Mitoxantrone hydrochloride ion pair self-assembly nanoparticles, preparation method and application thereof

Abstract

The application discloses ion pair self-assembly nanoparticles of mitoxantrone hydrochloride and a preparation method and application thereof, and belongs to the field of new excipients and new dosage forms of pharmaceutical preparations. Hydrophilic ion drugs mitoxantrone hydrochloride and hydrophobic auxiliary counterions form ion pairs, and self-assembly is realized by balancing the interaction between the two. Experimental results show that the intermolecular force between the hydrophilic ion drugs and the hydrophobic auxiliary counterions has a significant influence on the pharmaceutical properties, cytotoxicity and other aspects of the ion pair self-assembly nanoparticles. The ion pair self-assembly nanoparticles of mitoxantrone hydrochloride retain the curative effect of a mitoxantrone hydrochloride solution, significantly reduce the systemic toxicity of the mitoxantrone hydrochloride solution, and exhibit better tolerability. The ion pair self-assembly nanoparticles provide a new idea and more choices for the delivery of hydrophilic ion drugs, which will meet the urgent needs of clinical high-efficiency-low-toxicity chemotherapy preparations.

Description

Technical Field

[0001] This invention relates to novel excipients and dosage forms in the field of pharmaceutical formulations, specifically to a mitoxantrone hydrochloride ion-paired self-assembled nanoparticle, its preparation method, and its application in the preparation of antitumor drugs. Background Technology

[0002] The incidence and mortality rates of cancer are rising annually, posing a serious threat to people's health. Chemotherapy is used as a primary treatment for cancer. However, the non-specific delivery mode of cytotoxic chemotherapy drugs limits their clinical application and leads to severe side effects. Nanomedicine delivery systems have shown great promise in the biomedical field, helping to target anticancer drugs to tumor sites, thereby improving treatment efficacy while reducing systemic toxicity. Mitoxantrone hydrochloride (MTO) is a DNA topoisomerase II inhibitor that causes DNA damage by inserting into the DNA strand and increasing the concentration of the DNA-topoisomerase II covalent complex, as shown in the diagram. MTO is effective against various tumors, including breast cancer, prostate cancer, acute myeloid leukemia, and non-Hodgkin's lymphoma. Despite its great therapeutic potential, commercially available mitoxantrone hydrochloride injections suffer from poor tumor selectivity, and their widespread distribution in the body after intravenous injection can cause serious adverse reactions such as cardiotoxicity, bone marrow suppression, renal insufficiency, hematuria, and alopecia. The hydrophilicity of MTO (logP = -3.1) also presents challenges for its formulation development and clinical application. Therefore, there is an urgent need to develop MTO nanodelivery systems to achieve efficient delivery of MTO.

[0003]

[0004] Self-assembled nanodelivery systems have attracted considerable attention due to their high drug loading capacity and low carrier toxicity. However, most drug molecules do not exhibit good self-assembly properties; typically, these anticancer drugs either form large aggregates in aqueous solutions or dissolve completely. Achieving efficient self-assembly of a single anticancer drug is a significant challenge. One current approach is to develop prodrugs through chemical modification to promote drug assembly rather than aggregation or dissolution. However, the sites suitable for structural modification in anticancer drug molecules are relatively limited, with most located within the pharmacophore. Therefore, after structural modification, additional drug release steps are often required, thus affecting efficacy. Based on the structural characteristic of water-soluble cationic drug MTO containing positively charged secondary amine groups on its side chains, we propose a novel strategy combining hydrophobic ion pairing with a self-assembled nanodelivery system. This method utilizes hydrophobic auxiliary counterions, such as sodium cholesterol sulfate, to form ion pairs with hydrophilic ionic drugs through electrostatic interactions. The ion pairing strategy shields the intrinsic charge of the drug molecule, expands the hydrophobic region of the drug molecule, and ultimately achieves self-assembly through the balance of interactions between the drug molecule and the auxiliary molecule. Sodium cholesterol sulfate (SCS) is a natural cholesterol metabolite widely distributed in human tissues and has a high safety profile. Based on this, this invention utilizes negatively charged auxiliary molecules to enhance the hydrophobicity of the water-soluble cationic drug mitoxantrone hydrochloride, constructing mitoxantrone hydrochloride ion-paired self-assembled nanoparticles. Amphiphilic lipids are added to increase the in vivo circulation time of the self-assembled nanoparticles. The interaction between ion pairs formed by different molar ratios of drug molecules and auxiliary molecules varies, thus affecting the encapsulation efficiency, stability, and cytotoxicity of the self-assembled nanoparticles. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides mitoxantrone hydrochloride ion-paired self-assembled nanoparticles and their preparation method. Specifically, mitoxantrone hydrochloride, a hydrophilic ionic drug, is paired with a hydrophobic auxiliary counterion such as sodium cholesterol sulfate to form ion pairs, and self-assembly is achieved by balancing the interaction between the two. Mitoxantrone hydrochloride ion-paired self-assembled nanoparticles have advantages such as small particle size, uniform particle size distribution, high encapsulation efficiency, good stability, good antitumor effect, and high safety. The ratio of the hydrophilic ionic drug to the hydrophobic auxiliary counterion affects the intermolecular forces, thereby influencing the stability, cytotoxicity, and therapeutic efficacy of the self-assembled nanoparticles. This strategy provides new ideas and more options for the development of nanodelivery systems based on hydrophilic ionic drugs, which will meet the urgent clinical need for highly effective and low-toxicity chemotherapy agents.

[0006] A mitoxantrone hydrochloride ion-paired self-assembled nanoparticle comprises mitoxantrone hydrochloride, a hydrophobic auxiliary counterion, and an amphiphilic lipid. The hydrophobic auxiliary counterion is selected from sodium cholesterol sulfate, cholic acid, deoxycholic acid, chenodeoxycholic acid, lithocholic acid, glycocholic acid, taurocholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, deoxycholic acid lysine derivative, oleanolic acid, and ursolic acid. The amphiphilic lipid is selected from phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, and methoxy-polyethylene glycol 2000-distearate phosphatidylethanolamine (DSPE-PEG). 2K ) and cholesterol-polyethylene glycol 2000 (CH-PEG).

[0007] In the mitoxantrone hydrochloride ion-paired self-assembled nanoparticles, the molar ratio of mitoxantrone hydrochloride : hydrophobic auxiliary counterion : amphiphilic lipid = 1 : (1-10) : (0.1-1).

[0008] This invention provides a method for preparing mitoxantrone hydrochloride ion-paired self-assembled nanoparticles, comprising the following steps:

[0009] a. Dissolve mitoxantrone hydrochloride, a hydrophobic auxiliary counterion, and an amphiphilic lipid in dimethyl sulfoxide (DMSO);

[0010] b. Under ultrasound probe, the solution obtained in step a is slowly added dropwise to water to form a uniform nanocolloid solution, namely mitoxantrone hydrochloride ion-paired self-assembled nanoparticles.

[0011] In step a, the hydrophobic auxiliary counterion is selected from one of the following: sodium cholesterol sulfate, cholic acid, deoxycholic acid, chenodeoxycholic acid, lithocholic acid, glycocholic acid, taurocholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, deoxycholic acid lysine derivative, oleanolic acid, and ursolic acid.

[0012] The molar ratio of mitoxantrone hydrochloride, hydrophobic auxiliary counterion, and amphiphilic lipid in step a is 1:(1-10):(0.1-1).

[0013] The amphiphilic lipids mentioned in step a are selected from phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, and methoxy polyethylene glycol 2000-distearate phosphatidylethanolamine (DSPE-PEG). 2K ) and cholesterol-polyethylene glycol 2000 (CH-PEG).

[0014] In step b, the ultrasonic power is 100-400W and the ultrasonic time is 1-15min;

[0015] In step b, the volume of water is 8-20 times the volume of dimethyl sulfoxide (DMSO) in step a;

[0016] The mitoxantrone hydrochloride ion-paired self-assembled nanoparticles described in step b have a particle size of 80–200 nm, uniform particle size distribution, and a drug loading of 5–30%.

[0017] The present invention also provides the application of the mitoxantrone hydrochloride ion-paired self-assembled nanoparticles in the preparation of antitumor drugs.

[0018] The beneficial effects of this invention include:

[0019] (1) Self-assembled mitoxantrone hydrochloride ion-pairing nanoparticles with small particle size and uniform particle size distribution were prepared by a simple and easy method. These nanoparticles are comparable to mitoxantrone hydrochloride solution in efficacy, while significantly reducing toxic side effects.

[0020] (2) The effects of intermolecular forces between hydrophilic ionic drugs and hydrophobic auxiliary counterions on the formulation properties and cytotoxicity of ion-paired self-assembled nanoparticles were investigated.

[0021] (3) It provides more options for the development of nanodelivery systems for hydrophilic ionic drugs, meeting the urgent clinical need for highly efficient, low-toxicity, and highly stable chemotherapeutic agents. Attached Figure Description

[0022] Figure 1 The graph shows the tumor volume change in the in vivo antitumor experiment of mitoxantrone hydrochloride ion-paired self-assembled nanoparticles in Example 2 of this invention, where *: P<0.05; ****: P<0.0001 (both are two-tailed t-tests).

[0023] Figure 2 The graph shows the weight change of the mitoxantrone hydrochloride ion-paired self-assembled nanoparticles in the in vivo antitumor experiment of Example 2 of the present invention, where *: P<0.05; ****: P<0.0001 (both are two-tailed t-tests).

[0024] Figure 3 This is a graph showing the change in body weight during the acute toxicity experiment of mitoxantrone hydrochloride ion-paired self-assembled nanoparticles in Example 2 of the present invention.

[0025] Figure 4 This is a survival curve diagram in the acute toxicity experiment of mitoxantrone hydrochloride ion-paired self-assembled nanoparticles of Example 2 of the present invention. Detailed Implementation

[0026] The present invention will be further described in detail below with reference to the embodiments, but the present invention is not limited to the scope of the embodiments described herein.

[0027] This embodiment investigates the formulation characterization, in vitro cytotoxicity, and in vivo antitumor experiments of mitoxantrone hydrochloride ion-paired self-assembled nanoparticles.

[0028] Example 1

[0029] Preparation of Mitoxanthraquinone Hydrochloride Ion-Paired Self-Assembled Nanoparticles (NAs)

[0030] Accurately weigh mitoxantrone hydrochloride and sodium cholesterol sulfate (molar ratios of 1:1, 1:2, 1:3, 1:4, 1:5, and 1:10, respectively), dissolve them in DMSO, and use as the organic phase. Ten times the volume of deionized water is used as the aqueous phase in a container. Under ultrasonic probe conditions, the organic phase is slowly added dropwise to the aqueous phase, and ultrasonication continues to occur, resulting in the formation of ion-paired self-assembled nanoparticles (ultrasonic power 150 W, ultrasonic time 12 min). See Table 1.

[0031] Table 1. Particle size, particle size distribution, encapsulation efficiency, and drug loading of mitoxantrone hydrochloride ion-paired self-assembled nanoparticles.

[0032]

[0033] Encapsulation efficiency determination method: Nanoparticles and free drugs were separated by ultrafiltration centrifugation, and then quantified by enzyme-linked immunosorbent assay (ELISA).

[0034] The results showed that mitoxantrone hydrochloride and sodium cholesterol sulfate at different molar ratios (1:1, 1:2, 1:3, 1:4, 1:5) could co-assemble into nanoparticles with uniform particle size, around 130 nm. When the molar ratio of mitoxantrone hydrochloride to sodium cholesterol sulfate increased from 1:1 to 1:10, the encapsulation efficiency of the formed mitoxantrone hydrochloride ion-paired self-assembled nanoparticles increased from 60.5% to nearly 100%. However, when the molar ratio of mitoxantrone hydrochloride to sodium cholesterol sulfate was 1:10, the particle size distribution of the nanoparticles was uneven. When the molar ratios of mitoxantrone hydrochloride to sodium cholesterol sulfate were 1:3, 1:4, and 1:5, the formed nanoparticles had uniform particle size distribution and encapsulation efficiencies greater than 97%, indicating high drug loading. This facilitates the targeted accumulation of nanoparticles through the high permeability and retention effect of solid tumors. Therefore, ion-paired self-assembled nanoparticles with a molar ratio of mitoxantrone hydrochloride to sodium cholesterol sulfate of 1:3 to 1:5 are preferred.

[0035] Example 2

[0036] Preparation of PEG-modified mitoxantrone hydrochloride ion-paired self-assembled nanoparticles

[0037] To increase the blood circulation time of mitoxantrone hydrochloride ion-paired self-assembled nanoparticles, we selected the amphiphilic lipid DSPE-PEG. 2KCH-PEG was used to modify the nanoparticles.

[0038] Accurately weigh mitoxantrone hydrochloride, sodium cholesterol sulfate, and DSPE-PEG. 2K Alternatively, CH-PEG (molar ratios of 1:1:0.2, 1:2:0.2, 1:3:0.2, 1:4:0.2, 1:5:0.2, and 1:10:0.2) was dissolved in DMSO as the organic phase. Ten times the volume of deionized water was used as the aqueous phase in a container. The organic phase was slowly added dropwise to the aqueous phase under ultrasonic probe conditions, and ultrasonication was continued for an appropriate time to form PEGylated ion-paired self-assembled nanoparticles (ultrasonic power 150 W, ultrasonic time 12 min), as shown in Table 2.

[0039] Table 2. Particle size, particle size distribution, encapsulation efficiency, and drug loading of PEG-modified mitoxantrone hydrochloride ion-paired self-assembled nanoparticles.

[0040]

[0041] Encapsulation efficiency determination method: Nanoparticles and free drugs were separated by ultrafiltration centrifugation, and then quantified by enzyme-linked immunosorbent assay (ELISA).

[0042] The particle size of the PEGylated ion-paired self-assembled nanoparticles changed, and the encapsulation efficiency was also improved, which confirms the successful co-assembly of MTO, SCS and PEG.

[0043] Example 3

[0044] Cytotoxicity of mitoxantrone ion-paired self-assembled nanoparticles

[0045] The cytotoxicity of mitoxantrone hydrochloride solution (MTO Sol) and mitoxantrone hydrochloride ion-paired self-assembled nanoparticles to mouse breast cancer cells (4T1) was investigated using the MTT assay. First, cell suspension was added to 96-well plates at a concentration of 2000 cells / well and incubated at 37°C, 5% CO2 for 24 hours to allow cell adhesion. After cell adhesion, mitoxantrone hydrochloride solution or mitoxantrone hydrochloride ion-paired self-assembled nanoparticles prepared in Example 2 (molar ratios 1:3:0.2, 1:4:0.2, 1:5:0.2, DSPE-PEG) was used. 2K Replace the original culture medium with culture medium modified with CH-PEG (or CH-PEG modified) or blank culture medium, and incubate in an incubator for 48 hours. Then add 20 μL of MTT solution (5 mg / mL) to each well, and continue incubation for 4 hours. Discard the liquid, add 200 μL of DMSO to each well to dissolve the blue-purple formazan, and measure the absorbance of the solution at 570 nm using a microplate reader.

[0046] The results of cytotoxicity experiments were analyzed, and the half-maximal inhibitory concentration (IC50) of mitoxantrone hydrochloride solution and ion-paired self-assembled nanoparticles was calculated using GraphPad Prism 8. 50 The cytotoxicity of mitoxantrone hydrochloride ion-paired self-assembled nanoparticles was enhanced compared to mitoxantrone hydrochloride solution. This is because ion pairing increases the hydrophobicity of mitoxantrone hydrochloride, which facilitates its uptake by cells. Furthermore, the IC50 value of mitoxantrone hydrochloride ion-paired self-assembled nanoparticles increased with increasing molar ratio of mitoxantrone hydrochloride to sodium cholesterol sulfate. 50 The value gradually decreases.

[0047] Table 3. Cytotoxicity of mitoxantrone hydrochloride ion-paired self-assembled nanoparticles

[0048]

[0049] Example 4

[0050] In vivo antitumor experiment of mitoxantrone ion-paired self-assembled nanoparticles

[0051] Mouse breast cancer cell suspension (4T1, 10) 6 (100 μL / cell) was injected subcutaneously on the dorsal side of female BALB / c mice (weighing 18-22 g). The inoculation continued until the tumor volume reached 100 mm². 3 Around 1000 mice were randomly divided into 7 groups of 5 mice each: a saline group, a mitoxantrone solution group, and a mitoxantrone ion-paired self-assembled nanoparticle group. The self-assembled nanoparticles used for drug administration were prepared in Example 2, with a molar ratio of mitoxantrone to sodium cholesterol sulfate of 1:4 and 1:5, respectively, using DSPE-PEG. 2K Modification. The dosages for the mitoxantrone solution group and the mitoxantrone ion-paired self-assembled nanoparticle group were 1 mg / kg and 4 mg / kg, respectively. Administration was performed via tail vein every 2 days for a total of 4 doses. Tumor volume and body weight changes in mice were assessed daily after administration.

[0052] The results are as follows Figure 1-2 As shown in the diagram, in the saline group, the tumor volume grew rapidly, reaching 1000 mm on day 10. 3 Around 500 mm. In contrast, the mitoxantrone solution group and the mitoxantrone ion-paired self-assembled nanoparticle group significantly inhibited tumor growth (<500 mm). 3 However, there were no significant differences between the groups. Figure 1At a dose of 1 mg / kg mitoxantrone solution, mice showed a decreasing body weight trend, with a 17% weight loss at the end of treatment. However, at 4 mg / kg, mice experienced a weight loss of nearly 20% or death by day 5 after administration, indicating severe toxicity of the mitoxantrone solution. In contrast, the weight loss of mitoxantrone ion-paired self-assembled nanoparticles was delayed, suggesting better safety. Figure 2 In summary, promoting the self-assembly of hydrophilic cationic drugs through ion pairing strategies can improve their safety and tolerability while preserving their efficacy.

[0053] Example 5

[0054] Acute toxicity test of mitoxantrone ion-paired self-assembled nanoparticles

[0055] Healthy female BALB / c mice (weighing 18-22g) were randomly divided into 9 groups of 3 mice each: (i) saline group, (ii) mitoxantrone solution group (10mg / kg), (iii) mitoxantrone solution group (20mg / kg), (iv) mitoxantrone solution group (40mg / kg), (v) mitoxantrone solution group (80mg / kg), (vi) mitoxantrone ion-paired self-assembled nanoparticle group (10mg / kg), (vii) mitoxantrone ion-paired self-assembled nanoparticle group (20mg / kg), (viii) mitoxantrone ion-paired self-assembled nanoparticle group (40mg / kg), and (ix) mitoxantrone ion-paired self-assembled nanoparticle group (80mg / kg). Mice that lost 20% or more of their body weight were euthanized. The self-assembled nanoparticles used for drug delivery were prepared in Example 2, wherein the molar ratio of mitoxantrone to sodium cholesterol sulfate was 1:5, and DSPE-PEG was used. 2K Modification. Following a single tail vein injection, the mice's weight and survival were monitored daily. Results are as follows: Figure 3-4 As shown, at the same dosage, mitoxantrone ion-paired self-assembled nanoparticles delayed weight loss and prolonged survival in mice compared to the solution formulation. The toxicity of mitoxantrone ion-paired self-assembled nanoparticles at 40 mg / kg was comparable to that of the solution formulation at 10 mg / kg.

Claims

1. A mitoxantrone hydrochloride ion-paired self-assembled nanoparticle, characterized in that, It is composed of mitoxantrone hydrochloride, a hydrophobic auxiliary counterion, and an amphiphilic lipid, wherein the hydrophobic auxiliary counterion is sodium cholesterol sulfate, and the amphiphilic lipid is methoxy polyethylene glycol 2000-distearate phosphatidylethanolamine or cholesterol-polyethylene glycol 2000; the molar ratio of mitoxantrone hydrochloride:hydrophobic auxiliary counterion:amphiphilic lipid is 1:(3-5):0.

2.

2. The mitoxantrone hydrochloride ion-paired self-assembled nanoparticles according to claim 1, characterized in that, The mitoxantrone hydrochloride ion-paired self-assembled nanoparticles have a particle size of 80-200 nm, uniform particle size distribution, and drug loading of 5-30%.

3. The use of the mitoxantrone hydrochloride ion-paired self-assembled nanoparticles according to claim 1 in the preparation of anti-breast cancer drugs.

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