CN115702902A - Adriamycin prodrug antitumor preparation - Google Patents
Adriamycin prodrug antitumor preparation Download PDFInfo
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- CN115702902A CN115702902A CN202110933636.1A CN202110933636A CN115702902A CN 115702902 A CN115702902 A CN 115702902A CN 202110933636 A CN202110933636 A CN 202110933636A CN 115702902 A CN115702902 A CN 115702902A
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Abstract
The invention belongs to the technical field of medicines, relates to an adriamycin prodrug anti-tumor preparation, and particularly relates to a pH-sensitive adriamycin-fatty acid prodrug (general formula I), construction of albumin nanoparticles of the adriamycin-fatty acid prodrug, and application of the adriamycin-fatty acid prodrug in a drug delivery system. The invention synthesizes the pH-sensitive hydrazone bond bridged adriamycin prodrug containing fatty acid side chains with different lengths and prepares prodrug albumin nanoparticles. Experimental results show that the length of the carbon chain of the fatty acid can obviously influence the pharmaceutical properties, pharmacokinetics, pharmacodynamics and safety of the adriamycin prodrug albumin nanoparticles. Adriamycin-n-decanoic acid prodrug and Adriamycin-myristic acid prodrugOn the basis of ensuring the curative effect, the systemic toxicity of the adriamycin is obviously reduced, and the adriamycin has better tolerance and higher tolerance dose.
Description
Technical Field
The invention belongs to the technical field of medicines, relates to an adriamycin prodrug anti-tumor preparation, and particularly relates to a pH-sensitive adriamycin-fatty acid prodrug, construction of albumin nanoparticles of the adriamycin-fatty acid prodrug, and application of the adriamycin-fatty acid prodrug in a drug delivery system.
Background
Cancer seriously threatens the health of the whole human body. Over the past several decades, as medical technology has advanced, the treatment of cancer has rapidly progressed, including surgical therapy, chemotherapy, radiation therapy, immunotherapy, and the like. Chemotherapy is the most common form of tumor treatment. Chemotherapy kills or controls the division of tumor cells by 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. Adriamycin is a broad-spectrum antitumor antibiotic, has strong cytotoxicity and has inhibiting effect on various tumors. However, the toxic and side effects are large, especially the serious cardiotoxicity, which limits the clinical application of the medicine. Meanwhile, small molecule drugs are easily removed by the metabolism of the body in the blood circulation process, and the drugs are difficult to reach the tumor cells due to the special environment near the tumor tissue, such as a complicated vascular network structure, a compact interstitial structure and high interstitial pressure. These limitations affect the antitumor efficacy of doxorubicin. How to improve the poor physicochemical properties of the adriamycin, improve the treatment effect and reduce the toxic and side effects is a pharmaceutic problem to be solved urgently.
In recent years, researchers have developed different types of doxorubicin drug delivery systems, the most successful of which is doxorubicin liposomes, which can effectively improve the pharmacokinetics and in vivo distribution of doxorubicin, and reduce the cardiotoxicity of doxorubicin. However, even if the adriamycin liposome adopts an active drug loading mode, the drug loading rate is only 11%, and the risk of acroerythema after drug administration is obviously improved, thus seriously affecting the treatment effect and the life quality of patients. Therefore, the need for constructing a novel high-efficiency low-toxicity adriamycin nano preparation is high.
Among the numerous serum proteins, albumin, which accounts for 50% of the total protein content, is an ideal carrier for drugs. Due to the rapid growth of tumor, the integrity of tumor blood vessel is not good, and the common nanometer preparation is easy to permeate out of the tumor blood vessel and difficult to return to the systemic circulation through a lymphatic route, and the enhanced permeability and retention effect is called high-permeability long-retention effect. The concentration of albumin in blood is about 40mg/mL, the concentration of albumin in tumor stroma is about 14mg/mL, and under the condition of the concentration difference, the albumin shows good tumor passive targeting through high-permeability long-retention effect. In addition, the 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 the effective accumulation of albumin in tumor tissues. However, the binding capacity of the adriamycin and the albumin is poor, the drug loading rate and the encapsulation rate of the formed nanoparticles are low, and the particle sizes are not uniform. The prodrug strategy can improve the adverse properties of the chemotherapeutic drug by skillful structural modification, such as improving the solubility of the drug, enhancing the targeting property, reducing the toxic and side effects of the drug, 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. The fatty acids with different carbon chain lengths have different affinities with albumin, so that the pharmaceutical properties, the in vivo fate and the anti-tumor effect of the prodrug albumin nanoparticles can be influenced.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides the pH-sensitive adriamycin-fatty acid prodrug and the albumin nanoparticle thereof, and the nanoparticle has the effects of high drug loading, high encapsulation efficiency, good stability, low toxic and side effects and intelligent intracellular drug release. The pH-sensitive adriamycin-fatty acid prodrug and the albumin nanoparticles thereof obviously reduce the toxic and side effects of adriamycin and improve the tolerance dose of adriamycin.
The invention designs and synthesizes the pH sensitive adriamycin prodrug containing fatty acids with different carbon chain lengths, and prepares prodrug albumin nanoparticles. Experimental results show that the length of the carbon chain of the fatty acid can influence the pharmaceutical properties, pharmacodynamics and safety of the prodrug albumin nanoparticles, so that a new strategy and more choices are provided for developing an albumin-based anti-tumor drug delivery system, and the urgent needs of high-efficiency and low-toxicity chemotherapy preparations in clinic are met.
In order to achieve the above objects, the present invention provides a pH-sensitive hydrazone bond-bridged doxorubicin-fatty acid prodrug represented by the general formula (I):
wherein R is C 10 -C 14 The saturated or unsaturated fatty acid contains no part having a carboxyl group.
Further, R is a part without carboxyl hydroxyl in n-decanoic acid, myristic acid, myristoleic acid and tetradecadienoic acid.
Specifically, the pH-sensitive doxorubicin-fatty acid prodrug or a pharmaceutically acceptable salt thereof is:
further, the invention provides a synthesis method of the pH-sensitive adriamycin-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 hydrazine intermediate product;
(3) And (3) reacting the fatty acid hydrazide intermediate product with doxorubicin hydrochloride carbonyl, and recrystallizing to obtain the pH-sensitive doxorubicin-fatty acid prodrug, wherein the yield is over 90 percent, and the purity is over 99 percent.
Wherein the fatty acid in the step (1) is C 10 -C 14 Saturated or unsaturated fatty acids;
fatty acids (moles): methanol (ml): the proportion of concentrated sulfuric acid (ml) is as follows: 1, from 2000 to 5000, preferably from 1;
in the step (2), the molar ratio of the fatty acid methyl ester to the hydrazine hydrate is 1;
in the step (3), doxorubicin hydrochloride: the molar ratio of the fatty acid hydrazide intermediate product is 1; 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 and recovering methanol, pouring the reaction solution into ice water, adding saturated sodium bicarbonate, neutralizing to neutrality, extracting, and concentrating to obtain the final product;
wherein the solvent for extraction is ethyl acetate or dichloromethane.
The fatty acid is C 10 -C 14 Saturated fatty acids or unsaturated fatty acids.
(2) Synthesis of fatty acid hydrazides: dissolving the fatty acid methyl ester obtained in the step (1) in ethanol, adding hydrazine hydrate, and refluxing for 8-12 hours. After concentration, the reaction liquid is poured into water, solid is separated out, and the fatty acid hydrazide intermediate product is obtained after suction filtration.
(3) Dissolving intermediate products of doxorubicin hydrochloride, glacial acetic acid and fatty acid hydrazine in absolute methanol, carrying out condensation reflux reaction at 50-60 ℃ for 12-24 hours, concentrating, recrystallizing and carrying out suction filtration to obtain the doxorubicin hydrochloride/glacial acetic acid/fatty acid hydrazine hydrate intermediate product.
Wherein, glacial acetic acid: the volume ratio of the anhydrous methanol is 1.
Wherein, the content of adriamycin hydrochloride: the molar ratio of the fatty acid hydrazide intermediate product is 1.
Wherein the recrystallization solvent is acetone.
Wherein R is C 9 -C 13 An alkyl group.
The adriamycin-n-decanoic acid prodrug and the adriamycin-myristic acid prodrug have good self physical and chemical properties, larger polarity and better solubility due to n-decanoic acid and myristic acid, and the purity can reach 99.4 percent after recrystallization. The purity of the adriamycin-stearic acid prodrug with longer carbon chain is only 85% after recrystallization, and the final product still needs to be obtained by separation and purification of a preparation liquid phase.
The invention provides a pH-sensitive adriamycin-fatty acid prodrug albumin nanoparticle, which comprises a pH-sensitive adriamycin-fatty acid prodrug and serum albumin, wherein the mass ratio of the pH-sensitive adriamycin-fatty acid prodrug to the serum albumin is (1-1).
Furthermore, the invention also provides a preparation method of the albumin nanoparticles of the series of pH-sensitive adriamycin-fatty acid prodrugs, and the albumin nanoparticles are prepared by an ultrasonic method or a high-pressure homogenization method.
Specifically, the preparation method of the pH-sensitive adriamycin-fatty acid prodrug albumin nanoparticle provided by the invention comprises the following steps:
weighing a pH sensitive adriamycin-fatty acid prodrug, dissolving the prodrug by using methanol, slowly dripping the obtained solution into a serum albumin aqueous solution under stirring, and then carrying out ultrasonic or high-pressure homogenization to form uniform albumin nanoparticles. Distilling at 25-30 deg.C under reduced pressure to remove organic solvent.
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 adriamycin-fatty acid prodrug to the serum albumin is 1.1-10, preferably 1.
In the ultrasonic method, the power of the ultrasonic wave is 30-100W, and preferably 60-80W.
The time of the ultrasound is 1 to 5 minutes, preferably 2 to 3 minutes.
In the high-pressure homogenization method, the pressure is 500-1000bar, preferably 600-800bar.
In the high-pressure homogenization method, the time is 10 to 30 minutes, preferably 10 to 15 minutes.
In the invention, because the prodrug is required to be converted into a parent drug in vivo to play a drug effect function, a hydrazone bond bridged pH-sensitive adriamycin-fatty acid prodrug and albumin nanoparticles thereof are constructed, after the nanoparticles are taken up by tumor cells, the nanoparticles firstly enter an endosome and a lysosome, the hydrazone bond is broken when prodrug molecules are in the low pH condition of the endosome and the lysosome, the prodrug is converted into the parent drug to play an anti-tumor effect, a new strategy and more choices are provided for developing an intelligent response type tumor microenvironment drug delivery system, and the urgent need of a high-efficiency and low-toxicity chemotherapy preparation in clinic is met.
The invention has the following beneficial effects: (1) The pH-sensitive adriamycin-fatty acid prodrug with different carbon chain lengths is designed and synthesized, the synthesis method is simple and feasible, and the purity of the product can reach more than 99 percent through recrystallization; (2) The adriamycin-fatty acid prodrug albumin nanoparticles are prepared, the preparation method is simple and feasible, and the adriamycin-fatty acid prodrug albumin nanoparticles have high drug loading rate and encapsulation efficiency; (3) The differences of prodrug albumin nanoparticles in the aspects of pharmaceutical properties, cytotoxicity, pharmacokinetics, pharmacodynamics and the like are investigated, and test results show that the adriamycin-fatty acid prodrug albumin nanoparticles have good anti-tumor effect, can effectively reduce the toxic and side effects of adriamycin, and the prodrug with different structures has different anti-tumor activity and safety, so that a new strategy is provided for developing high-efficiency and low-toxicity chemotherapy preparations.
Drawings
FIG. 1 is Doxorubicin-n-decanoic acid prodrug (DOX-C) of example 1 of the present invention 10 ) Mass spectrum of (2).
FIG. 2 is Doxorubicin-n-decanoic acid prodrug (DOX-C) of example 1 of the present invention 10 ) Is/are as follows 1 HNMR spectrogram.
FIG. 3 shows the prodrug of Doxorubicin-myristic acid (DOX-C) in example 2 of the present invention 14 ) Mass spectrum of (2).
FIG. 4 shows the prodrug of Doxorubicin-myristic acid (DOX-C) in example 2 of the present invention 14 ) Is/are as follows 1 HNMR spectrogram.
FIG. 5 is Doxorubicin-stearic acid prodrug (DOX-C) of example 3 of the present invention 18 ) Mass spectrum of (2).
FIG. 6 is Doxorubicin-stearic acid prodrug (DOX-C) of example 3 of the present invention 18 ) Is/are as follows 1 HNMR spectrogram.
Fig. 7 is a graph of the 4T1 cytotoxicity of doxorubicin-fatty acid prodrug albumin nanoparticles of example 6 of the invention.
Fig. 8 is a prodrug blood concentration-time curve of doxorubicin-fatty acid prodrug albumin nanoparticles of example 7 of the present invention.
Fig. 9 is a graph of the plasma concentration-time of the parent drug of the nanoparticles of adriamycin-fatty acid prodrug albumin of example 7 of the present invention.
FIG. 10 shows the doxorubicin-fatty acid prodrug albumin of example 7 of the present invention prodrug addition blood concentration-time curve graph of the nanoparticle.
Fig. 11 is a graph of the change of the tumor volume of the mice in the in vivo anti-tumor experiment of the doxorubicin-fatty acid prodrug albumin nanoparticles of example 8 of the present invention.
**：P<0.01；***：P<0.001；****：P<0.0001。
FIG. 12 is a mouse tumor comparison graph of doxorubicin-fatty acid prodrug albumin nanoparticles of example 8 of the present invention in an in vivo anti-tumor experiment.
Fig. 13 is a graph of the change in body weight of mice in an in vivo anti-tumor experiment of the doxorubicin-fatty acid prodrug albumin nanoparticles of example 8 of the present invention.
**：P<0.01；***：P<0.001；****：P<0.0001。
Fig. 14 is a tumor burden graph of doxorubicin-fatty acid prodrug albumin nanoparticles of example 8 of the present invention in an in vivo anti-tumor experiment.
* *: p <0.01; * **: p <0.001; ns: there was no significant difference.
Fig. 15 is a spleen weight graph of doxorubicin-fatty acid prodrug albumin nanoparticles of example 8 of the present invention in an in vivo anti-tumor experiment.
* *: p <0.01; * **: p <0.001; ns: there was no significant difference.
FIG. 16 is a comparative image of the spleen of the doxorubicin-fatty acid prodrug albumin nanoparticles of example 8 of the present invention in an in vivo antitumor experiment.
Fig. 17 is a comparison graph of leukocytes, lymphocytes, monocytes, and neutrophils in an in vivo anti-tumor experiment of the doxorubicin-fatty acid prodrug albumin nanoparticles of example 8 of the present invention.
FIG. 18 is a graph comparing the percentage of lymphocytes, monocytes and neutrophils in an in vivo anti-tumor assay of the doxorubicin-fatty acid prodrug albumin nanoparticles of example 8 of the present invention.
Fig. 19 is a comparison graph of erythrocytes, hemoglobin and related parameters in an in vivo antitumor experiment of the doxorubicin-fatty acid prodrug albumin nanoparticles of example 8 of the present invention.
Fig. 20 is a graph comparing the platelets, platelet volume, platelet distribution and platelet volume in the in vivo antitumor experiment of the doxorubicin-fatty acid prodrug albumin nanoparticles of example 8 of the present invention.
FIG. 21 is a comparison of glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, urea nitrogen, and creatinine in an in vivo antitumor assay performed with doxorubicin-fatty acid prodrug albumin nanoparticles of example 8 of the present invention.
Fig. 22 is a graph of the change of the tumor volume of the mice in the in vivo anti-tumor experiment of the doxorubicin-fatty acid prodrug albumin nanoparticles of example 9 of the present invention.
**：P<0.01；***：P<0.001；****：P<0.0001。
FIG. 23 is a mouse tumor comparison graph of doxorubicin-fatty acid prodrug albumin nanoparticles of example 9 of the present invention in an in vivo anti-tumor experiment.
Fig. 24 is a graph of the change in body weight of mice in an in vivo anti-tumor experiment of the doxorubicin-fatty acid prodrug albumin nanoparticles of example 9 of the present invention.
* *: p <0.01; * **: p <0.001; * ***: p <0.0001; ns: there was no significant difference.
Fig. 25 is a tumor burden graph of doxorubicin-fatty acid prodrug albumin nanoparticles of example 9 of the present invention in an in vivo anti-tumor experiment.
*：P<0.05；****：P<0.0001。
Fig. 26 is a survival curve diagram of the doxorubicin-fatty acid prodrug albumin nanoparticles of example 9 of the present invention in an in vivo antitumor experiment.
Fig. 27 is a comparison graph of leukocytes, lymphocytes, monocytes, and neutrophils in an in vivo anti-tumor assay of doxorubicin-fatty acid prodrug albumin nanoparticles of example 9 of the present invention.
FIG. 28 is a comparison of the percentage of lymphocytes, monocytes and neutrophils in the in vivo anti-tumor assay of doxorubicin-fatty acid prodrug albumin nanoparticles of example 9 of the present invention.
FIG. 29 is a comparison of erythrocytes, hemoglobin and related parameters in an in vivo anti-tumor experiment with doxorubicin-fatty acid prodrug albumin nanoparticles of example 9 of the present invention.
Fig. 30 is a comparison graph of platelets, platelet volume, platelet distribution and platelet volume in an in vivo anti-tumor experiment of doxorubicin-fatty acid prodrug albumin nanoparticles of example 9 of this invention.
Fig. 31 is a graph comparing glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, urea nitrogen, and creatinine in an in vivo anti-tumor experiment of doxorubicin-fatty acid prodrug albumin nanoparticles of example 9 of the present invention.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the invention thereto.
Example 1: doxorubicin-N-decanoic acid (DOX-C) 10 ) Synthesis of prodrugs
Adding 17.2g of n-decanoic acid (0.1 mol) and 300mL of methanol into a round-bottom flask, adding 5mL of concentrated sulfuric acid, and refluxing for 12 hours; after concentration, pouring the reaction liquid into ice water, adding saturated sodium bicarbonate, neutralizing to be neutral, extracting by ethyl acetate, and concentrating to obtain a fatty acid methyl ester intermediate product; dissolving the intermediate product in ethanol, adding 100mL of hydrazine hydrate, and refluxing for 12 hours; after concentration, pouring the reaction liquid into water, separating out solid, and performing suction filtration to obtain a fatty acid hydrazide intermediate product; dissolving 5.8g of doxorubicin hydrochloride (0.01 mol), 10mL of glacial acetic acid and 3.6g of fatty acid hydrazine intermediate product (0.015 mol) in anhydrous methanol, carrying out reflux reaction at 50-60 ℃ for 12 hours, concentrating, carrying out recrystallization by using acetone as a solvent, and carrying out suction filtration. Yield: 94%, purity: 99 percent.
The structure of the prodrug of example 1 was determined by mass spectrometry and nuclear magnetic resonance hydrogen spectroscopy, and the results are shown in fig. 1 and 2, and the results of the spectroscopy are as follows:
1 H NMR(600MHz,DMSO-d 6 )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 C 37 H 49 N 3 O 11 H[M+H] + :712.34327.
example 2: doxorubicin-myristic acid (DOX-C) 14 ) Synthesis of prodrugs
Adding 22.8g of myristic acid (0.1 mol) and 300mL of methanol into a round-bottom flask, adding 5mL of concentrated sulfuric acid, and refluxing for 12 hours; after concentration, pouring the reaction liquid into ice water, adding saturated sodium bicarbonate, neutralizing to be neutral, extracting by ethyl acetate, and concentrating to obtain a fatty acid methyl ester intermediate product; dissolving the intermediate product in ethanol, adding 100mL of hydrazine hydrate, and refluxing for 12 hours; after concentration, pouring the reaction liquid into water, separating out solid, and performing suction filtration to obtain a fatty acid hydrazide intermediate product; dissolving 5.8g of doxorubicin hydrochloride (0.01 mol), 10mL of glacial acetic acid and 3.6g of fatty acid hydrazine intermediate product (0.015 mol) in anhydrous methanol, carrying out reflux reaction at 50-60 ℃ for 12 hours, concentrating, carrying out recrystallization by using acetone as a solvent, and carrying out suction filtration. Yield: 92%, purity: 99 percent.
The structure of the prodrug of example 2 was determined by mass spectrometry and nmr hydrogen spectrometry, and the results are shown in fig. 3 and 4, and the results of the spectroscopy were as follows:
1 H NMR(600MHz,DMSO-d 6 )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 C 41 H 57 N 3 O 11 H[M+H] + :768.40531.
example 3: adriamycin-stearic acid (DOX-C) 18 ) Synthesis of 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 reflux was carried out for 12 hours; after concentration, pouring the reaction liquid into ice water, adding saturated sodium bicarbonate, neutralizing to be neutral, extracting by ethyl acetate, and concentrating to obtain a fatty acid methyl ester intermediate product; dissolving the intermediate product in ethanol, adding 100mL of hydrazine hydrate, and refluxing for 12 hours; after concentration, pouring the reaction liquid into water, separating out solid, and performing suction filtration to obtain a fatty acid hydrazide intermediate product; dissolving 5.8g of doxorubicin hydrochloride (0.01 mol), 10mL of glacial acetic acid and 3.6g of fatty acid hydrazine intermediate product (0.015 mol) in anhydrous methanol, carrying out reflux reaction at 50-60 ℃ for 12 hours, concentrating, carrying out recrystallization by using acetone as a solvent, and carrying out suction filtration. Yield: 80%, purity: 85 percent. The target product is separated and purified by preparative liquid chromatography, and the purity is 99%.
Mass spectrometry and hydrogen nuclear magnetic resonance spectroscopy were used to determine the structure of the prodrug of example 3, and the results are shown in FIGS. 5 and 6. The results of the spectrum analysis were as follows:
1 H NMR(600MHz,DMSO-d 6 )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 C 45 H 65 N 3 O 11 H[M+H] + :824.4696.
example 4: consideration of influence factors of pH-sensitive adriamycin-fatty acid prodrug albumin nanoparticles
TABLE 1 particle size, particle size distribution and encapsulation efficiency of Adriamycin-fatty acid prodrug and Albumin at different mass ratios
Precisely weighing a pH sensitive adriamycin prodrug, dissolving the prodrug by using 1mL of methanol, slowly dripping the methanol solution into 4mL of bovine serum albumin (or human serum albumin and canine serum albumin) aqueous solution under stirring, and carrying out ultrasonic treatment by using an ultrasonic cell disruptor to form uniform albumin nanoparticles with the ultrasonic treatment time of 2min and the ultrasonic power of 60W. The organic solvent was removed from the formulation using a 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 1-3, the particle size of the prodrug albumin nanoparticle is 50-200nm, and the particle size distribution is uniform; when the mass ratio is 1.
Example 5: preparation of pH-sensitive adriamycin-fatty acid prodrug albumin nanoparticles
Precisely weighing 4mg of a pH-sensitive adriamycin prodrug, dissolving the prodrug by using 1mL of methanol, slowly dripping the methanol solution into 4mL of 2mg/mL aqueous solution of bovine serum albumin (or human serum albumin and canine serum albumin) under stirring to ensure that the mass ratio of the prodrug to the albumin is 1. The organic solvent in the nanoformulation was removed by rotary evaporator at 25 ℃.
TABLE 2 characterization of pH-sensitive Adriamycin-fatty acid prodrug albumin nanoparticles
As shown in Table 2, the affinity between adriamycin and albumin is poor, the particle size of the formed nanoparticles is as high as 480nm, the particle size distribution is as high as 0.6, and the encapsulation efficiency is less than 20%. In contrast, the particle size of the three prodrug albumin nanoparticles is about 120nm, the particle size distribution is less than 0.2, the encapsulation rate is greater 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 the adriamycin-fatty acid prodrug albumin nanoparticles on mouse breast cancer (4T 1) cells is examined by adopting an MTT method. Digesting the cells in a good state, diluting the cells to 5000cells/mL by using a culture solution, uniformly blowing the cells, adding 100 mu L of cell suspension into each hole of a 96-hole plate, and placing the cells in an incubator for incubation for 24 hours to adhere to the walls. After the cells were attached to the wall, doxorubicin solution or doxorubicin-fatty acid prodrug albumin nanoparticles prepared in example 5 were added and incubated for a further 48 hours, using untreated cells as control. At the end of the incubation, 20. Mu.L of MTT (5 mg/mL) was added to each well and incubated at 37 ℃ for 4 hours. The medium was discarded and 200. Mu.L DMSO per well was added and shaken on a shaker for 10min. The absorbance was measured at 570nm using a microplate reader.
Cytotoxicity results are shown in fig. 7, and the cytotoxicity of prodrug albumin nanoparticles is reduced compared to doxorubicin solution. This is because doxorubicin needs to be released from the prodrug nanoparticle, 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 nanoparticles are.
Example 7: pharmacokinetics research of pH-sensitive adriamycin-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 access to water. Doxorubicin solution and the three doxorubicin-fatty acid prodrug albumin nanoparticles prepared in example 5 were injected intravenously, respectively. The dose of doxorubicin was 4mg/kg. Blood was collected from the orbit at the prescribed time points and separated to obtain plasma. The drug concentration in plasma was determined by liquid chromatography-mass spectrometer.
The experimental results are shown in fig. 8-10, compared with the adriamycin solution preparation group, the prodrug albumin nanoparticles have prolonged retention time in blood, and the area under the drug-time curve (AUC) of adriamycin is remarkably improved 0-24 h ) And the method provides guarantee for the tumor targeted accumulation of the adriamycin. Meanwhile, the length of the fatty acid carbon chain has a significant influence on the pharmacokinetic behavior of the prodrug albumin nanoparticles. Total AUC of adriamycin-n-decanoic acid prodrug albumin nanoparticles, adriamycin-myristic acid prodrug albumin nanoparticles and adriamycin-stearic acid prodrug albumin nanoparticles 0-24 h (sum of prodrug and parent drug) is 1.37 times, 5.18 times and 13.73 times of adriamycin solution respectively.
TABLE 3 pharmacokinetic parameters of pH sensitive Doxorubicin-fatty acid prodrug Albumin nanoparticles
Example 8: anti-tumor experiment of pH-sensitive adriamycin-fatty acid prodrug albumin nanoparticles
Establishing 4T1 cell tumor-bearing mouse model, and collecting 4T1 cells (100 μ L containing 5 × 10 cells) 6 Individual cells) were inoculated subcutaneously into BALB/c mice. When the tumor volume grows to 100-150mm 3 In the left and right, they were randomly divided into 7 groups (5 mice per group). The doxorubicin-fatty acid prodrug albumin nanoparticles of example 5 were administered into the tail vein every other day, doxorubicin solution, commercially available doxorubicin hydrochloride liposome (Doxil), doxorubicin maleimide prodrug (DOXO-EMCH), and physiological saline were set as a control group, and the total injection was performed 5 times at a dose of 10mg/kg of equivalent doxorubicin, and the tumor volume and mouse body weight were measured daily.
The results are shown in FIGS. 11-16, where doxorubicin liposomes had the best antitumor effect, but the mice also had the most significant weight loss compared to the saline group, showing severe toxic side effects. The doxorubicin solution group and the DOXO-EMCH group also have good antitumor effects, but after the fifth administration, mice all die, and the toxic and side effects are very strong. All three prodrug albumin nanoparticles have significant anti-tumor effects (fig. 11, 12). Fig. 14 shows that the tumor-bearing rates of the doxorubicin-n-decanoic acid prodrug, the doxorubicin-myristic acid prodrug and the doxorubicin-stearic acid prodrug are not significantly different. However, the body weight of the doxorubicin-stearic acid prodrug was significantly reduced, while the body weight of mice in the doxorubicin-n-decanoic acid prodrug and doxorubicin-myristic acid prodrug group did not decrease, and there was no significant difference compared with the normal saline group, indicating that the mice were safer and less toxic (fig. 13). FIGS. 15-16 show that spleen shrinkage occurs in mice with doxorubicin liposome group compared to normal non-tumorigenic mice, indicating that they have stronger myelosuppressive toxicity, while spleen shrinkage does not occur in mice with doxorubicin-n-decanoic acid prodrug and doxorubicin-myristic acid prodrug, indicating that they are safer. The results of routine blood and liver and kidney function tests are shown in FIGS. 17-21. Therefore, compared with the adriamycin-stearic acid prodrug with longer carbon chain length, the adriamycin-n-decanoic acid prodrug and the adriamycin-myristic acid prodrug albumin nanoparticles have better tolerance and safety while having good anti-tumor effect.
Example 9: anti-tumor experiment of pH-sensitive adriamycin-fatty acid prodrug albumin nanoparticles
Establishing KB cell tumor-bearing nude mouse model, and culturing KB cell (100 mu L medium contains 5 × 10) 6 Individual cells) were inoculated subcutaneously into BALB/c-Nu nude mice. When the tumor volume grows to 100-150mm 3 On the left and right, they were randomly divided into 5 groups (5 mice per group). The doxorubicin-myristic acid prodrug albumin nanoparticles of example 5 were administered every two days in tail vein, and commercially available doxorubicin hydrochloride liposome (Doxil) and normal saline were set as control groups, wherein the normal saline group and the doxorubicin-myristic acid prodrug albumin nanoparticle group were injected 4 times in total, and the commercially available doxorubicin hydrochloride liposome (Doxil) group was injected 2 times in total, at doses of 15mg/kg and 20mg/kg (doxorubicin equivalents 15mg/kg, 20 mg/kg), and tumor volume and mouse body weight were measured daily.
The results are shown in FIGS. 22-26, and at the administration dose of 20mg/kg, the doxorubicin liposome group died on day eight, all died on day nine, and the toxic side effects were very strong. At the 15mg/kg dose, the doxorubicin liposome group died on day ten, and all died on day eleven (fig. 26). While the adriamycin-myristic acid prodrug albumin nanoparticle group is administrated four times, the prodrug albumin nanoparticle has a remarkable antitumor effect under the administration doses of 15mg/kg and 20mg/kg (figures 22 and 23), mice do not die, the body weight does not drop, and compared with the normal saline group, the prodrug albumin nanoparticle has no remarkable difference, which indicates that the prodrug albumin nanoparticle is better in safety and lower in toxicity (figure 24). The blood routine and liver and kidney function test results of each group are shown in fig. 27-31, and the leucocytes and lymphocytes of the adriamycin-n-decanoic acid prodrug and the adriamycin-myristic acid prodrug are not reduced, so that obvious liver and kidney injury is not caused. Especially, the leucocytes and lymphocytes of mice with adriamycin-myristic acid prodrug albumin nanoparticle groups are not reduced, which shows that the mice do not have bone marrow suppression, and the liver and kidney function indexes show that the mice do not damage the liver and kidney. As can be seen, the adriamycin-n-decanoic acid prodrug and the adriamycin-myristic acid prodrug have not only remarkable anti-tumor effect, but also small toxic and side effects and high safety. The adriamycin myristate prodrug albumin nanoparticle has the advantages of obvious anti-tumor effect, low toxicity and high safety.
Claims (10)
1. A pH-sensitive doxorubicin-fatty acid prodrug represented by the general formula (I):
wherein R is C 10 -C 14 A portion of the saturated or unsaturated fatty acid not containing a carboxyl group; preferably, the moiety does not contain a carboxyl group in n-decanoic acid, myristic acid (n-tetradecanoic acid), myristoleic acid, and tetradecadienoic acid.
2. The pH-sensitive doxorubicin-fatty acid prodrug or a salt thereof of claim 1 having the structure:
3. the method of preparing the pH-sensitive doxorubicin-fatty acid prodrug of claim 1, comprising the steps of:
(1) Refluxing fatty acid, methanol and concentrated sulfuric acid to obtain fatty acid methyl ester;
(2) Refluxing the fatty acid methyl ester obtained in the step (1) with hydrazine hydrate to obtain a fatty acid hydrazine intermediate product;
(3) And (3) reacting the fatty acid hydrazine intermediate product with doxorubicin hydrochloride carbonyl, and recrystallizing to obtain the fatty acid hydrazine intermediate product.
4. The method for preparing the pH-sensitive doxorubicin-fatty acid prodrug according to claim 3, wherein the fatty acid in the step (1) is C 10 -C 14 Saturated or unsaturated fatty acids; fatty acids (moles): methanol (ml): the ratio of concentrated sulfuric acid (ml) is: 1; in the step (2), the molar ratio of the fatty acid methyl ester to the hydrazine hydrate is 1; in the step (3), doxorubicin hydrochloride: the molar ratio of the fatty acid hydrazide intermediate product is 1.
5. The method for preparing a pH-sensitive doxorubicin-fatty acid prodrug of claim 3, wherein in step (3), the recrystallization solvent is acetone.
6. The albumin nanoparticle of the pH-sensitive doxorubicin-fatty acid prodrug of claim 1 or 2, wherein:
the albumin nanoparticle comprises a pH-sensitive adriamycin-fatty acid prodrug and serum albumin, wherein the mass ratio of the pH-sensitive adriamycin-fatty acid prodrug to the serum albumin is (1).
7. The method for preparing albumin nanoparticles of a pH-sensitive doxorubicin-fatty acid prodrug according to claim 6, wherein:
weighing a pH-sensitive adriamycin-fatty acid prodrug, dissolving the prodrug by using an organic solvent, slowly dripping the obtained solution into a serum albumin water solution under stirring, and carrying out ultrasonic or high-pressure homogenization to form uniform albumin nanoparticles.
8. Use of the pH-sensitive doxorubicin-fatty acid prodrug of claim 1 or 2 or the pH-sensitive doxorubicin-fatty acid prodrug albumin nanoparticles of claim 6 for the preparation of a drug delivery system.
9. The use of the pH-sensitive doxorubicin-fatty acid prodrug of claim 1 or 2 or the pH-sensitive doxorubicin-fatty acid prodrug albumin nanoparticle of claim 6 in the preparation of an anti-tumor medicament.
10. Use of the pH-sensitive doxorubicin-fatty acid prodrug of claim 1 or 2 or the pH-sensitive doxorubicin-fatty acid prodrug albumin nanoparticles of claim 6 for the preparation of an injectable, oral or topical delivery system.
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| 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 |
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| 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 |
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| CN113713117A (en) * | 2021-09-10 | 2021-11-30 | 山东大学 | Albumin-binding tumor environment-responsive antitumor prodrug and preparation method and application thereof |
| 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 |
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