Paclitaxel prodrugs and albumin nanoparticle pharmaceutical compositions thereof

Abstract

A kind of epirubicin prodrug, albumin nanoparticle pharmaceutical composition comprising the prodrug and its preparation method and application.The long-chain fatty acid is connected with epirubicin by using acyl hydrazone bond sensitive to tumor acid microenvironment to construct epirubicin prodrug, so as to improve the binding capacity of epirubicin and albumin.The prepared epirubicin prodrug can realize the sufficient dissolution of prodrug using water as solvent, then mixed with albumin aqueous solution, and after high pressure homogenization, the uniformly distributed epirubicin-fatty acid prodrug albumin nanoparticle pharmaceutical composition can be obtained.The preparation process provided does not need to use organic solvent, breaks through the technical obstacle that existing albumin preparation product must use organic solvent to prepare, simplifies the preparation process, and at the same time avoids the risk of organic solvent residue and inducing albumin denaturation.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical formulation technology, specifically to the field of epirubicin pharmaceutical compositions, and more specifically to epirubicin prodrug albumin nanoparticle pharmaceutical compositions, their preparation methods, and applications. Background Technology

[0002] Cancer is a disease caused by the excessive proliferation of cells in the body, seriously endangering human health. Chemotherapy is an important clinical treatment for cancer, showing good results in treating primary and metastatic tumors throughout the body. Epirubicin, as a third-generation anthracycline antitumor drug, exerts its antitumor effect by intercalating into adjacent base pairs in DNA, interfering with cellular transcription, and thus inhibiting DNA and RNA synthesis. Compared with the second-generation anthracycline antitumor drug doxorubicin, epirubicin has comparable anticancer activity, stronger hydrophilicity, and lower cardiotoxicity, showing broad application prospects. Its commercially available formulation, epirubicin hydrochloride injection, has broad-spectrum antitumor activity and is widely used in the treatment of breast cancer, malignant lymphoma, soft tissue sarcoma, gastric cancer, and malignant melanoma. However, epirubicin hydrochloride injection still faces challenges such as poor tumor targeting, low tumor selectivity, short in vivo circulation time, and significant toxic side effects, which seriously restrict its efficacy and application prospects. Therefore, developing a highly effective and low-toxicity epirubicin tumor-targeting formulation is an urgent pharmaceutical challenge to be solved.

[0003] Human serum albumin (HSA), the most abundant protein in blood plasma (approximately 40 mg / mL), possesses advantages such as low immunogenicity, biodegradability, and a long half-life (up to 19 days). Furthermore, albumin exhibits excellent stability and tumor targeting, thus being considered an ideal delivery carrier for anti-tumor drugs. Injectable albumin-bound paclitaxel was approved by the FDA in 2005. This is one of the most successful applications of albumin as a carrier. However, albumin has strict requirements on drug properties and is mainly suitable for drugs that are highly hydrophobic and have high albumin-binding ability. For drugs like epirubicin, which have some hydrophilicity and poor albumin-binding ability, effective binding is difficult to achieve. Therefore, improving the binding ability of drugs to albumin is of great research significance.

[0004] Fatty acids are natural delivery substrates for albumin and have a strong binding affinity to it. Several products utilizing fatty acid-bound albumin, such as liraglutide and semaglutide, have been approved for marketing by the FDA. Currently, there are also studies on modifying doxorubicin with fatty acids to improve its affinity for albumin, thereby preparing albumin nanoparticles. CN 115212185 A prepared doxorubicin-fatty acid prodrug albumin nanoparticles using an ultrasonic method and investigated the effect of fatty acid saturation on the formulation properties and in vivo fate of the doxorubicin-fatty acid prodrug nanoparticles. The results showed that the doxorubicin prodrug albumin nanoparticles modified with saturated fatty acids had the most significant toxicity-reducing and efficacy-enhancing effect. CN 115702902A also reported an antitumor formulation of doxorubicin prodrug, synthesizing a short-chain fatty acid-modified doxorubicin prodrug and preparing prodrug albumin nanoparticles. Although epirubicin and doxorubicin have similar plasma protein binding rates, epirubicin is more hydrophilic. Therefore, the inventors hypothesize that medium- and long-chain fatty acid modification is needed to improve its hydrophobicity and affinity for albumin, thereby preparing albumin nanoparticles.

[0005] In existing technologies, methods for preparing albumin nanoparticles (including...) The preparation methods mentioned above, including CN 115212185 A and CN 115702902 A, all require the use of organic solvents to dissolve the drug. The drug-containing organic solvent is then added to an aqueous albumin solution to form a proemulsion. After homogenization by ultrasound or high pressure, the organic solvent is removed by vacuum distillation, dialysis, or high-temperature methods to obtain albumin nanoparticles. However, this method has certain limitations, such as complex preparation processes, residual organic solvents, and the risk of inducing albumin deformability. In the early stages of research, this invention attempted to prepare an epirubicin-fatty acid prodrug albumin nanoparticle drug composition based on existing technology, and repeatedly replicated the key embodiments of the aforementioned patents, particularly Examples 4 and 5 of CN 115212185 A and Examples 4 and 5 of CN 115702902 A. However, none of these attempts successfully prepared an albumin nanoparticle drug composition with uniform particle size. The inventors then conducted in-depth research and optimized key parameters in the preparation process, including the type and ratio of organic solvents used to dissolve the prodrug, the mass ratio of the prodrug to albumin, the mixing method of the prodrug and albumin, and the parameters of high-pressure homogenization. However, even after multiple adjustments, they were still unable to successfully prepare epirubicin-fatty acid prodrug albumin nanoparticles with uniform particle size.

[0006] Through repeated exploration, the inventors discovered that epirubicin retains a certain degree of water solubility even after being prepared as a fatty acid prodrug. Therefore, this invention proposes a novel preparation strategy: directly adding an aqueous albumin solution to an epirubicin prodrug dissolved in water, mixing thoroughly, and then homogenizing under high pressure to prepare an epirubicin-fatty acid prodrug albumin nanoparticle drug composition with uniform particle size and good stability. This method significantly improves the binding efficiency between the drug and albumin, while greatly simplifying the preparation process and overcoming the bottleneck of traditional methods that require the use of organic solvents. The advantages of this invention are obvious: 1) It avoids the use of organic solvents, fundamentally eliminating the risk of albumin denaturation that may be caused by organic solvents; 2) It eliminates the need to detect and control residual organic solvents, conforming to the concept of green pharmaceuticals; 3) It simplifies the preparation steps, helping to reduce production costs; 4) The epirubicin prodrug albumin nanoparticles significantly improve the pharmacokinetic behavior of epirubicin, increasing its accumulation and anti-tumor effect at tumor sites, while reducing toxic side effects.

[0007] Furthermore, in the preparation of albumin nanoparticles, protein structure elucidating agents can be added to disrupt the native conformation of albumin, breaking its intramolecular and intermolecular hydrogen bonds and hydrophobic interactions. This allows albumin molecules to fully unfold, exposing more hydrophobic regions to promote nanoparticle formation and improve drug encapsulation efficiency and drug loading. Commonly used protein structure elucidating agents in this field include 2-mercaptoethanol, 2-thiothreitol, guanidine hydrochloride, urea, perchloric acid, tributylphosphine, methimazole-propionylproline, and performic acid. Although protein structure elucidating agents can improve the encapsulation efficiency of albumin nanoparticles to some extent, their application has many safety risks and technical limitations. Excessive or over-elucidation of albumin may damage the mechanical strength of nanoparticles, increasing the risk of drug leakage. Simultaneously, the properties of unfolded albumin molecules change, making them more easily recognized as foreign substances by the immune system and rapidly cleared, and they may also possess certain sensitizing properties. In addition, protein structure elucidating agents themselves may also have certain toxicity or biosafety risks. For example, 2-mercaptoethanol is not only cytotoxic but also highly irritating and may cause allergic reactions, therefore it is not suitable for use in pharmaceutical formulations. In processes using protein structure eluents, removing residual eluent is crucial; incomplete removal can severely compromise formulation safety. This process typically requires additional impurity removal steps, leading to process complexity and increased production costs. In contrast, the preparation method described in this application does not involve the use of protein structure eluents, resulting in a simpler process. It avoids the toxicity issues caused by structure eluents and also eliminates potential problems such as decreased albumin nanoparticle structural strength, drug leakage, and sensitization that structure eluents may cause.

[0008] The epirubicin prodrug synthesized in this invention is obtained by modifying medium- and long fatty acid chains after desalting epirubicin, and its water solubility was expected to decrease significantly. However, the inventors unexpectedly discovered that epirubicin, despite its high water solubility, retains a certain degree of water solubility after being prepared as a prodrug, and can fully dissolve in water at a certain concentration. Those skilled in the art would not typically consider using water instead of organic solvents to dissolve prodrugs; therefore, this invention provides an innovative preparation technique, opening up a new direction for the development of albumin nanomedicines. Summary of the Invention

[0009] The first objective of this invention is to provide a drug composition for epirubicin prodrug albumin nanoparticles. A medium- and long-chain fatty acid is linked to epirubicin via acylhydrazone bonds, which are sensitive to the acidic tumor microenvironment, to construct an epirubicin prodrug. This enhances the binding affinity of epirubicin to albumin, which is then used to prepare albumin nanoparticles. The invention also explores the influence of fatty acid length on the binding affinity of the prodrug to albumin, as well as its effects on in vitro and in vivo fate and therapeutic efficacy, in order to screen for the optimal epirubicin prodrug albumin nanoparticle drug composition.

[0010] The second objective of this invention is to provide a method for preparing an epirubicin prodrug albumin nanoparticle drug composition. The epirubicin prodrug synthesized in this invention is formed by desalting epirubicin and then modifying it with medium- and long fatty acids, resulting in a significant decrease in the water solubility of the prodrug. Those skilled in the art would not anticipate that water could still be used as a solvent to dissolve a drug with increased hydrophobicity. By directly adding an aqueous albumin solution to the epirubicin prodrug dissolved in water, mixing thoroughly, and then homogenizing under high pressure, a homogeneous epirubicin prodrug albumin nanoparticle drug composition can be obtained. The preparation process provided by this invention does not require the use of organic solvents, overcoming the technical barrier that existing albumin formulations must be prepared using organic solvents, simplifying the preparation process, and avoiding the risks of organic solvent residue and albumin denaturation.

[0011] A third objective of this invention is to provide an application of an epirubicin prodrug albumin nanoparticle pharmaceutical composition.

[0012] To achieve the first objective mentioned above, the present invention adopts the following technical solution:

[0013] This invention provides an epirubicin-fatty acid prodrug as shown in general formula (I) or a pharmaceutically acceptable salt thereof:

[0014]

[0015] Where R is C 14 -C 26 The portion of saturated or unsaturated straight-chain fatty acids that does not contain carboxyl hydroxyl groups.

[0016] Furthermore, R is the portion of myristic acid, stearic acid, behenic acid, ceric acid, myristoleic acid, oleic acid, behenoleic acid, tetradecadienoic acid, octadecadienoic acid, and dodecadienoic acid that does not contain a carboxyl hydroxyl group.

[0017] Specifically, the epirubicin-fatty acid prodrug or its pharmaceutically acceptable salt is:

[0018]

[0019] Furthermore, the present invention provides a method for synthesizing the epirubicin-fatty acid prodrug, comprising the following steps:

[0020] (1) Reflux fatty acids, methanol and concentrated sulfuric acid to obtain fatty acid methyl esters;

[0021] (2) The fatty acid methyl ester obtained in step (1) is refluxed with hydrazine hydrate to obtain fatty acid acyl hydrazine intermediate;

[0022] (3) The fatty acid hydrazide intermediate in step (2) is reacted with epirubicin hydrochloride carbonyl group and recrystallized to obtain crude epirubicin-fatty acid prodrug.

[0023] (4) The crude product obtained is separated and purified by preparative liquid chromatography and then analyzed by analytical liquid chromatography. The purity can reach more than 99%.

[0024] The fatty acid mentioned in step (1) is C. 14 -C 22 Saturated fatty acids or unsaturated fatty acids;

[0025] The ratio of fatty acid (moles): methanol (mL): concentrated sulfuric acid (mL) is 1:500-10000:10-200, preferably 1:1000-4000:20-50;

[0026] In step (2), the molar ratio of fatty acid methyl ester to hydrazine hydrate is 1:5-50, preferably 1:10-30;

[0027] In step (3), the molar ratio of epirubicin hydrochloride to fatty acid acylhydrazine intermediate is 1:1-3;

[0028] The recrystallization solvent is acetone.

[0029] Furthermore, the preparation method of the epirubicin-fatty acid prodrug includes the following steps:

[0030] (1) Synthesis of fatty acid methyl ester: Add fatty acids and methanol to a round-bottom flask, add concentrated sulfuric acid, and reflux for 12 hours; concentrate and recover methanol, pour the reaction solution into ice water, add saturated sodium bicarbonate, neutralize to neutral, extract, and concentrate to obtain the product;

[0031] The solvent used for extraction is ethyl acetate or dichloromethane.

[0032] The fatty acid mentioned is C. 14 -C 26 Saturated fatty acids or unsaturated fatty acids.

[0033] (2) Synthesis of fatty acid hydrazide: Dissolve the fatty acid methyl ester from step (1) in ethanol, add hydrazine hydrate, and reflux for 8-12 hours. After concentration, pour the reaction solution into water, precipitate the solid, and filter to obtain the fatty acid hydrazide intermediate;

[0034] (3) Dissolve epirubicin hydrochloride, glacial acetic acid and fatty acid hydrazide intermediates in anhydrous methanol, reflux in an oil bath at 50-60℃ for 12-24h, concentrate, recrystallize, filter and dry to obtain crude product.

[0035] (4) The crude product obtained is then separated and purified by preparative liquid chromatography, and then analyzed by analytical liquid chromatography. The purity can reach more than 99%.

[0036] The volume ratio of glacial acetic acid to anhydrous methanol is 1:100-300.

[0037] The molar ratio of epirubicin hydrochloride to fatty acid acylhydrazine intermediate is 1:1-5.

[0038] The recrystallization solvent is acetone.

[0039]

[0040] Where R is C 13 -C 25 alkyl.

[0041] The present invention provides an epirubicin-fatty acid prodrug albumin nanoparticle pharmaceutical composition, wherein the albumin nanoparticles comprise epirubicin-fatty acid prodrug and serum albumin, and the mass ratio of epirubicin-fatty acid prodrug to serum albumin is 1:0.1-10, preferably 1:0.5-5, and more preferably 1:1-3.

[0042] The epirubicin-fatty acid prodrug albumin nanoparticle drug composition has a particle size of 70-200 nm, preferably 80-150 nm, and more preferably 80-120 nm.

[0043] No organic solvents are used in the preparation of the albumin nanoparticles.

[0044] Epirubicin-fatty acid prodrug was dissolved in deionized water, and human serum albumin was diluted with deionized water.

[0045] To achieve the second objective mentioned above, the present invention adopts the following technical solution:

[0046] This invention provides a method for preparing the aforementioned series of epirubicin-fatty acid prodrug albumin nanoparticle pharmaceutical compositions. The albumin nanoparticles are prepared by ultrasonication or high-pressure homogenization. The epirubicin-fatty acid prodrug is dissolved in deionized water, and human serum albumin is diluted with deionized water.

[0047] Specifically, the preparation method of the epirubicin-fatty acid prodrug albumin nanoparticle drug composition provided by the present invention is as follows:

[0048] (1) Dissolve epirubicin-fatty acid prodrug in deionized water to obtain an aqueous drug solution;

[0049] (2) Dilute the human serum albumin solution with deionized water to obtain a diluted albumin solution;

[0050] (3) While stirring continuously, slowly add the albumin diluent obtained in step (2) to the drug solution obtained in step (1);

[0051] (4) After continuous stirring for a period of time, a nano-colloidal solution without any organic solvent can be directly obtained by ultrasonic or high-pressure homogenization.

[0052] (5) After sterile filtration, a sterile and uniform nano-formulation can be obtained, which is the epirubicin-fatty acid prodrug albumin nanoparticle drug composition.

[0053] The stirring time is 5-30 minutes, preferably 10-20 minutes.

[0054] The serum albumin mentioned is human serum albumin or bovine serum albumin, preferably human serum albumin.

[0055] The concentration of the albumin diluent is 0.1-100 mg / mL, preferably 30-60 mg / mL.

[0056] The mass ratio of the epirubicin prodrug to serum albumin is 1:0.1-10, preferably 1:0.5-5, and more preferably 1:1-3.

[0057] In the ultrasonic method, the ultrasonic power is 50-500W, preferably 100-300W.

[0058] In the ultrasound method, the ultrasound duration is 0.5-10 minutes, preferably 1-5 minutes, and more preferably 1-3 minutes.

[0059] In the high-pressure homogenization method, the pressure is 2000-25000 psi, preferably 10000-20000 psi.

[0060] In the high-pressure homogenization method, the number of homogenization cycles is 1-15, preferably 3-10.

[0061] The sterilization filtration uses a 0.22μm polyethersulfone water membrane.

[0062] The purpose of this invention is to provide a formulation with stable physicochemical properties, preferably a lyophilized powder injection.

[0063] The lyophilization protectant used in the powder injection is selected from one or more of trehalose, sucrose, mannitol, glucose and dextran, preferably trehalose, sucrose and dextran, and more preferably trehalose and sucrose;

[0064] The weight ratio of the lyophilization protectant to the epirubicin-fatty acid prodrug is 10.0-20.0:1, preferably 15.0-18.0:1;

[0065] The reconstitution formulation for the lyophilized preparation is selected from water for injection, 5% glucose solution or 0.9% sodium chloride solution, preferably 5% glucose solution.

[0066] To achieve the third objective mentioned above, the technical solution adopted by the present invention is: the application of epirubicin-fatty acid prodrug or epirubicin-fatty acid prodrug albumin nanoparticle drug composition in the preparation of drug delivery systems and the application of epirubicin-fatty acid prodrug or epirubicin-fatty acid prodrug albumin nanoparticle drug composition in the preparation of antitumor drugs.

[0067] This invention has the following advantages:

[0068] (1) Epirubicin was selected as the raw material drug. As an improved version of doxorubicin, it has similar anticancer activity but significantly reduced toxicity, especially in the heart.

[0069] (2) A medium- and long-chain fatty acid modified epirubicin-fatty acid prodrug that responds to the acidic microenvironment of tumors was designed and synthesized. The synthesis method is simple and easy to implement.

[0070] (3) Water was selected as the solvent to dissolve the designed epirubicin prodrug. The epirubicin prodrug was directly added to the water-dissolved epirubicin prodrug, and after thorough mixing, homogenized under high pressure to obtain a homogeneous epirubicin prodrug albumin nanoparticle drug composition. This preparation process avoids the use of organic solvents, overcoming the technical barrier of requiring organic solvents in the preparation of existing albumin formulations, simplifying the preparation process, and avoiding the risks of organic solvent residue and albumin denaturation. The resulting albumin nanoparticle formulation has a higher drug loading capacity compared to the doxorubicin prodrug albumin nanoparticles in Example 5 of CN 115702902A.

[0071] (4) The method for preparing albumin nanoparticles in this application does not involve the use of protein structure emulsifiers. The preparation process is simple, and there are no toxicity problems caused by structure emulsifiers. It also does not involve problems such as decreased strength of albumin nanoparticle structure and drug leakage, as well as potential sensitization, which may be caused by structure emulsifiers.

[0072] (5) The influence of the length of medium- and long-chain fatty acids on the binding ability of the prodrug to albumin, as well as its effect on in vitro and in vivo fate and efficacy, was investigated to screen the optimal epirubicin prodrug albumin nanoparticle drug composition. Experimental results showed that, compared with commercially available epirubicin hydrochloride injection, the optimal epirubicin prodrug albumin nanoparticle drug composition had better antitumor effects and could effectively reduce the toxic side effects of epirubicin. Furthermore, compared with the doxorubicin prodrug albumin nanoparticles prepared in CN 115702902A, the epirubicin prodrug albumin nanoparticles constructed in this invention exhibited improved pharmacokinetic behavior, as well as better tumor inhibition rate and safety, showing better prospects for clinical translation. Attached Figure Description

[0073] Figure 1 The epirubicin-myristic acid prodrug (EPI-C) of Example 1 of this invention 14 The structure of the spectrum is confirmed.

[0074] A: EPI-C 14 of 1 H-NMR spectrum.

[0075] B: EPI-C 14 The mass spectrum.

[0076] Figure 2 Epirubicin-stearic acid prodrug (EPI-C) of Example 2 of this invention 18 The structure of the spectrum is confirmed.

[0077] A: EPI-C 18 of 1 H-NMR spectrum.

[0078] B: EPI-C 18 The mass spectrum.

[0079] Figure 3 The epirubicin-behenicol prodrug (EPI-C) of Example 3 of this invention 22 The structure of the spectrum is confirmed.

[0080] A: EPI-C 22 of 1 H-NMR spectrum.

[0081] B: EPI-C 22 The mass spectrum.

[0082] Figure 4 This is an in vitro release test diagram of the epirubicin-fatty acid prodrug albumin nanoparticle drug composition of Example 12 of the present invention.

[0083] A: In vitro release diagram of the epirubicin-fatty acid prodrug albumin nanoparticle drug composition at pH 5.0.

[0084] B: In vitro release diagram of the epirubicin-fatty acid prodrug albumin nanoparticle drug composition at pH 6.5.

[0085] C: In vitro release diagram of the epirubicin-fatty acid prodrug albumin nanoparticle drug composition at pH 7.4.

[0086] *: P < 0.05; **: P < 0.01.

[0087] Figure 5 This is a 4T1 cytotoxicity diagram of the epirubicin-fatty acid prodrug albumin nanoparticle drug composition of Example 13 of the present invention.

[0088] Figure 6 This is a blood concentration-time curve of the epirubicin-fatty acid prodrug albumin nanoparticle drug composition of Example 14 of the present invention.

[0089] A: Plasma concentration-time curve of epirubicin in SD rats.

[0090] B: Plasma concentration-time curve of prodrug in SD rats.

[0091] C: Plasma concentration-time curve of prodrug and total epirubicin in SD rats.

[0092] Figure 7 This is a diagram showing the accumulation of the epirubicin-fatty acid prodrug albumin nanoparticle drug composition in tumors according to Example 15 of the present invention.

[0093] **: P<0.01; ****: P<0.0001.

[0094] Figure 8 This describes the antitumor effect of the epirubicin-fatty acid prodrug albumin nanoparticle drug composition of Example 16 of the present invention in a 4T1 tumor model (administered concentration: 2.5 mg / kg).

[0095] A: Effect of epirubicin-fatty acid prodrug albumin nanoparticle drug composition on the growth of subcutaneous breast cancer tumors in Balb / C mice.

[0096] B: Effect of epirubicin-fatty acid prodrug albumin nanoparticle drug composition on body weight in tumor-bearing mice.

[0097] C: Effect of epirubicin-fatty acid prodrug albumin nanoparticle drug composition on tumor bearing rate in Balb / C mice.

[0098] *: P<0.05; ***: P<0.001; ns: No significant difference.

[0099] Figure 9 This describes the antitumor effect of the epirubicin-fatty acid prodrug albumin nanoparticle drug composition of Example 16 of the present invention in a 4T1 tumor model (administered concentration: 5 mg / kg).

[0100] A: Effect of epirubicin-fatty acid prodrug albumin nanoparticle drug composition on the growth of subcutaneous breast cancer tumors in Balb / C mice.

[0101] B: Effect of epirubicin-fatty acid prodrug albumin nanoparticle drug composition on body weight in tumor-bearing mice.

[0102] C: Effect of epirubicin-fatty acid prodrug albumin nanoparticle drug composition on tumor bearing rate in Balb / C mice.

[0103] *: P<0.05; **: P<0.01; ***: P<0.001; ns: No significant difference.

[0104] Figure 10 This describes the antitumor effect of the epirubicin-fatty acid prodrug albumin nanoparticle drug composition of Example 16 of the present invention in a 4T1 tumor model (administered concentration: 10 mg / kg).

[0105] A: Effect of epirubicin-fatty acid prodrug albumin nanoparticle drug composition on the growth of subcutaneous breast cancer tumors in Balb / C mice.

[0106] B: Effect of epirubicin-fatty acid prodrug albumin nanoparticle drug composition on body weight in tumor-bearing mice.

[0107] C: Effect of epirubicin-fatty acid prodrug albumin nanoparticle drug composition on tumor bearing rate in Balb / C mice.

[0108] *: P<0.05; ***: P<0.001; ****: P<0.0001; ns: No significant difference. Detailed Implementation

[0109] The present invention will be further illustrated by way of embodiments below, but the invention is not limited to the scope of the embodiments described herein.

[0110] Example 1: Epirubicin-Myristic Acid (EPI-C) 14 Synthesis of prodrugs

[0111] 14.0 g of myristic acid (0.1 mol) and 150 mL of methanol were added to a round-bottom flask, followed by 3 mL of concentrated sulfuric acid, and refluxed for 12 h. After concentration, the reaction solution was poured into ice water, saturated sodium bicarbonate was added, and the mixture was neutralized to neutral. Extraction was performed with ethyl acetate, and the solution was concentrated to obtain a fatty acid methyl ester intermediate. The intermediate was dissolved in ethanol, and 60 mL of hydrazine hydrate was added, followed by reflux for 12 h. After concentration, the reaction solution was poured into water, and a solid precipitated. Filtering was performed to obtain a fatty acid acyl hydrazine intermediate. 2.9 g of epirubicin hydrochloride, 5 mL of glacial acetic acid, and 1.8 g of the fatty acid acyl hydrazine intermediate were dissolved in anhydrous methanol, refluxed at 50-60 °C for 12 h, concentrated, recrystallized in acetone, and filtered. The target product was purified by preparative liquid chromatography.

[0112] The structure of the prodrug in Example 1 was determined using mass spectrometry and proton nuclear magnetic resonance spectroscopy, and the results are as follows: Figure 1 As shown. The spectral analysis results are as follows:

[0113] 1 H NMR(600MHz,DMSO-d6)δ14.11(s,1H,13-OH),13.25(s,1H,6-OH),10.29(s,1H,-CO NH-),7.93-7.89(m,2H,1,2-H),7.66(dd,J=6.8,2.9Hz,1H,18-H),5.77(t,J=4.9H z,1H,10'-OH),5.71(d,J=5.9Hz,1H,11-H),5.59(s,1H,9-OH),5.29(d,J=3.5Hz,1 H,22-OH),4.93(t,J=7.3Hz,1H,19-H),4.40(qd,J=14.4,4.5Hz,2H,10'-CH2),3.9 7(s,3H,-OCH3),3.78(p,J=6.5Hz,1H,23-H),3.36(d,J=17.9Hz,1H,8a-CH2),3.15 -3.07(m,2H,21,22-H),2.71(d,J=17.2Hz,1H,8b-CH2),2.56-2.51(m,1H,10a-CH2 ),2.18-2.01(m,4H,12'-CH2,10b-CH2,20a-CH2),1.78(td,J=12.8,3.8Hz,1H,20b -CH2),1.36-0.99(m,25H,23-CH3,13'~23'-CH2),0.84(t,J=6.9Hz,3H,24'-CH3).

[0114] Example 2: Epirubicin-Stearic Acid (EPI-C) 18 Synthesis of prodrugs

[0115] 16.9 g of stearic acid and 150 mL of methanol were added to a round-bottom flask, followed by 3 mL of concentrated sulfuric acid. The mixture was refluxed for 12 h. After concentration, the reaction solution was poured into ice water, saturated sodium bicarbonate was added, and the mixture was neutralized to neutral. Extraction was performed with ethyl acetate, and the solution was concentrated to obtain a fatty acid methyl ester intermediate. The intermediate was dissolved in ethanol, and 60 mL of hydrazine hydrate was added. The mixture was refluxed for 12 h. After concentration, the reaction solution was poured into water, and a solid precipitated. Filtering was performed to obtain a fatty acid acyl hydrazine intermediate. 2.9 g of epirubicin hydrochloride, 5 mL of glacial acetic acid, and 1.8 g of the fatty acid acyl hydrazine intermediate were dissolved in anhydrous methanol and refluxed at 50-60 °C for 12 h. The mixture was concentrated, recrystallized in acetone, and filtered. The target product was purified by preparative liquid chromatography.

[0116] The structure of the prodrug in Example 2 was determined using mass spectrometry and proton nuclear magnetic resonance spectroscopy, and the results are as follows: Figure 2 As shown. The spectral analysis results are as follows:

[0117] 1 H NMR(600MHz,DMSO-d6)δ14.12(s,1H,13-OH),13.32(s,1H,6-OH),10.29(s,1H,-CON H-),7.93-7.89(m,2H,1,2-H),7.66(dd,J=7.0,2.6Hz,1H,18-H),5.77(t,J=4.8Hz, 1H,10'-OH),5.72(d,J=5.9Hz,1H,11-H),5.59(s,1H,9-OH),5.29(d,J=3.7Hz,1H,2 2-OH),4.93(t,J=7.3Hz,1H,19-H),4.40(qd,J=14.4,4.7Hz,2H,10'-CH2),3.97(s,3 H,-OCH3),3.78(dt,J=12.8,6.4Hz,1H,23-H),3.36(d,J=17.6Hz,1H,8a-CH2),3.14 -3.06(m,2H,21,22-H),2.72(d,J=17.2Hz,1H,8b-CH2),2.56-2.52(m,1H,10a-CH2) ,2.19-2.00(m,4H,12'-CH2,10b-CH2,20a-CH2),1.78(td,J=12.7,3.8Hz,1H,20b-C H2), 1.23 (d, J=13.3Hz, 33H, 23-CH3, 13'~27'-CH2), 0.84 (t, J=6.9Hz, 3H, 28'-CH3).

[0118] Example 3: Epirubicin-Behenicol (EPI-C)22 Synthesis of prodrugs

[0119] 19.6 g of behenic acid (0.05 mol) and 150 mL of methanol were added to a round-bottom flask, followed by 3 mL of concentrated sulfuric acid. The mixture was refluxed for 12 h. After concentration, the reaction solution was poured into ice water, saturated sodium bicarbonate was added, and the mixture was neutralized to neutral. The solution was extracted with ethyl acetate and concentrated to obtain a fatty acid methyl ester intermediate. The intermediate was dissolved in ethanol, and 60 mL of hydrazine hydrate was added. The mixture was refluxed for 12 h. After concentration, the reaction solution was poured into water, and a solid precipitated. The solid was filtered to obtain a fatty acid acyl hydrazine intermediate. 2.9 g of epirubicin hydrochloride, 5 mL of glacial acetic acid, and 1.8 g of the fatty acid acyl hydrazine intermediate were dissolved in anhydrous methanol and refluxed at 50-60 °C for 12 h. The mixture was concentrated, recrystallized in acetone, and filtered. The target product was purified by preparative liquid chromatography.

[0120] The structure of the prodrug in Example 3 was determined using mass spectrometry and proton nuclear magnetic resonance spectroscopy, and the results are as follows: Figure 3 As shown. The spectral analysis results are as follows:

[0121] 1 H NMR(600MHz,DMSO-d6)δ14.11(s,1H,13-OH),13.26(s,1H,6-OH),10.29(s,1H,-CO NH-),7.93-7.89(m,2H,1,2-H),7.66(dd,J=7.2,2.5Hz,1H,18-H),5.76(t,J=4.8H z,1H,10'-OH),5.71(d,J=5.7Hz,1H,11-H),5.58(s,1H,9-OH),5.29(d,J=3.7Hz,1 H,22-OH),4.93(t,J=7.3Hz,1H,19-H),4.40(qd,J=14.4,4.5Hz,2H,10'-CH2),3.9 7(s,3H,-OCH3),3.78(p,J=6.6Hz,1H,23-H),3.36(d,J=18.1Hz,1H,8a-CH2),3.14 -3.07(m,2H,21,22-H),2.72(d,J=15.1Hz,1H,8b-CH2),2.59-2.52(m,1H,10a-CH2 ),2.18-2.01(m,4H,12'-CH2,10b-CH2,20a-CH2),1.77(td,J=12.7,4.0Hz,1H,20b -CH2),1.31-1.10(m,41H,23-CH3,13'~31'-CH2),0.84(t,J=6.9Hz,3H,32'-CH3).

[0122] Example 4: Investigation of influencing factors of epirubicin-fatty acid prodrug albumin nanoparticle drug composition

[0123] Accurately weigh 6 mg of epirubicin prodrug and dissolve it in 5.7 mL of deionized water. While continuously stirring, slowly add 0.3 mL of human serum albumin dropwise to the prodrug aqueous solution. Then, use an ultrasonic cell disruptor to sonicate the solution to form uniform albumin nanoparticles. The sonication time was 1 minute and the ultrasonic power was 200 W. The mass ratio of epirubicin prodrug to albumin was varied, and the results are shown in Table 1. When the mass ratio of prodrug to albumin was 1:1-2, the particle size of the prodrug albumin nanoparticles was between 80-130 nm; when the mass ratio was 1:2, the particle size, particle size distribution, and binding rate were even better.

[0124] Table 1. Formulation characterization of rubicin-fatty acid prodrug and albumin at different mass ratios.

[0125]

[0126] Example 5: Preparation of Epirubicin-Fatty Acid Prodrug Albumin Nanoparticle Drug Composition by Probe Ultrasonic Method

[0127] Accurately weigh 6 mg of epirubicin prodrug and dissolve it in 5.7 mL of deionized water. While stirring continuously, slowly add 0.3 mL of human serum albumin dropwise to the prodrug solution. Then, use an ultrasonic cell disruptor to sonicate the solution to form uniform albumin nanoparticles. The sonication time is 1 minute and the ultrasonic power is 200 W.

[0128] As shown in Table 2, epirubicin has poor affinity for albumin, resulting in nanoparticles with a diameter as high as 491.9 nm, a particle size distribution as high as 0.6, and a binding rate of less than 10%. In contrast, among the three prodrugs, EPI-C... 22 The affinity for albumin is poor, making it difficult to form uniform albumin nanoparticles (particle size distribution > 0.3, binding rate < 60%). The other two prodrug albumin nanoparticles have a particle size of approximately 120 nm, a particle size distribution less than 0.2, a binding rate greater than 90%, and a drug loading of over 20%. Compared to the doxorubicin prodrug albumin nanoparticles (< 20%) prepared in Example 5 of CN115702902A, the epirubicin-fatty acid prodrug albumin nanoparticle drug composition prepared in this example has a higher drug loading.

[0129] Table 2. Formulation characterization of the rorupricin-fatty acid prodrug albumin nanoparticle drug composition

[0130]

[0131] Example 6: Preparation of Epirubicin-Fatty Acid Prodrug Albumin Nanoparticle Drug Composition by High Pressure Homogenization

[0132] 40 mg of epirubicin prodrug was accurately weighed and dissolved in 18.5 mL of deionized water. After incubation at 25 °C for 10 minutes, 1.5 mL of human serum albumin aqueous solution was slowly added dropwise to the prodrug aqueous solution. The mixture was stirred continuously for 10 minutes, and then homogenized in a high-pressure homogenizer to obtain uniform albumin nanoparticles. The homogenization pressure was 10000 psi, and the homogenization was performed 3 times. The results are shown in Table 3. The obtained albumin nanoparticles had a particle size of approximately 96 nm, a particle size distribution of less than 0.25, a binding rate greater than 90%, and a drug loading of 25%, which is significantly higher than the doxorubicin prodrug albumin nanoparticles (<20%) prepared in Example 5 of CN 115702902 A.

[0133] Table 3. Formulation characterization of the rorupricin-fatty acid prodrug albumin nanoparticle drug composition

[0134]

[0135] Example 7: Preparation of Epirubicin-Fatty Acid Prodrug Albumin Nanoparticle Drug Composition by High Pressure Homogenization

[0136] Accurately weigh 60 mg of epirubicin prodrug and dissolve it in 17.75 mL of deionized water. Slowly add 2.25 mL of human serum albumin aqueous solution to the prodrug aqueous solution and stir continuously for 10 minutes. Then, transfer the solution to a high-pressure homogenizer for cyclic homogenization to obtain uniform albumin nanoparticles. The homogenization pressure was 20,000 psi, and the homogenization was performed 5 times. The results are shown in Table 4. The obtained albumin nanoparticles had a particle size of about 90 nm, a particle size distribution of less than 0.2, a binding rate as high as 95%, and a drug loading of about 25%, which is much higher than the doxorubicin prodrug albumin nanoparticles (<20%) prepared in Example 5 of CN 115702902A.

[0137] Table 4. Formulation characterization of the rorupricin-fatty acid prodrug albumin nanoparticle drug composition

[0138]

[0139] Example 8: Preparation of Epirubicin-Fatty Acid Prodrug Albumin Nanoparticle Drug Composition by High Pressure Homogenization

[0140] Accurately weigh 80 mg of epirubicin prodrug and dissolve it in 17 mL of deionized water. Slowly add 3 mL of human serum albumin aqueous solution to the prodrug aqueous solution and stir continuously for 10 minutes. Then, transfer the solution to a high-pressure homogenizer for cyclic homogenization to obtain uniform albumin nanoparticles. The homogenization pressure was 20,000 psi, and the homogenization was performed 10 times. The results are shown in Table 5. The obtained albumin nanoparticles had a particle size of about 80 nm, a particle size distribution of less than 0.2, a binding rate of more than 90%, and a drug loading of about 25%, which is much higher than that of the doxorubicin prodrug albumin nanoparticles (<20%) prepared in Example 5 of CN 115702902A.

[0141] Table 5. Formulation characterization of the rorbicin-fatty acid prodrug albumin nanoparticle drug composition

[0142]

[0143] Example 9: Lyophilization and reconstitution of epirubicin-fatty acid prodrug albumin nanoparticle drug composition

[0144] The epirubicin-myristate prodrug albumin nanoparticles and epirubicin-stearate prodrug albumin nanoparticles prepared by ultrasonication in Example 5 were freeze-dried. 5% anhydrous trehalose was used as a cryoprotectant at a ratio of 1:1 (v / v). After 24 hours of freeze-drying, the lyophilized formulations were obtained and reconstituted with 5% glucose solution. The results are shown in Tables 6 and 7. Using 5% anhydrous trehalose as a cryoprotectant, the formulation properties of the epirubicin-myristate prodrug albumin nanoparticles and epirubicin-stearate prodrug albumin nanoparticles remained essentially unchanged after freeze-drying and reconstitution, and they exhibited good storage stability.

[0145] Table 6. Formulation characterization of the reconstituted xyloside-fatty acid prodrug albumin nanoparticle drug composition.

[0146]

[0147] Table 7. Stability Study of the Ruboxine-Fatty Acid Prodrug Albumin Nanoparticle Drug Composition after Reconstitution

[0148]

[0149] Example 10: Lyophilization and reconstitution of epirubicin-stearic acid prodrug albumin nanoparticle drug composition

[0150] The epirubicin-stearic acid prodrug albumin nanoparticles prepared by high-pressure homogenization in Example 7 were freeze-dried. 5% anhydrous trehalose was used as a cryoprotectant at a ratio of 1:1 (v / v). After 24 hours of freeze-drying, the lyophilized formulation was obtained and then reconstituted with 5% glucose solution. The results are shown in Tables 8 and 9. Using 5% anhydrous trehalose as a cryoprotectant, the formulation properties of the epirubicin-stearic acid prodrug albumin nanoparticles remained essentially unchanged after freeze-drying and reconstitution, and they exhibited good storage stability.

[0151] Table 8. Formulation characterization of the reconstituted xylobicin-stearic acid prodrug albumin nanoparticle drug composition.

[0152]

[0153] Table 9. Stability Study of Ruboxine-Stearic Acid Prodrug Albumin Nanoparticle Drug Composition after Reconstitution

[0154]

[0155] Example 11: Lyophilization and reconstitution of epirubicin-stearic acid prodrug albumin nanoparticle drug composition

[0156] The epirubicin-stearic acid prodrug albumin nanoparticles prepared by high-pressure homogenization in Example 7 were freeze-dried. 5% sucrose was used as a cryoprotectant at a ratio of 1:1 (v / v). After 24 hours of freeze-drying, the lyophilized formulation was obtained and then reconstituted with 5% glucose solution. The results are shown in Tables 10 and 11. Using 5% sucrose as a cryoprotectant, the formulation properties of the epirubicin-stearic acid prodrug albumin nanoparticles remained essentially unchanged after freeze-drying and reconstitution, and they exhibited good storage stability.

[0157] Table 10. Formulation characterization of the reconstituted xylobicin-stearic acid prodrug albumin nanoparticle drug composition.

[0158]

[0159] Table 11. Stability study of the reconstituted xarabic-stearic acid prodrug albumin nanoparticle drug composition after rehydration.

[0160]

[0161] Example 12: In vitro release experiment of epirubicin-fatty acid prodrug albumin nanoparticle drug composition

[0162] The drug release of epirubicin-fatty acid prodrug albumin nanoparticles was investigated in normal tissues, acidic tumor microenvironments, and lysosomes / endosomes. Epirubicin-myristate prodrug albumin nanoparticles and epirubicin-stearate prodrug albumin nanoparticles prepared in Example 5 were placed in phosphate buffer solutions (containing 30% ethanol (v / v)) at different pH values ​​(7.4, 6.5, or 5.0) and gently stirred at 37°C. Subsequently, 200 μL samples were collected at predetermined time intervals to study the in vitro drug release of epirubicin-fatty acid prodrug albumin nanoparticles under different pH conditions.

[0163] In vitro release results by Figure 4 As shown, after 48 hours of incubation, both epirubicin-myristate prodrug albumin nanoparticles and epirubicin-stearic acid prodrug albumin nanoparticles released the parent drug under acidic environments (pH 5.0 and 6.5), with the release rate increasing with stronger acidity. This is mainly because the acylhydrazone bonds in the epirubicin prodrug structure are unstable under acidic conditions and rapidly hydrolyze. The epirubicin-stearic acid prodrug albumin nanoparticles released the parent drug at a faster rate than the epirubicin-myristate prodrug albumin nanoparticles, possibly because the longer acylhydrazone bonds are more easily hydrolyzed under acidic conditions. Under neutral conditions (pH 7.4), the acylhydrazone bonds remained stable and were difficult to hydrolyze; after 48 hours of incubation, the release rates of both prodrugs were very low (<15%).

[0164] The above results indicate that the epirubicin-fatty acid prodrug albumin nanoparticle drug composition can only rapidly release the parent drug in an acidic pH environment at the tumor site, endosomes, and lysosomes, thereby avoiding drug leakage in systemic circulation.

[0165] Example 13: Cytotoxicity test of epirubicin-fatty acid prodrug albumin nanoparticle drug composition

[0166] The toxicity of the epirubicin-fatty acid prodrug albumin nanoparticle drug composition to mouse breast cancer (4T1) cells was investigated using the MTT assay. Healthy cells were digested, diluted with culture medium to 10,000 cells / mL, and seeded at a density of 2,000 cells / well in 96-well plates. After 12 h of culture, the culture medium was discarded. Epirubicin hydrochloride injection or the epirubicin-myristate prodrug albumin nanoparticles and epirubicin-stearic acid prodrug albumin nanoparticles prepared in Example 5 were added, and the cells were incubated for another 48 h. Untreated cells were used as a control. At the end of incubation, 35 μL of MTT (5 mg / mL) was added to each well, and the cells were incubated at 37°C for another 4 h. The culture medium was then removed, and the resulting blue crystals were dissolved in 200 μL of dimethyl sulfoxide (DMSO). The absorbance was measured at 570 nm using a microplate reader.

[0167] Table 12. Cytotoxicity of the Ruboxine-Fatty Acid Prodrug Albumin Nanoparticle Drug Composition on 4T1 Cells

[0168]

[0169] Cytotoxicity results such as Figure 5 As shown in Table 12, compared with epirubicin hydrochloride injection, the prodrug albumin nanoparticles exhibited reduced cytotoxicity, and the longer the fatty acid carbon chain length, the stronger the cytotoxicity of the prodrug albumin nanoparticles.

[0170] Example 14: Pharmacokinetic Study of Epirubicin-Fatty Acid Prodrug Albumin Nanoparticle Drug Composition

[0171] Pharmacokinetic studies were conducted using SD rats (200-250g). Rats were randomly assigned to groups and fasted for 12 hours prior to administration, with free access to water. Epirubicin hydrochloride injection, epirubicin-myristate prodrug albumin nanoparticles, and epirubicin-stearic acid prodrug albumin nanoparticles prepared in Example 5 were administered intravenously, respectively. The dose of epirubicin was 4 mg / kg. Blood was collected from the orbital sinus at specified time points, and plasma was obtained. The drug concentration in the plasma was determined by liquid chromatography-mass spectrometry.

[0172] Experimental results are as follows Figure 6 As shown in Table 13, compared with the epirubicin hydrochloride injection group, the prodrug albumin nanoparticles had a prolonged retention time in the blood and significantly increased the area under the drug-time curve (AUC0-24 h) of epirubicin, ensuring the tumor-targeted accumulation of epirubicin. The overall AUC0-24 h (sum of prodrug and parent drug) of epirubicin-myristate prodrug albumin nanoparticles and epirubicin-stearate prodrug albumin nanoparticles were 14.0 times and 38.72 times that of epirubicin hydrochloride injection, respectively. These values ​​are significantly higher than those of DOX-C in Example 7 of CN 115702902 A. 10 Nanoparticle group (1.37 times that of DOX solution), DOX-C 14 Nanoparticle group (5.18 times that of DOX solution) and DOX-C 18 The nanoparticle group (13.73 times that of the DOX solution) indicates that the epirubicin prodrug albumin nanoparticle pharmaceutical composition of the present invention significantly improves the pharmacokinetic behavior of epirubicin, with higher AUC and slower clearance.

[0173] Table 13. Pharmacokinetic parameters of the rorbixin prodrug albumin nanoparticle drug composition

[0174]

[0175] Example 15: Tumor accumulation experiment of epirubicin prodrug albumin nanoparticle drug composition

[0176] A 4T1 cell-bearing mouse model was established by subcutaneously injecting 75 μL of 4T1 cell suspension into the right back of the mouse. When the tumor volume reached 400 mm², the tumor was successfully treated. 3 Mice were randomly divided into groups of 3 at approximately 24 hours post-treatment. Epirubicin hydrochloride injection and the epirubicin-myristate prodrug albumin nanoparticles and epirubicin-stearic acid prodrug albumin nanoparticles prepared in Example 5 were injected via tail vein. The dose was equivalent to 10 mg / kg of epirubicin. Tumors were collected at 1, 4, and 24 hours post-administration. The concentrations of the prodrug and parent drug in the tissues were determined using UPLC-MS-MS.

[0177] The results are as follows Figure 7 As shown, the accumulation of the two prodrug albumin nanoparticles at the tumor site was significantly higher than that of epirubicin hydrochloride injection. Furthermore, the drugs primarily existed in the form of epirubicin prodrugs, enabling the gradual release of epirubicin into the acidic environment of the tumor tissue.

[0178] Example 16: Antitumor Experiment of Epirubicin Prodrug Albumin Nanoparticle Drug Composition

[0179] A 4T1 cell-bearing mouse model was established by subcutaneously injecting 75 μL of 4T1 cell suspension into the right back of the mouse. When the tumor volume reached 100 mm... 3 At approximately 10:00 AM, mice were randomly divided into 9 groups (5 mice per group), receiving saline, epirubicin hydrochloride injection (2.5 mg / kg and 5 mg / kg), epirubicin-myristate prodrug albumin nanoparticles prepared in Example 5 (2.5 mg / kg, 5 mg / kg and 10 mg / kg), and epirubicin-stearic acid prodrug albumin nanoparticles prepared in Example 5 (2.5 mg / kg, 5 mg / kg and 10 mg / kg), all at equivalent doses of epirubicin. Administered every two days for a total of 5 times, with tumor volume and mouse weight measured daily.

[0180] The results are as follows Figure 8-10 As shown, at a dosage concentration of 5 mg / kg, mice in the epirubicin hydrochloride injection group experienced a significant decrease in body weight during administration. However, in the epirubicin-myristate prodrug albumin nanoparticle group and the epirubicin-stearic acid prodrug albumin nanoparticle group, no significant decrease in body weight was observed even at a dosage of 10 mg / kg, indicating that the epirubicin-fatty acid prodrug albumin nanoparticle group improved the tolerable dose of epirubicin and demonstrated good safety. Furthermore, compared to the saline group, all treatment groups showed significant antitumor effects, with the epirubicin-stearic acid prodrug albumin nanoparticle group exhibiting the most significant antitumor effect.

[0181] To further compare the antitumor effects of epirubicin prodrug albumin nanoparticles, the tumor inhibition rate and weight loss rate of each administration group in this example and Example 8 of CN115702902A were calculated. The results are shown in Table 14. At the same dosage of 10 mg / kg, the tumor inhibition rate of the epirubicin-stearic acid prodrug albumin nanoparticle group of this invention was significantly higher than that of all doxorubicin-fatty acid prodrug albumin nanoparticle groups in Example 8 of CN115702902A. In addition, the epirubicin-stearic acid prodrug albumin nanoparticle group of this invention had a comparable tumor inhibition rate to the doxorubicin liposome (Doxil) group in Example 8 of CN115702902A, but its safety profile was significantly higher than that of the Doxil group, the doxorubicin (DOX) solution group, and the doxorubicin maleimide prodrug (DOXO-EMCH, Phase III clinical trial) group. Even more surprisingly, the epirubicin-fatty acid prodrug albumin nanoparticles of the present invention, at a dosage of 5 mg / kg, exhibited antitumor efficacy comparable to the various treatment groups in Example 8 of CN 115702902A, with improved safety. These results demonstrate that the epirubicin prodrug albumin nanoparticles constructed in this invention not only possess better antitumor efficacy but also significantly reduce chemotherapy-related toxic side effects.

[0182] Table 14. Tumor inhibition rate and body weight loss rate of each drug administration group in this example and Example 8 of CN 115702902 A in the 4T1 cell tumor-bearing mouse model.

[0183]

Claims

1. Epirubicin-fatty acid prodrug of general formula (I) or a pharmaceutically acceptable salt thereof: in, R is C 14 -C 26 The portion of saturated or unsaturated fatty acids that does not contain carboxyl hydroxyl groups is selected from the portions of myristic acid, stearic acid, behenic acid, waxy acid, myristoleic acid, oleic acid, behenoleic acid, tetradecanoic acid, octadecanoic acid, and docosahexaenoic acid that do not contain carboxyl hydroxyl groups.

2. The epirubicin-fatty acid prodrug of claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, The structure is as follows:

3. The method for synthesizing epirubicin-fatty acid prodrug as described in claim 1 or 2, characterized in that, Prepared using the following steps: (1) Reflux fatty acids, methanol and concentrated sulfuric acid to obtain fatty acid methyl esters; (2) The fatty acid methyl ester obtained in step (1) is refluxed with hydrazine hydrate to obtain fatty acid acyl hydrazine intermediate; (3) The fatty acid hydrazide intermediate in step (2) is reacted with epirubicin hydrochloride carbonyl group and recrystallized to obtain crude epirubicin-fatty acid prodrug. (4) The crude product obtained is separated and purified by preparative liquid chromatography and then analyzed by analytical liquid chromatography. The purity can reach more than 99%.

4. The method for synthesizing epirubicin-fatty acid prodrug as described in claim 3, characterized in that: The fatty acid mentioned in step (1) is C 14 -C 22 Saturated fatty acids or unsaturated fatty acids; The ratio of fatty acid (moles): methanol (mL): concentrated sulfuric acid (mL) is 1:500-10000:10-200, preferably 1:1000-4000:20-50; In step (2), the molar ratio of fatty acid methyl ester to hydrazine hydrate is 1:5-50, preferably 1:10-30; In step (3), the molar ratio of epirubicin hydrochloride to fatty acid acylhydrazine intermediate is 1:1-3; The recrystallization solvent is acetone.

5. The epirubicin-fatty acid prodrug albumin nanoparticle pharmaceutical composition according to claim 1 or 2, characterized in that: The albumin nanoparticles comprise epirubicin-fatty acid prodrug and human serum albumin, wherein the mass ratio of epirubicin-fatty acid prodrug to albumin is 1:0.1-10, preferably 1:0.5-5, more preferably 1:1-3, and the albumin nanoparticles have a particle size of 70-200 nm, preferably 80-150 nm, more preferably 80-120 nm.

6. The epirubicin-fatty acid prodrug albumin nanoparticle pharmaceutical composition as described in claim 5, characterized in that: No organic solvents are used in the preparation of the albumin nanoparticles.

7. The method for preparing the epirubicin-fatty acid prodrug albumin nanoparticle drug composition as described in claim 5, characterized in that: The albumin nanoparticles were prepared using an ultrasonic method or a high-pressure homogenization method; wherein, epirubicin-fatty acid prodrug was dissolved in deionized water, and human serum albumin was diluted with deionized water; the preparation process was carried out using the following steps: (1) Dissolve epirubicin-fatty acid prodrug in deionized water to obtain an aqueous drug solution; (2) Dilute the human serum albumin solution with deionized water to obtain a diluted albumin solution; (3) While stirring continuously, slowly add the albumin diluent obtained in step (2) to the drug solution obtained in step (1); (4) After continuous stirring for a period of time, a nano-colloidal solution without any organic solvent can be directly obtained by ultrasonic or high-pressure homogenization. (5) After sterile filtration, a sterile and uniform nano-formulation can be obtained, which is the epirubicin-fatty acid prodrug albumin nanoparticle drug composition.

8. The method for preparing epirubicin-fatty acid prodrug albumin nanoparticles as described in claim 7, characterized in that: The stirring time is 5-30 minutes, preferably 10-20 minutes; The serum albumin mentioned is human serum albumin or bovine serum albumin, preferably human serum albumin; The concentration of the albumin diluent is 0.1-100 mg / mL, preferably 30-60 mg / mL; The mass ratio of the pH-sensitive epirubicin-fatty acid prodrug to serum albumin is 1:0.1-10, preferably 1:0.5-5, and more preferably 1:1-3; In the ultrasonic method, the ultrasonic power is 50-500W, preferably 100-300W; In the ultrasound method, the ultrasound duration is 0.5-10 minutes, preferably 1-5 minutes, and more preferably 1-3 minutes; In the high-pressure homogenization method, the pressure is 2000-25000 psi, preferably 10000-20000 psi; In the high-pressure homogenization method, the number of homogenization cycles is 1-15, preferably 3-10. The sterilization filtration uses a 0.22μm polyethersulfone water membrane.

9. A lyophilized formulation made from the epirubicin-fatty acid prodrug albumin nanoparticle pharmaceutical composition of claim 5, characterized in that: The lyophilization protectant is selected from one or more of trehalose, sucrose, mannitol, glucose, and dextran, preferably trehalose, sucrose, and dextran, and most preferably trehalose and sucrose; the weight ratio of the lyophilization protectant to the epirubicin-fatty acid prodrug is (10.0-20.0):1, preferably (15.0-18.0):1; the reconstitution solvent for the lyophilized preparation is selected from water for injection, 5% glucose solution, or 0.9% sodium chloride solution, preferably 5% glucose solution.

10. The use of the epirubicin-fatty acid prodrug of claim 1 or 2, or the epirubicin-fatty acid prodrug albumin nanoparticle drug composition of claims 5-6, or the lyophilized formulation of claim 9, in the preparation of a drug delivery system, preferably the drug delivery system being an antitumor drug.

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