fourth, detection
Pumping off piperidine solution, taking more than ten resin particles, washing with ethanol for three times, adding ninhydrin, KCN and phenol solution into the resin particles, heating the mixture at 105-110 ℃ for 5min, and turning deep blue into positive reaction.
Fifth, washing
DMF (10 ml/g) was twice, methanol (10 ml/g) was twice, and DMF (10 ml/g) was twice.
Sixth, condensation
3-fold molar excess Fmoc protected amino acid, 3-fold molar excess HBTU, 10-fold molar excess DIEA and finally DMF were added for dissolution and shaking for 45min.
Seventhly, detection
Taking more than ten pieces of resin, washing the resin with ethanol for three times, adding ninhydrin, pyridine and phenol solution into the resin, heating the resin at 105-110 ℃ for 5min, and taking colorless negative reaction.
Eighth step of washing
DMF (10 ml/g) was taken once, methanol (10 ml/g) was taken twice, and DMF (10 ml/g) was taken twice.
Nine. Synthesis of CORM401-R9 (CRs)
Repeating three to eight steps, sequentially connecting amino acids in the sequence from right to left, adding 3 times molar excess CORM-401,3 times molar excess HBTU, adding 10 times molar excess DIEA, adding DMF, dissolving, and oscillating for 45min.
Ten times of pumping
The resin was washed twice with DMF (10 ml/g), three times with DCM (10 ml/g) and four times with methanol (10 ml/g) and drained for 10min.
Eleven cutting
Cleavage (10/g) TFA95%; hydrating 2%; EDT 2%; TIS 1%, cutting time: 180min.
Twelve, blow-drying and washing
Drying the lysate with nitrogen as much as possible, precipitating diethyl ether, centrifuging to remove supernatant, washing precipitate with diethyl ether for six times, and volatilizing at normal temperature.
Thirteenth, purifying and preparing.
1) Taking a small amount of crude product, and dissolving the crude product by H2O/ACN.
2) And taking a small amount of sample, and analyzing and judging the peak time corresponding to the target peak on an HPLC analysis instrument.
3) Preparing a system by using C18 reverse phase chromatography, wherein the wave length is 220nm; flow Rate 15m/min; inj.Vol 20mL Column Temp:25 DEG C
Buffer A0.1% TFAin water; buffer B0.1%TFA in Acetonitrile; the target peak solution was collected.
4) A few target peak solutions were taken with a 1.5ml centrifuge tube for mass spectrometry and purity detection.
Fourteen freeze drying
And freeze-drying the qualified target peak solution to obtain a finished product, as shown in figure 3A.
Fifteen, authentication
A small amount of the final polypeptide was taken and subjected to molecular weight characterization by MS and purity characterization by HPLC analysis, respectively, as shown in fig. 3B and 3C.
Sixteen, packaging and preserving
Sealing and packaging the polypeptide powder, and preserving at-20deg.C.
3. Use of the carbon monoxide releasing molecule-transmembrane peptide complex CORM401-R9 (CRs) according to claim 1 for the preparation of a medicament for the prophylaxis and treatment of age-related cataracts.
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