CN115702902B - Anti-tumor preparation of doxorubicin prodrug - Google Patents
Anti-tumor preparation of doxorubicin prodrug Download PDFInfo
- Publication number
- CN115702902B CN115702902B CN202110933636.1A CN202110933636A CN115702902B CN 115702902 B CN115702902 B CN 115702902B CN 202110933636 A CN202110933636 A CN 202110933636A CN 115702902 B CN115702902 B CN 115702902B
- Authority
- CN
- China
- Prior art keywords
- doxorubicin
- fatty acid
- prodrug
- albumin
- sensitive
- 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.)
- Active
Links
- 229940002612 prodrug Drugs 0.000 title claims abstract description 147
- 239000000651 prodrug Substances 0.000 title claims abstract description 147
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 title abstract description 85
- 229960004679 doxorubicin Drugs 0.000 title abstract description 37
- 230000000259 anti-tumor effect Effects 0.000 title abstract description 18
- 239000000194 fatty acid Substances 0.000 claims abstract description 101
- 102000009027 Albumins Human genes 0.000 claims abstract description 95
- 108010088751 Albumins Proteins 0.000 claims abstract description 95
- 239000002105 nanoparticle Substances 0.000 claims abstract description 90
- 238000012377 drug delivery Methods 0.000 claims abstract description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 57
- 239000013067 intermediate product Substances 0.000 claims description 19
- 239000000243 solution Substances 0.000 claims description 17
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical group CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 12
- MWWSFMDVAYGXBV-RUELKSSGSA-N Doxorubicin hydrochloride Chemical compound Cl.O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 MWWSFMDVAYGXBV-RUELKSSGSA-N 0.000 claims description 12
- 108010071390 Serum Albumin Proteins 0.000 claims description 11
- 102000007562 Serum Albumin Human genes 0.000 claims description 11
- 229960002918 doxorubicin hydrochloride Drugs 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 10
- 238000010992 reflux Methods 0.000 claims description 10
- NWZSZGALRFJKBT-KNIFDHDWSA-N (2s)-2,6-diaminohexanoic acid;(2s)-2-hydroxybutanedioic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O.NCCCC[C@H](N)C(O)=O NWZSZGALRFJKBT-KNIFDHDWSA-N 0.000 claims description 9
- IKDUDTNKRLTJSI-UHFFFAOYSA-N hydrazine monohydrate Substances O.NN IKDUDTNKRLTJSI-UHFFFAOYSA-N 0.000 claims description 9
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 8
- 238000001953 recrystallisation Methods 0.000 claims description 7
- 239000002904 solvent Substances 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- 238000000265 homogenisation Methods 0.000 claims description 4
- 239000003960 organic solvent Substances 0.000 claims description 4
- 238000003756 stirring Methods 0.000 claims description 4
- TWJNQYPJQDRXPH-UHFFFAOYSA-N 2-cyanobenzohydrazide Chemical compound NNC(=O)C1=CC=CC=C1C#N TWJNQYPJQDRXPH-UHFFFAOYSA-N 0.000 claims description 3
- 235000021360 Myristic acid Nutrition 0.000 claims description 3
- TUNFSRHWOTWDNC-UHFFFAOYSA-N Myristic acid Natural products CCCCCCCCCCCCCC(O)=O TUNFSRHWOTWDNC-UHFFFAOYSA-N 0.000 claims description 3
- 239000002246 antineoplastic agent Substances 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 239000000047 product Substances 0.000 claims description 3
- TUNFSRHWOTWDNC-HKGQFRNVSA-N tetradecanoic acid Chemical compound CCCCCCCCCCCCC[14C](O)=O TUNFSRHWOTWDNC-HKGQFRNVSA-N 0.000 claims description 3
- 229940041181 antineoplastic drug Drugs 0.000 claims description 2
- ZAZKJZBWRNNLDS-UHFFFAOYSA-N methyl tetradecanoate Chemical compound CCCCCCCCCCCCCC(=O)OC ZAZKJZBWRNNLDS-UHFFFAOYSA-N 0.000 claims 8
- KXTZPAQXYKUVLD-UHFFFAOYSA-N tetradecanehydrazide Chemical compound CCCCCCCCCCCCCC(=O)NN KXTZPAQXYKUVLD-UHFFFAOYSA-N 0.000 claims 3
- 230000000699 topical effect Effects 0.000 claims 1
- 235000014113 dietary fatty acids Nutrition 0.000 abstract description 26
- 229930195729 fatty acid Natural products 0.000 abstract description 26
- 150000004665 fatty acids Chemical group 0.000 abstract description 25
- 230000000694 effects Effects 0.000 abstract description 18
- 239000003814 drug Substances 0.000 abstract description 17
- 229940079593 drug Drugs 0.000 abstract description 16
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 7
- 229940009456 adriamycin Drugs 0.000 abstract description 4
- 230000003285 pharmacodynamic effect Effects 0.000 abstract description 3
- 238000010276 construction Methods 0.000 abstract description 2
- 231100000057 systemic toxicity Toxicity 0.000 abstract 1
- 206010028980 Neoplasm Diseases 0.000 description 26
- 230000005917 in vivo anti-tumor Effects 0.000 description 21
- 238000002474 experimental method Methods 0.000 description 20
- 241000699670 Mus sp. Species 0.000 description 16
- 210000004027 cell Anatomy 0.000 description 15
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 12
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 10
- 239000000203 mixture Substances 0.000 description 10
- 239000002245 particle Substances 0.000 description 10
- 231100000331 toxic Toxicity 0.000 description 10
- 230000002588 toxic effect Effects 0.000 description 10
- 239000008117 stearic acid Substances 0.000 description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 8
- 235000019387 fatty acid methyl ester Nutrition 0.000 description 8
- 231100000135 cytotoxicity Toxicity 0.000 description 7
- 230000003013 cytotoxicity Effects 0.000 description 7
- 238000005538 encapsulation Methods 0.000 description 7
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 6
- GHVNFZFCNZKVNT-UHFFFAOYSA-N decanoic acid Chemical compound CCCCCCCCCC(O)=O GHVNFZFCNZKVNT-UHFFFAOYSA-N 0.000 description 6
- 210000004698 lymphocyte Anatomy 0.000 description 6
- 239000002504 physiological saline solution Substances 0.000 description 6
- 238000004611 spectroscopical analysis Methods 0.000 description 6
- 241000699666 Mus <mouse, genus> Species 0.000 description 5
- 229960000583 acetic acid Drugs 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 229940042317 doxorubicin liposome Drugs 0.000 description 5
- 239000012362 glacial acetic acid Substances 0.000 description 5
- 210000004185 liver Anatomy 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- 231100000419 toxicity Toxicity 0.000 description 5
- 230000001988 toxicity Effects 0.000 description 5
- 210000004881 tumor cell Anatomy 0.000 description 5
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical class [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 description 4
- 238000002841 anti-cancer assay Methods 0.000 description 4
- 230000037396 body weight Effects 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 239000012295 chemical reaction liquid Substances 0.000 description 4
- 238000002512 chemotherapy Methods 0.000 description 4
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 description 4
- 238000009472 formulation Methods 0.000 description 4
- 239000005457 ice water Substances 0.000 description 4
- 231100000053 low toxicity Toxicity 0.000 description 4
- 210000001616 monocyte Anatomy 0.000 description 4
- 230000003472 neutralizing effect Effects 0.000 description 4
- 210000000440 neutrophil Anatomy 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 210000000952 spleen Anatomy 0.000 description 4
- 238000011282 treatment Methods 0.000 description 4
- 238000005160 1H NMR spectroscopy Methods 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 108091006905 Human Serum Albumin Proteins 0.000 description 3
- 102000008100 Human Serum Albumin Human genes 0.000 description 3
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 3
- 238000005481 NMR spectroscopy Methods 0.000 description 3
- 229940098773 bovine serum albumin Drugs 0.000 description 3
- 229940063519 doxorubicin hydrochloride liposome Drugs 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 239000001257 hydrogen Substances 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 238000011068 loading method Methods 0.000 description 3
- 230000014759 maintenance of location Effects 0.000 description 3
- 238000004949 mass spectrometry Methods 0.000 description 3
- 238000001819 mass spectrum Methods 0.000 description 3
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 3
- 230000001376 precipitating effect Effects 0.000 description 3
- 150000004671 saturated fatty acids Chemical class 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- 230000008685 targeting Effects 0.000 description 3
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 3
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 3
- 238000005303 weighing Methods 0.000 description 3
- YWWVWXASSLXJHU-AATRIKPKSA-N (9E)-tetradecenoic acid Chemical compound CCCC\C=C\CCCCCCCC(O)=O YWWVWXASSLXJHU-AATRIKPKSA-N 0.000 description 2
- 102100036475 Alanine aminotransferase 1 Human genes 0.000 description 2
- 108010082126 Alanine transaminase Proteins 0.000 description 2
- 108010003415 Aspartate Aminotransferases Proteins 0.000 description 2
- 102000004625 Aspartate Aminotransferases Human genes 0.000 description 2
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 2
- 241000282465 Canis Species 0.000 description 2
- 206010048610 Cardiotoxicity Diseases 0.000 description 2
- 102000001554 Hemoglobins Human genes 0.000 description 2
- 108010054147 Hemoglobins Proteins 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- PNNCWTXUWKENPE-UHFFFAOYSA-N [N].NC(N)=O Chemical compound [N].NC(N)=O PNNCWTXUWKENPE-UHFFFAOYSA-N 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- -1 carboxyl hydroxyl Chemical group 0.000 description 2
- 231100000259 cardiotoxicity Toxicity 0.000 description 2
- 229940109239 creatinine Drugs 0.000 description 2
- 229940115080 doxil Drugs 0.000 description 2
- 210000001163 endosome Anatomy 0.000 description 2
- 210000003743 erythrocyte Anatomy 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000009592 kidney function test Methods 0.000 description 2
- 210000000265 leukocyte Anatomy 0.000 description 2
- 238000007449 liver function test Methods 0.000 description 2
- 210000003712 lysosome Anatomy 0.000 description 2
- 230000001868 lysosomic effect Effects 0.000 description 2
- OBMJQRLIQQTJLR-USGQOSEYSA-N n-[(e)-[1-[(2s,4s)-4-[(2r,4s,5s,6s)-4-amino-5-hydroxy-6-methyloxan-2-yl]oxy-2,5,12-trihydroxy-7-methoxy-6,11-dioxo-3,4-dihydro-1h-tetracen-2-yl]-2-hydroxyethylidene]amino]-6-(2,5-dioxopyrrol-1-yl)hexanamide Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(\CO)=N\NC(=O)CCCCCN1C(C=CC1=O)=O)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 OBMJQRLIQQTJLR-USGQOSEYSA-N 0.000 description 2
- 238000011580 nude mouse model Methods 0.000 description 2
- 238000000967 suction filtration Methods 0.000 description 2
- 238000001308 synthesis method Methods 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 238000009210 therapy by ultrasound Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 230000004614 tumor growth Effects 0.000 description 2
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
- YWWVWXASSLXJHU-UHFFFAOYSA-N 9E-tetradecenoic acid Natural products CCCCC=CCCCCCCCC(O)=O YWWVWXASSLXJHU-UHFFFAOYSA-N 0.000 description 1
- 238000011725 BALB/c mouse Methods 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 206010065553 Bone marrow failure Diseases 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 108050001049 Extracellular proteins Proteins 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 206010067125 Liver injury Diseases 0.000 description 1
- PEEHTFAAVSWFBL-UHFFFAOYSA-N Maleimide Chemical compound O=C1NC(=O)C=C1 PEEHTFAAVSWFBL-UHFFFAOYSA-N 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 206010061481 Renal injury Diseases 0.000 description 1
- 235000021355 Stearic acid Nutrition 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000003972 antineoplastic antibiotic Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000013043 chemical agent Substances 0.000 description 1
- 230000000973 chemotherapeutic effect Effects 0.000 description 1
- 229940044683 chemotherapy drug Drugs 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001647 drug administration Methods 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 230000003907 kidney function Effects 0.000 description 1
- 208000037806 kidney injury Diseases 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 230000003908 liver function Effects 0.000 description 1
- 230000001926 lymphatic effect Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 1
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 238000005191 phase separation Methods 0.000 description 1
- 230000036470 plasma concentration Effects 0.000 description 1
- 238000004262 preparative liquid chromatography Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000001839 systemic circulation Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- WNLVXSRACLAFOL-UHFFFAOYSA-N tetradeca-2,4-dienoic acid Chemical compound CCCCCCCCCC=CC=CC(O)=O WNLVXSRACLAFOL-UHFFFAOYSA-N 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 230000004580 weight loss Effects 0.000 description 1
Abstract
The invention belongs to the technical field of medicines, relates to an anti-tumor preparation of an adriamycin prodrug, and in particular relates to construction of a pH sensitive adriamycin-fatty acid prodrug (general formula I) and albumin nanoparticles thereof, and application of the pH sensitive adriamycin-fatty acid prodrug in a drug delivery system. The invention synthesizes the pH sensitive hydrazone bond bridged doxorubicin prodrug containing fatty acid side chains with different lengths, and prepares prodrug albumin nanoparticles. Experimental results show that the carbon chain length of fatty acid can significantly influence the pharmaceutics, pharmacokinetics, pharmacodynamics and safety of the doxorubicin prodrug albumin nanoparticle. The doxorubicin-n-capric acid prodrug and the doxorubicin-myristic acid prodrug obviously reduce the systemic toxicity of the doxorubicin on the basis of ensuring the curative effect, and have better tolerance and higher tolerance dose.
Description
Technical Field
The invention belongs to the technical field of medicines, relates to an anti-tumor preparation of an adriamycin prodrug, and in particular relates to construction of pH-sensitive adriamycin-fatty acid prodrug and albumin nanoparticles thereof, and application of the pH-sensitive adriamycin-fatty acid prodrug and albumin nanoparticles in a drug delivery system.
Background
Cancer is a serious threat to the health of all humans. In the past decades, with advances in medical technology, cancer treatments have evolved rapidly, including surgical therapies, chemotherapy, radiation therapy, immunotherapy, and the like. Chemotherapy is the most common mode of tumor treatment. Chemotherapy kills tumor cells or controls the division of tumor cells by means of one or more chemical agents that interfere with DNA synthesis, affect DNA structure and function, interfere with RNA transcription processes, interfere with protein synthesis, and the like. Doxorubicin is a broad-spectrum antitumor antibiotic with strong cytotoxicity and has inhibition effect on various tumors. However, the toxic and side effects are large, especially the serious cardiotoxicity, which limits the clinical application of the traditional Chinese medicine. Meanwhile, small molecular drugs are easy to be metabolized and cleared by organisms in the blood circulation process, and the drugs are difficult to reach into tumor cells due to special environments near tumor tissues, such as a complicated vascular network structure, a compact interstitial structure and higher interstitial pressure. These limitations affect the antitumor effects of doxorubicin. How to improve the bad physicochemical properties of doxorubicin, improve the treatment effect and alleviate the toxic and side effects is a formulation difficult problem to be solved urgently.
In recent years, researchers have developed different types of doxorubicin drug delivery systems, the most successful of which are represented by doxorubicin liposomes, which can effectively improve the pharmacokinetics and in vivo distribution of doxorubicin and reduce the cardiotoxicity of doxorubicin. However, even if the doxorubicin liposome adopts an active drug-carrying mode, the drug-carrying quantity is only 11%, and the risk of acroerythema after drug administration is obviously improved, so that the treatment effect and the life quality of patients are seriously affected. Therefore, there is a need to construct novel high-efficiency low-toxicity doxorubicin nano-formulations.
Among the numerous serum proteins, albumin is 50% of the total protein content and is an ideal carrier for drugs. Due to the rapid growth of tumors, the integrity of tumor vessels is not good, and common nanoformulations are easily penetrated from tumor vessels while also being difficult to re-circulate back to the systemic circulation via the lymphatic route, this enhanced permeability and retention effect being termed a high penetration long retention effect. The concentration of albumin in blood is about 40mg/mL, the concentration of albumin in tumor interstitium is about 14mg/mL, and under the condition of the concentration difference, the albumin shows good tumor passive targeting through the high permeation long retention effect. Furthermore, rapid metabolism and growth of tumor cells requires active uptake of large amounts of extracellular proteins (including albumin) as a source of amino acids, which also enables albumin to be efficiently accumulated in tumor tissue. However, the binding capacity of the doxorubicin and the albumin is poor, the drug loading rate and the encapsulation rate of the formed nanoparticle are low, and the particle size is uneven. The prodrug strategy can improve the adverse properties of the chemotherapeutic drugs through ingenious structural modification, such as improving the solubility of the drugs, enhancing the targeting property, reducing the toxic and side effects of the drugs and the like. Albumin has 7 fatty acid binding sites and therefore, doxorubicin-fatty acid prodrugs are expected to enhance the affinity of doxorubicin for albumin. Fatty acids of different carbon chain lengths have different affinities for albumin and therefore have an impact on the pharmaceutics, in vivo fate and anti-tumor effects of the prodrug albumin nanoparticles.
Disclosure of Invention
In order to overcome the defects of the prior art, the technical problem to be solved by the invention is to provide the pH sensitive doxorubicin-fatty acid prodrug and the albumin nanoparticle thereof, wherein the nanoparticle has the effects of high drug loading capacity, high encapsulation efficiency, good stability, low toxic and side effects and intelligent intracellular drug release. The pH sensitive doxorubicin-fatty acid prodrug and the albumin nanoparticle thereof obviously reduce the toxic and side effects of the doxorubicin and improve the tolerance dose of the doxorubicin.
The invention designs and synthesizes the pH sensitive doxorubicin prodrug containing fatty acids with different carbon chain lengths, and prepares prodrug albumin nano-particles. Experimental results show that the carbon chain length of fatty acid can influence the pharmaceutics, pharmacodynamics and safety of prodrug albumin nanoparticles, provide a new strategy and more choices for developing an albumin-based antitumor drug delivery system, and meet urgent requirements of high-efficiency and low-toxicity chemotherapy preparations in clinic.
In order to achieve the above object, the present invention provides a pH-sensitive hydrazone-bridged doxorubicin-fatty acid prodrug represented by the general formula (I):
Wherein R is a part of C 10-C14 saturated or unsaturated fatty acid which does not contain carboxyl hydroxyl.
Further, R is a part which does not contain carboxyl hydroxyl in n-capric acid, myristic acid, myristoleic acid and tetradecadienoic acid.
Specifically, the pH-sensitive doxorubicin-fatty acid prodrug or pharmaceutically acceptable salt thereof is:
Further, the invention provides a synthesis method of the pH sensitive doxorubicin-fatty acid prodrug, which comprises the following steps:
(1) Refluxing fatty acid, methanol and concentrated sulfuric acid to obtain fatty acid methyl ester;
(2) Refluxing the fatty acid methyl ester and hydrazine hydrate in the step (1) to obtain a fatty acid hydrazide intermediate product;
(3) The fatty acid hydrazide intermediate product reacts with the carbonyl of the doxorubicin hydrochloride, and the pH sensitive doxorubicin-fatty acid prodrug is obtained by recrystallization, wherein the yield is more than 90% and the purity is more than 99%.
Wherein the fatty acid in the step (1) is C 10-C14 saturated fatty acid or unsaturated fatty acid;
Fatty acid (moles): methanol (ml): the proportion of concentrated sulfuric acid (milliliters) is as follows: 1:2000-5000:20-100, preferably 1:2000-4000:40-60;
in the step (2), the molar ratio of fatty acid methyl ester to hydrazine hydrate is 1:5-30, preferably 1:10-20;
in the step (3), doxorubicin hydrochloride: the molar ratio of the fatty acid hydrazide intermediate products is 1:1-5; the recrystallization solvent is acetone.
Further, the preparation method of the pH sensitive doxorubicin-fatty acid prodrug comprises the following steps:
(1) Synthesis of fatty acid methyl ester: adding fatty acid and methanol into a round-bottom flask, adding concentrated sulfuric acid, and refluxing for 12-18 hours; concentrating to recover methanol, pouring the reaction solution into ice water, adding saturated sodium bicarbonate, neutralizing to neutrality, extracting, and concentrating;
wherein the solvent for extraction is ethyl acetate or dichloromethane.
The fatty acid is C 10-C14 saturated fatty acid or unsaturated fatty acid.
(2) Synthesis of fatty acid hydrazide: dissolving the fatty acid methyl ester in the step (1) in ethanol, adding hydrazine hydrate, and refluxing for 8-12 hours. After concentration, the reaction liquid is poured into water to separate out solid, and the fatty acid hydrazide intermediate product is obtained through suction filtration.
(3) Dissolving doxorubicin hydrochloride, glacial acetic acid and fatty acid hydrazide intermediate products in absolute methanol, condensing and refluxing at 50-60 ℃ for reaction for 12-24 hours, concentrating, recrystallizing and carrying out suction filtration to obtain the doxorubicin hydrochloride and fatty acid hydrazide intermediate product.
Wherein, glacial acetic acid: the volume ratio of the anhydrous methanol is 1:100-200.
Wherein, doxorubicin hydrochloride: the molar ratio of the fatty acid hydrazide intermediate product is 1:1-5.
Wherein the recrystallization solvent is acetone.
Wherein R is C 9-C13 alkyl.
The adriamycin-n-capric acid prodrug and the adriamycin-myristic acid prodrug have good physicochemical properties of n-capric acid and myristic acid, have larger polarity and better solubility, and the purity after recrystallization can reach 99.4 percent. The doxorubicin-stearic acid prodrug with longer carbon chain has purity of 85% after recrystallization, and the final product is still obtained by preparation of liquid phase separation and purification.
The invention provides pH-sensitive doxorubicin-fatty acid prodrug albumin nanoparticles, which comprise a pH-sensitive doxorubicin-fatty acid prodrug and serum albumin, wherein the mass ratio of the pH-sensitive doxorubicin-fatty acid prodrug to the serum albumin is 1:0.1-10, preferably 1:1-3.
Furthermore, the invention also provides a preparation method of the albumin nanoparticle of the series of pH-sensitive doxorubicin-fatty acid prodrugs, and the albumin nanoparticle is prepared by adopting an ultrasonic method or a high-pressure homogenization method.
Specifically, the preparation method of the pH sensitive doxorubicin-fatty acid prodrug albumin nanoparticle provided by the invention comprises the following steps:
Weighing pH sensitive doxorubicin-fatty acid prodrug, dissolving the prodrug with methanol, slowly dripping the obtained solution into serum albumin aqueous solution under stirring, and homogenizing under ultrasonic or high pressure to form uniform albumin nanoparticles. Distilling under reduced pressure at 25-30deg.C to remove organic solvent in the nanometer preparation.
The serum albumin is bovine serum albumin or human serum albumin.
The concentration of the serum albumin is 0.1mg/mL-100mg/mL.
The mass ratio of the pH sensitive doxorubicin-fatty acid prodrug to serum albumin is 1:0.1-10, preferably 1:1-3.
In the ultrasonic method, the power of ultrasonic waves is 30-100W, preferably 60-80W.
The time of the ultrasound is 1 to 5 minutes, preferably 2 to 3 minutes.
In the high-pressure homogenization process, the pressure is 500 to 1000bar, preferably 600 to 800bar.
In the high pressure homogenization method, the time is 10 to 30 minutes, preferably 10 to 15 minutes.
In the invention, the prodrug must be converted into the parent drug in vivo to exert the drug effect function, so that the pH sensitive doxorubicin-fatty acid prodrug bridged by hydrazone bonds and albumin nanoparticles thereof are constructed, and the nanoparticles firstly enter endosomes and lysosomes after being taken up by tumor cells, so that the hydrazone bonds of prodrug molecules are broken under the low pH conditions of the endosomes and lysosomes, the prodrug is converted into the parent drug, the anti-tumor effect of the prodrug is exerted, a new strategy and more choices are provided for developing an intelligent response type drug delivery system in tumor microenvironment, and the urgent requirements of clinical high-efficiency and low-toxicity chemotherapeutic preparations are met.
The invention has the following beneficial effects: (1) The pH sensitive doxorubicin-fatty acid prodrugs with different carbon chain lengths are designed and synthesized, the synthesis method is simple and easy to implement, and the purity of the product can reach more than 99% through recrystallization; (2) The preparation method is simple and feasible, and has higher drug-loading rate and encapsulation rate; (3) The differences of the prodrug albumin nanoparticle in the aspects of pharmaceutics, cytotoxicity, pharmacokinetics, pharmacodynamics and the like are examined, and test results show that the doxorubicin-fatty acid prodrug albumin nanoparticle has good anti-tumor effect, can effectively reduce toxic and side effects of the doxorubicin, has different anti-tumor activities and safety of the prodrug with different structures, and provides a new strategy for developing high-efficiency and low-toxicity chemical therapy preparations.
Drawings
FIG. 1 is a mass spectrum of doxorubicin-n-decanoic acid prodrug (DOX-C 10) of example 1 of the present invention.
FIG. 2 is a 1 HNMR spectrum of doxorubicin-n-decanoic acid prodrug (DOX-C 10) of example 1 of the present invention.
FIG. 3 is a mass spectrum of doxorubicin-myristic acid prodrug (DOX-C 14) of example 2 of the present invention.
FIG. 4 is a 1 HNMR spectrum of the doxorubicin-myristic acid prodrug (DOX-C 14) of example 2 of the present invention.
FIG. 5 is a mass spectrum of doxorubicin-stearic acid prodrug (DOX-C 18) of example 3 of the present invention.
FIG. 6 is a 1 HNMR spectrum of the doxorubicin-stearic acid prodrug (DOX-C 18) of example 3 of the present invention.
FIG. 7 is a 4T1 cytotoxicity profile of doxorubicin-fatty acid prodrug albumin nanoparticles of example 6 of the invention.
FIG. 8 is a graph of prodrug blood concentration versus time for doxorubicin-fatty acid prodrug albumin nanoparticles of example 7 of the invention.
FIG. 9 is a graph of the plasma concentration versus time of the parent drug for the doxorubicin-fatty acid pro-drug albumin nanoparticle of example 7 of the invention.
FIG. 10 is a graph of prodrug drug addition versus time for doxorubicin-fatty acid prodrug albumin nanoparticles of example 7 of the invention.
FIG. 11 is a graph showing the tumor volume change of mice in an in vivo anti-tumor experiment of doxorubicin-fatty acid prodrug-albumin nanoparticle of example 8 of the present invention.
**：P<0.01；***：P<0.001；****：P<0.0001。
FIG. 12 is a graph showing comparison of mouse tumors in an in vivo anti-tumor experiment of doxorubicin-fatty acid prodrug-albumin nanoparticle of example 8 of the present invention.
FIG. 13 is a graph showing the weight change of mice in an in vivo antitumor experiment of doxorubicin-fatty acid prodrug-albumin nanoparticle of example 8 of the present invention.
**：P<0.01；***：P<0.001；****：P<0.0001。
FIG. 14 is a graph showing tumor burden in vivo anti-tumor experiments for doxorubicin-fatty acid prodrug albumin nanoparticles of example 8 of the invention.
* *: P <0.01; * **: p <0.001; ns: no significant differences.
FIG. 15 is a graph showing spleen weight in an in vivo antitumor experiment of doxorubicin-fatty acid prodrug albumin nanoparticle of example 8 of the invention.
* *: P <0.01; * **: p <0.001; ns: no significant differences.
FIG. 16 is a comparison of spleen in an in vivo anti-tumor experiment of doxorubicin-fatty acid prodrug albumin nanoparticle of example 8 of the invention.
FIG. 17 is a graph showing the comparison of leukocytes, lymphocytes, monocytes and neutrophils in an in vivo anti-tumor assay of doxorubicin-fatty acid pro-drug albumin nanoparticle of example 8 of the invention.
FIG. 18 is a graph showing the percentage comparison of lymphocytes, monocytes and neutrophils in an in vivo anti-tumor assay of doxorubicin-fatty acid pro-drug albumin nanoparticle of example 8 of the invention.
FIG. 19 is a graph showing the comparison of red blood cells, hemoglobin and related parameters in an in vivo anti-tumor experiment of doxorubicin-fatty acid pro-drug albumin nanoparticle of example 8 of the present invention.
FIG. 20 is a graph showing comparison of platelets, platelet volume, platelet distribution and platelet volume in an in vivo anti-tumor experiment of doxorubicin-fatty acid prodrug albumin nanoparticle of example 8 of the present invention.
FIG. 21 is a graph showing the comparison of glutamic-oxaloacetic transaminase, glutamic-pyruvic transaminase, urea nitrogen and creatinine in an in vivo anti-tumor experiment of doxorubicin-fatty acid prodrug albumin nanoparticle of example 8 of the present invention.
FIG. 22 is a graph showing the tumor volume change of mice in an in vivo anti-tumor experiment of doxorubicin-fatty acid prodrug-albumin nanoparticle of example 9 of the present invention.
**：P<0.01；***：P<0.001；****：P<0.0001。
FIG. 23 is a graph showing comparison of mouse tumors in an in vivo anti-tumor experiment of doxorubicin-fatty acid prodrug-albumin nanoparticle of example 9 of the present invention.
FIG. 24 is a graph showing the weight change of mice in an in vivo antitumor experiment of doxorubicin-fatty acid prodrug-albumin nanoparticle of example 9 of the present invention.
* *: P <0.01; * **: p <0.001; * ***: p <0.0001; ns: no significant differences.
FIG. 25 is a graph showing tumor burden in an in vivo anti-tumor experiment of doxorubicin-fatty acid prodrug albumin nanoparticle of example 9 of the invention.
*：P<0.05；****：P<0.0001。
FIG. 26 is a graph showing survival in an in vivo anti-tumor experiment of doxorubicin-fatty acid prodrug albumin nanoparticle of example 9 of the invention.
FIG. 27 is a graph showing the comparison of leukocytes, lymphocytes, monocytes and neutrophils in an in vivo anti-tumor assay of doxorubicin-fatty acid pro-drug albumin nanoparticle of example 9 of the invention.
FIG. 28 is a graph showing the percentage of lymphocytes, monocytes and neutrophils in an in vivo anti-tumor assay of doxorubicin-fatty acid pro-drug albumin nanoparticle of example 9 of the invention.
FIG. 29 is a graph showing the comparison of red blood cells, hemoglobin and related parameters in an in vivo anti-tumor experiment of doxorubicin-fatty acid pro-drug albumin nanoparticle of example 9 of the present invention.
FIG. 30 is a graph showing comparison of platelets, platelet volume, platelet distribution and platelet volume in an in vivo anti-tumor experiment of doxorubicin-fatty acid prodrug albumin nanoparticle of example 9 of the present invention.
FIG. 31 is a graph showing the comparison of glutamic-oxaloacetic transaminase, glutamic-pyruvic transaminase, urea nitrogen and creatinine in an in vivo anti-tumor experiment of doxorubicin-fatty acid prodrug albumin nanoparticle of example 9 of the present invention.
Detailed Description
The invention is further illustrated by way of examples which follow, but are not thereby limited to the scope of the examples described.
Example 1: synthesis of doxorubicin-n-decanoic acid (DOX-C 10) prodrugs
17.2G of n-decanoic acid (0.1 mol) and 300mL of methanol were added to a round bottom flask, 5mL of concentrated sulfuric acid was added, and the mixture was refluxed for 12 hours; after concentration, pouring the reaction liquid into ice water, adding saturated sodium bicarbonate, neutralizing to neutrality, extracting with ethyl acetate, and concentrating to obtain fatty acid methyl ester intermediate; dissolving the intermediate product in ethanol, adding 100mL of hydrazine hydrate, and refluxing for 12 hours; concentrating, pouring the reaction solution into water, precipitating solid, and filtering to obtain a fatty acid hydrazide intermediate product; 5.8g of doxorubicin hydrochloride (0.01 mol), 10mL of glacial acetic acid and 3.6g of fatty acid hydrazide intermediate product (0.015 mol) are dissolved in absolute methanol, and the mixture is refluxed at 50-60 ℃ for 12 hours, concentrated, recrystallized by using acetone as a solvent and filtered by suction. Yield: 94%, purity: 99%.
The structure of the prodrug in example 1 was determined by mass spectrometry and nuclear magnetic resonance hydrogen spectrometry, and the results are shown in fig. 1 and 2, and the results of the spectroscopic analysis are as follows:
1H NMR(600MHz,DMSO-d6)δ14.12(s,1H,13-OH),13.34(s,1H,6-OH),10.29(s,1H,11'-NH),7.95-7.91(m,2H,H-1,H-2),7.85(s,3H,21-NH3),7.70-7.64(m,1H,H-18),5.77(s,1H,9-OH),5.56(s,1H,10'-OH),5.47(s,1H,19-H),5.30(d,J＝3.6Hz,1H,22-OH),4.93(t,J＝7.2Hz,1H,H-21),4.48-4.35(m,2H,H-10'),4.02(q,J＝6.7Hz,1H,23-H),3.98(s,3H,17-OCH3),3.57(s,1H,H-11),3.36(s,1H,H-22),3.33(m,1H,H-8),2.72(d,J＝17.2Hz,1H,H-8),2.21-2.10(m,3H,H-10,H-12'),2.06(ddd,J＝15.2,8.4,6.8Hz,1H,H-10),1.89(td,J＝12.7,3.8Hz,1H,H-20),1.74(dd,J＝12.5,4.5Hz,1H,H-20),1.51(t,J＝7.2Hz,2H,H-13'),1.27-1.22(m,12H,H-(13'-19')),1.17(d,J＝6.5Hz,3H,H-23),0.86(t,J＝7.0Hz,3H,H-20').MS(ESI)m/z for C37H49N3O11H[M+H]+:712.34327.
example 2: synthesis of doxorubicin-myristic acid (DOX-C 14) prodrugs
22.8G of myristic acid (0.1 mol) and 300mL of methanol were added to a round bottom flask, 5mL of concentrated sulfuric acid was added, and the mixture was refluxed for 12 hours; after concentration, pouring the reaction liquid into ice water, adding saturated sodium bicarbonate, neutralizing to neutrality, extracting with ethyl acetate, and concentrating to obtain fatty acid methyl ester intermediate; dissolving the intermediate product in ethanol, adding 100mL of hydrazine hydrate, and refluxing for 12 hours; concentrating, pouring the reaction solution into water, precipitating solid, and filtering to obtain a fatty acid hydrazide intermediate product; 5.8g of doxorubicin hydrochloride (0.01 mol), 10mL of glacial acetic acid and 3.6g of fatty acid hydrazide intermediate product (0.015 mol) are dissolved in absolute methanol, and the mixture is refluxed at 50-60 ℃ for 12 hours, concentrated, recrystallized by using acetone as a solvent and filtered by suction. Yield: 92%, purity: 99%.
The structure of the prodrug in example 2 was determined by mass spectrometry and nuclear magnetic resonance hydrogen spectrometry, and the results are shown in fig. 3 and 4, and the results of the spectroscopic analysis are as follows:
1H NMR(600MHz,DMSO-d6)δ10.29(s,1H,11'-NH),7.94-7.90(m,2H,H-1,H-2),7.69-7.64(m,1H,H-18),5.76(t,J＝4.9Hz,1H,9-OH),5.53(s,1H,10'-OH),5.42(d,J＝6.0Hz,1H,19-H),5.30(d,J＝3.6Hz,1H,22-OH),4.92(t,J＝7.1Hz,1H,H-21),4.46-4.36(m,2H,H-10'),4.03(q,J＝6.7Hz,1H,23-H),3.98(s,3H,17-OCH3),3.55(s,1H,H-11),3.36(s,1H,H-22),3.33(s,1H,H-8),2.73(d,J＝17.2Hz,1H,H-8),2.21-2.10(m,2H,H-10,H-12'),2.06(ddd,J＝15.2,8.3,6.7Hz,1H,H-10),1.87(dt,J＝12.8,6.4Hz,1H,H-20),1.73(dd,J＝12.3,4.4Hz,1H,H-20),1.35-0.92(m,26H,H-(12'-23'),H-23),0.85(t,J＝7.0Hz,3H,H-24').MS(ESI)m/zfor C41H57N3O11H[M+H]+:768.40531.
Example 3: synthesis of doxorubicin-stearic acid (DOX-C 18) prodrugs
28.0G of stearic acid (0.1 mol) and 300mL of methanol were added to a round bottom flask, 5mL of concentrated sulfuric acid was added, and the mixture was refluxed for 12 hours; after concentration, pouring the reaction liquid into ice water, adding saturated sodium bicarbonate, neutralizing to neutrality, extracting with ethyl acetate, and concentrating to obtain fatty acid methyl ester intermediate; dissolving the intermediate product in ethanol, adding 100mL of hydrazine hydrate, and refluxing for 12 hours; concentrating, pouring the reaction solution into water, precipitating solid, and filtering to obtain a fatty acid hydrazide intermediate product; 5.8g of doxorubicin hydrochloride (0.01 mol), 10mL of glacial acetic acid and 3.6g of fatty acid hydrazide intermediate product (0.015 mol) are dissolved in absolute methanol, and the mixture is refluxed at 50-60 ℃ for 12 hours, concentrated, recrystallized by using acetone as a solvent and filtered by suction. Yield: 80%, purity: 85%. The target product is separated and purified by preparative liquid chromatography, and the purity is 99%.
The structure of the prodrug in example 3 was determined by mass spectrometry and nuclear magnetic resonance hydrogen spectrometry, and the results are shown in fig. 5 and 6. The results of the spectroscopic analysis are as follows:
1H NMR(600MHz,DMSO-d6)δ10.31(s,1H,NH),7.89(dd,J＝9.1Hz,5.1Hz,2H,Ph-H),7.65(ddd,J＝9.6Hz,6.2Hz and 3.2Hz,1H,Ph-H),5.63(s,1H,9-OH),5.58(s,1H,4'-OH),5.51-5.28(s,1H,14-OH),5.28(s,1H,1'-H),4.90(t,J＝6.9Hz,1H,7-H),4.41(q,J＝14.4Hz,2H,14-H),4.06-3.99(m,1H,5'-H),3.97(d,J＝9.8Hz,3H,4-OCH3),3.54(s,1H,4'-H),3.32(s,1H,3'-H),2.73(d,J＝17.2Hz,1H,10-H),2.16(ddd,J＝23.3Hz,15.6Hz and 7.5Hz,2H,2"-H),2.06(dd,J＝15.3Hz and 7.9Hz,1H,8-H),2.03-1.88(m,1H,8-H),1.84(d,J＝12.7Hz,1H,2'-H),1.68-1.57(m,1H,2'-H),1.33-0.97(m,33H),0.84(t,J＝7.0Hz,3H,18"-CH3).MS(ESI)m/z for C45H65N3O11H[M+H]+:824.4696.
Example 4: investigation of influence factors of pH-sensitive doxorubicin-fatty acid prodrug albumin nanoparticles
TABLE 1 particle size, particle size distribution and encapsulation efficiency of Adriamycin-fatty acid prodrugs and albumin at different Mass ratios
Accurately weighing the pH sensitive doxorubicin prodrug, dissolving the pH sensitive doxorubicin prodrug with 1mL of methanol, slowly dripping the methanol solution into 4mL of aqueous solution of bovine serum albumin (or human serum albumin and canine serum albumin) under stirring, and performing ultrasonic treatment by using an ultrasonic cell disruption instrument to form uniform albumin nanoparticles, wherein the ultrasonic treatment time is 2min, and the ultrasonic power is 60W. The organic solvent in the formulation was removed by rotary evaporator at 25 ℃. The mass ratio of doxorubicin-fatty acid prodrug to albumin was varied and the results are shown in table 1. When the mass ratio of the prodrug to the albumin is 1:1-3, the particle size of the prodrug albumin nanoparticle is 50-200nm, and the particle size distribution is uniform; when the mass ratio is 1:2-3, the particle size distribution and the encapsulation efficiency are optimal.
Example 5: preparation of pH sensitive type adriamycin-fatty acid prodrug albumin nanoparticle
Accurately weighing 4mg of the pH sensitive doxorubicin prodrug, dissolving the prodrug with 1mL of methanol, slowly dripping the methanol solution into 4mL of bovine serum albumin (or human serum albumin and canine serum albumin) water solution with the concentration of 2mg/mL under stirring, enabling the mass ratio of the prodrug to albumin to be 1:2, and then forming uniform albumin nanoparticles by ultrasonic waves of an ultrasonic cell disruption instrument, wherein the ultrasonic time is 2min and the ultrasonic power is 60W. The organic solvent in the nanofabricated formulation was removed with a rotary evaporator at 25 ℃.
Characterization of pH sensitive Adriamycin-fatty acid prodrug Albumin nanoparticles
As shown in Table 2, the affinity of doxorubicin and albumin is poor, the particle size of the formed nanoparticle is up to 480nm, the particle size distribution is up to 0.6, and the encapsulation efficiency is less than 20%. In contrast, the particle size of the albumin nanoparticle of the three prodrugs is about 120nm, the particle size distribution is less than 0.2, the encapsulation efficiency is more than 85%, and the balling rate is more than 90%.
Example 6: cytotoxicity experiment of pH sensitive Adriamycin-fatty acid prodrug Albumin nanoparticles
The toxicity of doxorubicin-fatty acid prodrug albumin nanoparticle on mouse breast cancer (4T 1) cells was examined using the MTT method. The cells in good condition are digested, diluted to 5000cells/mL by culture solution, 100 mu L of cell suspension is added to each well of a 96-well plate after the cells are uniformly blown, and the cells are placed in an incubator for incubation for 24 hours to adhere to the cells. Adriamycin solution or the doxorubicin-fatty acid pro-drug albumin nanoparticle prepared in example 5 was added after cell attachment and incubated for a further 48 hours, using untreated cells as controls. At the end of incubation, 20. Mu.L MTT (5 mg/mL) was added to each well and incubated at 37℃for 4 hours. The medium was discarded and 200. Mu.L of DMSO was added to each well and shaken on a shaker for 10min. Absorbance was measured at 570nm using a microplate reader.
The cytotoxicity results are shown in fig. 7, where the prodrug albumin nanoparticle has reduced cytotoxicity compared to doxorubicin solution. This is because doxorubicin needs to be released from the prodrug nanoparticles, and the cytotoxicity of doxorubicin is limited by the drug release process. The longer the fatty acid carbon chain length, the more cytotoxic the prodrug albumin nanoparticle.
Example 7: pharmacokinetic study of pH-sensitive doxorubicin-fatty acid prodrug albumin nanoparticles
Pharmacokinetic studies were performed using SD rats (200-250 g). Rats were randomized and fasted for 12 hours prior to dosing, with free water. The doxorubicin solutions and the three doxorubicin-fatty acid pro-drug albumin nanoparticles prepared in example 5 were each intravenously injected. The dosage of doxorubicin was 4mg/kg. The orbit was bled at the prescribed time points and plasma was isolated. The drug concentration in the plasma was determined by liquid chromatography-mass spectrometry.
The experimental results are shown in fig. 8-10, compared with the doxorubicin solution group, the residence time of the prodrug albumin nanoparticle in blood is prolonged, the area under the drug-time curve (AUC 0-24 h) of doxorubicin is remarkably improved, and the guarantee is provided for tumor targeting accumulation of doxorubicin. At the same time, the length of the fatty acid carbon chain has a significant impact on the pharmacokinetic behavior of the prodrug albumin nanoparticle. The total AUC 0-24 h (sum of prodrug and parent) of doxorubicin-n-decanoic acid prodrug albumin nanoparticle, doxorubicin-myristic acid prodrug albumin nanoparticle and doxorubicin-stearic acid prodrug albumin nanoparticle was 1.37 times, 5.18 times and 13.73 times, respectively, that of doxorubicin solution.
Pharmacokinetic parameters of pH sensitive doxorubicin-fatty acid prodrug albumin nanoparticles
Example 8: antitumor experiment of pH-sensitive doxorubicin-fatty acid prodrug albumin nanoparticle
A4T 1 cell tumor-bearing mouse model was established and 4T1 cells (5X 10 6 cells in 100. Mu.L) were inoculated subcutaneously into BALB/c mice. When the tumor volume was as long as about 100-150mm 3, it was randomly divided into 7 groups (5 mice per group). Adriamycin-fatty acid pro-drug albumin nanoparticle of example 5 was administered every other day tail vein, and doxorubicin solution, commercially available doxorubicin hydrochloride liposome (Doxil), doxorubicin maleimide pro-drug (DOXO-EMCH) and physiological saline were set as control groups, and injected 5 times in total, with a dose of 10mg/kg of equivalent doxorubicin, and tumor volume and mouse body weight were measured daily.
As shown in FIGS. 11-16, the doxorubicin liposome had the best antitumor effect, but the mice had the most significant weight loss compared with the physiological saline group, and exhibited serious toxic and side effects. The doxorubicin solution group and DOXO-EMCH group also have good anti-tumor effect, but after the fifth administration, all mice die and the toxic and side effects are very strong. The three pro-drugs albumin nanoparticles all had significant anti-tumor effects (fig. 11, 12). Fig. 14 shows that there is no significant difference in tumor burden rate between doxorubicin-n-decanoic acid prodrug, doxorubicin-myristic acid prodrug and doxorubicin-stearic acid prodrug. However, the body weight of the doxorubicin-stearic acid prodrug was significantly reduced, whereas the body weight of the mice in the doxorubicin-n-decanoic acid prodrug and doxorubicin-myristic acid prodrug groups were not reduced, and there was no significant difference from the physiological saline group, indicating that the safety was better and the toxicity was lower (fig. 13). Figures 15-16 show that compared with normal mice without tumor, the spleen of the doxorubicin liposome group mice is shrunken, which shows that the mice have stronger myelosuppression toxicity, while the spleen of the doxorubicin-n-decanoic acid prodrug and doxorubicin-myristic acid prodrug group mice is not shrunken, which shows that the safety is better. The results of the blood routine and liver and kidney function tests for each group are shown in FIGS. 17-21. Compared with the adriamycin-stearic acid prodrug with longer carbon chain length, the adriamycin-n-capric acid prodrug and the adriamycin-myristic acid prodrug albumin nanoparticle have good anti-tumor effect and better tolerance and safety.
Example 9: antitumor experiment of pH-sensitive doxorubicin-fatty acid prodrug albumin nanoparticle
A KB cell tumor-bearing nude mouse model was established and KB cells (5X 10 6 cells in 100. Mu.L) were inoculated subcutaneously into BALB/c-Nu nude mice. When the tumor volume was as long as about 100-150mm 3, it was randomly divided into 5 groups (5 mice per group). Adriamycin-myristic acid prodrug albumin nanoparticle of example 5 was administered every two days, a commercial doxorubicin hydrochloride liposome (Doxil) and physiological saline were set as control groups, wherein the physiological saline group and the doxorubicin-myristic acid prodrug albumin nanoparticle group were injected 4 times in total, and the commercial doxorubicin hydrochloride liposome (Doxil) group was injected 2 times in total, at doses of 15mg/kg and 20mg/kg (equivalent doxorubicin 15mg/kg, 20 mg/kg), and tumor volumes and mouse weights were measured daily.
As shown in FIGS. 22-26, the doxorubicin liposome group died on the eighth day and all died on the ninth day at the dose of 20mg/kg, and the toxic and side effects were very strong. At a dose of 15mg/kg, the doxorubicin liposome group died on day ten, all of which died on day eleventh (fig. 26). Whereas the doxorubicin-myristic acid prodrug albumin nanoparticle group was administered four times, the prodrug albumin nanoparticle had a significant antitumor effect at the administration doses of 15mg/kg and 20mg/kg (fig. 22, 23), and the mice did not die, did not have a decrease in body weight, and did not have a significant difference from the physiological saline group, indicating a better safety and lower toxicity (fig. 24). The results of blood routine and liver and kidney function tests of each group are shown in figures 27-31, and neither leucocytes nor lymphocytes of the doxorubicin-n-decanoic acid prodrug nor doxorubicin-myristic acid prodrug group are reduced, nor cause obvious liver and kidney injury. Especially, the leucocyte, the lymphocyte and the like of the mice of the adriamycin-myristic acid prodrug albumin nanoparticle group are not reduced, which indicates that bone marrow suppression does not occur, and liver and kidney function indexes indicate that the mice are not damaged to the liver and the kidney. Therefore, the adriamycin-n-capric acid prodrug and the adriamycin-myristic acid prodrug have remarkable anti-tumor effect, small toxic and side effects and high safety. While the adriamycin myristic acid prodrug albumin nanoparticle has obvious anti-tumor effect, the toxicity is the lowest, and the safety is the highest.
Claims (9)
- Albumin nanoparticle of pH sensitive doxorubicin-fatty acid prodrug, wherein the albumin nanoparticle comprises pH sensitive doxorubicin-fatty acid prodrug and serum albumin, and the mass ratio of the pH sensitive doxorubicin-fatty acid prodrug to the serum albumin is 1:1-3; the pH sensitive doxorubicin-fatty acid prodrug has the following structure:。
- 2. albumin nanoparticle of a pH sensitive doxorubicin-fatty acid prodrug according to claim 1, wherein said pH sensitive doxorubicin-fatty acid prodrug is prepared by the steps of:(1) Reflux of myristic acid, methanol and concentrated sulfuric acid to obtain methyl myristate;(2) Refluxing the methyl myristate and hydrazine hydrate in the step (1) to obtain a myristic acid hydrazide intermediate product;(3) And (3) reacting the myristic acid hydrazide intermediate product with doxorubicin hydrochloride carbonyl, and recrystallizing to obtain the product.
- 3. Albumin nanoparticle of a pH sensitive doxorubicin-fatty acid prodrug according to claim 2, wherein in step (1), myristic acid: methanol: the ratio of the concentrated sulfuric acid is as follows: 2000-5000 ml of 1 mol and 20-100 ml of water; in the step (2), the molar ratio of the methyl myristate to the hydrazine hydrate is 1:5-30; in the step (3), doxorubicin hydrochloride: the molar ratio of the myristic acid hydrazide intermediate product is 1:1-5.
- 4. Albumin nanoparticle of a pH sensitive doxorubicin-fatty acid prodrug according to claim 2, wherein in step (2) the molar ratio of methyl myristate to hydrazine hydrate is 1:10-20.
- 5. Albumin nanoparticle of a pH sensitive doxorubicin-fatty acid prodrug according to claim 2, wherein in step (3), the recrystallization solvent is acetone.
- 6. The method for preparing albumin nanoparticle of pH sensitive doxorubicin-fatty acid prodrug according to claim 1, wherein: the pH sensitive doxorubicin-fatty acid prodrug is weighed, dissolved by an organic solvent, and the obtained solution is slowly dripped into a serum albumin aqueous solution under stirring, and uniform albumin nano-particles are formed through ultrasonic or high-pressure homogenization.
- 7. Use of the pH sensitive doxorubicin-fatty acid pro-drug albumin nanoparticle of claim 1 in the preparation of a drug delivery system.
- 8. The use of the pH sensitive doxorubicin-fatty acid pro-drug albumin nanoparticle of claim 1 in the preparation of an antitumor drug.
- 9. Use of the pH sensitive doxorubicin-fatty acid pro-drug albumin nanoparticle of claim 1 in the preparation of an injectable, oral or topical delivery system.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110933636.1A CN115702902B (en) | 2021-08-14 | 2021-08-14 | Anti-tumor preparation of doxorubicin prodrug |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110933636.1A CN115702902B (en) | 2021-08-14 | 2021-08-14 | Anti-tumor preparation of doxorubicin prodrug |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115702902A CN115702902A (en) | 2023-02-17 |
| CN115702902B true CN115702902B (en) | 2024-04-26 |
Family
ID=85181186
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110933636.1A Active CN115702902B (en) | 2021-08-14 | 2021-08-14 | Anti-tumor preparation of doxorubicin prodrug |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115702902B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113713117B (en) * | 2021-09-10 | 2024-01-19 | 山东大学 | Albumin-binding type tumor environment response type antitumor prodrug and preparation method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001068142A1 (en) * | 2000-03-13 | 2001-09-20 | Ktb Tumorforschungsgesellschaft Mbh | Therapeutic and diagnostic ligand systems comprising transport molecule binding properties and medicaments containing the same |
| WO2018013783A1 (en) * | 2016-07-13 | 2018-01-18 | Cephalon, Inc. | Pharmaceutical prodrugs and methods of their preparation and use |
| CN112386586A (en) * | 2020-12-01 | 2021-02-23 | 苏州大学 | Preparation method of albumin nanoparticles |
-
2021
- 2021-08-14 CN CN202110933636.1A patent/CN115702902B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001068142A1 (en) * | 2000-03-13 | 2001-09-20 | Ktb Tumorforschungsgesellschaft Mbh | Therapeutic and diagnostic ligand systems comprising transport molecule binding properties and medicaments containing the same |
| WO2018013783A1 (en) * | 2016-07-13 | 2018-01-18 | Cephalon, Inc. | Pharmaceutical prodrugs and methods of their preparation and use |
| CN112386586A (en) * | 2020-12-01 | 2021-02-23 | 苏州大学 | Preparation method of albumin nanoparticles |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115702902A (en) | 2023-02-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108670954B (en) | Chemotherapeutic drug co-loaded glycyrrhetinic acid prodrug micelle and preparation method thereof | |
| KR101597338B1 (en) | Novel compositions and processes for preparing 5-amino or substituted amino 1,2,3-triazoles and triazole orotate formulations | |
| CN115702902B (en) | Anti-tumor preparation of doxorubicin prodrug | |
| CN109350598B (en) | Sugar-polyethylene glycol-DSPE coupling compound and preparation method and application thereof | |
| CN111298132B (en) | Tree-shaped molecule gemcitabine self-assembled nano prodrug and preparation method and application thereof | |
| CN110433292A (en) | A kind of double targeting materials and its application in drug delivery | |
| CN108586551B (en) | Preparation and application of IR 780-L A/CPT-ss-CPT nanoparticles | |
| CN103360590B (en) | Preparation method of methoxy polyethylene glycol-bi-fatty acryl phosphatidyl ethanolamine | |
| CN112979755A (en) | polypeptide capable of forming hydrogel through pH response drug-loading self-assembly, preparation method and application | |
| CN115212185B (en) | Albumin nanoparticles of pH-sensitive doxorubicin-fatty acid prodrugs | |
| CN113461754B (en) | Base-modified adriamycin prodrug and preparation method and application thereof | |
| CN108434457B (en) | Adriamycin polyethylene glycol epothilone B conjugate and preparation method thereof | |
| CN110917212A (en) | Graphene base targeting DNA major groove and inhibiting topoisomerase and preparation method and application thereof | |
| CN113398276B (en) | Preparation and application of brain glioma targeted berberine and folic acid modified lipid material | |
| CN115487308A (en) | Antisense nucleic acid glycosyl conjugate, preparation method thereof and application thereof in liver cancer treatment | |
| CN110540551B (en) | Liposome, preparation method thereof, liposome assembly and loaded liposome complex | |
| KR20120126356A (en) | Nanoparticles comprising amphiphilic low molecular weight hyaluronic acid complex and a process for the preparation thereof | |
| CN116120333B (en) | Podophyllotoxin nano prodrug and preparation method and application thereof | |
| CN106554329A (en) | Water-soluble paclitaxel anti-cancer drug compounds and its preparation method and application | |
| CN108210933B (en) | Conjugate of dezocine and polyethylene glycol | |
| CN113072582B (en) | Pi-pi conjugated pyridyl lipid derivative and preparation method and application thereof | |
| CN107998403B (en) | PEG-modified water-soluble prodrugs of triacontanol | |
| CN111840547B (en) | Preparation method of pemetrexed magnetic self-assembled nano composite particles for injection | |
| CN117756752A (en) | Prodrug anti-tumor preparation for improving safety of paclitaxel and application thereof | |
| WO2022227556A1 (en) | Docetaxel-fatty alcohol small molecule prodrug and construction of self-assembled nanoparticles thereof |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |