Emulsifying-assisting hydrophobic auxiliary material as well as synthesis method and application thereof

Abstract

The invention discloses an emulsification-assisted hydrophobic auxiliary material and a synthesis method and application thereof, the auxiliary material is composed of Fmoc-amino acid and a derivative thereof and a fatty acid chain formed on the Fmoc-amino acid and the derivative thereof, the auxiliary material is used as the emulsification-assisted hydrophobic auxiliary material, and the Fmoc-amino acid and the derivative thereof are partially used for being combined with a hydrophobic active drug. Fatty acid chains are used for being embedded into molecules needing assistant emulsification. The emulsion-assisted hydrophobic auxiliary material can effectively improve the stability of the albumin nano preparation of docetaxel and other drugs, so that the preparation of corresponding high-concentration products is facilitated, and the albumin nano preparation does not contain polysorbate 80, ethanol and other easily hemolytic auxiliary materials, so that the hemolysis risk does not exist; the medicine effect can be better exerted.

Description

Technical Field

[0001] The invention belongs to the technical field of anti-tumor drug nano-preparations, and relates to an emulsification-assisting hydrophobic auxiliary material and a synthesis method and application thereof. Background Art

[0002] Docetaxel is a taxane drug, which has stronger microtubule inhibition activity than paclitaxel and is a class of anti-tumor drugs widely used in clinical practice. Due to its strong hydrophobicity, the excipients of its injection solution need to use high concentrations of polysorbate 80 and ethanol to aid dissolution, which brings unnecessary safety issues such as hemolysis. In order to improve such problems, docetaxel can be prepared into a nanoformulation for injection. Existing docetaxel nanoformulations include docetaxel albumin, docetaxel micelles, etc. From the perspective of not bringing in the safety of excipients, docetaxel albumin nanoformulations have always attracted much attention and are expected to become the next albumin nanoformulation of paclitaxel albumin preparations. However, it is difficult to prepare docetaxel using the same, similar or improved process as paclitaxel albumin preparations. This is due to the structural characteristics and hydrophobicity of docetaxel, which leads to a large particle size of the albumin nanosolution formed, poor stability, and easier coagulation, and it is particularly difficult to prepare a high-concentration product.

[0003] The existing preparation methods of albumin nano can be divided into two types. One is to dissolve the hydrophobicity through an organic solvent, and then prepare it into colostrum, shear it through a high-shear instrument such as a high-pressure homogenizer to form an albumin nano preparation, and then remove the organic solvent by rotary evaporation or filtration to finally obtain an albumin nano preparation; the second is to destroy the structure of albumin by reducing agents, heating, etc., and then mix and stir with the drug solution for a long time to form an albumin nano preparation, and after filtering and concentrating, the final product is obtained. At present, these two methods are also commonly used methods for preparing docetaxel albumin, among which the first method is more commonly used because this is also the original research method of paclitaxel albumin nano preparation. However, the in vitro stability of docetaxel albumin prepared by these methods is poor and easy to precipitate. Therefore, researchers add different excipients to stabilize docetaxel albumin nano preparations on the basis of the original method. For example, CN113577305A discloses stabilizing docetaxel albumin nano preparations by adding cationic lipids. But in fact, the addition of these excipients may change the original starting point. It does not completely change the hemolysis problem of these excipients. It only reduces the risk of hemolysis by switching to excipients with lower hemolysis risk or reducing the amount used. The addition of safer excipients, such as poloxamer, has high requirements for process stability and can only be applied to a single process prescription or process. The stability time of the prepared docetaxel albumin nanoformulation is not ideal.

[0004] Based on this, the present invention designs a new type of hydrophobic excipient that can assist in the emulsification of albumin. Different from the common ionic amphiphilic excipients that easily cause hemolysis risks, according to a specific embodiment of the present invention, the excipient is composed of Fmoc-amino acid as the head and fatty acid chain as the tail. The Fmoc-amino acid at the head can be well combined with the hydrophobic drug, and the fatty acid chain at the tail can be embedded in the albumin molecule to form an auxiliary bridge, which is beneficial for the preparation of drugs such as docetaxel into stable albumin nanoformulations, thereby achieving the purpose of improving the safety and effectiveness of the preparations. Summary of the invention

[0005] The purpose of the present invention is to provide an emulsification-aiding hydrophobic excipient, a synthesis method and application thereof in view of the deficiencies in the prior art. The emulsification-aiding hydrophobic excipient can effectively improve the stability of hydrophobic drug albumin nanoformulations such as docetaxel albumin nanoformulations, thereby facilitating the preparation of corresponding high-concentration products.

[0006] The technical solution adopted by the present invention is as follows:

[0007] An emulsification-aiding hydrophobic auxiliary material is composed of amino acids and derivatives thereof protected by Fmoc groups and fatty acid chains formed thereon, and is used as an emulsification-aiding hydrophobic auxiliary material, wherein the amino acids and derivatives thereof protected by Fmoc groups are used to combine with hydrophobic active drugs, and the fatty acid chains are used to be embedded in molecules that need emulsification-aiding.

[0008] The general structural formula of the hydrophobic auxiliary material for emulsification is:

[0009]

[0010] Wherein, R1 is a group formed by amino acids and their derivatives losing hydrogen on the amino group and hydroxyl on the carboxyl group, with the general formula -NH-CHR3-CO-, R3 represents a variable group (i.e., a side chain group used to represent different amino acids), and the amino acids and their derivatives are selected from glycine, alanine, valine, leucine, isoleucine, methionine (methionine), proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine, BOC-valine, BOC-asparagine, BOC-glutamine, BOC- Serine, BOC-threonine, BOC-arginine, BOC-lysine, preferably lysine, BOC-lysine, most preferably BOC-lysine; wherein the R2 group is -XHn-R4; wherein X is selected from O or N, n=0 when X=O, and n=1 or 0 when X=N; R4 is selected from C1-C24 alkyl, unsubstituted C2-C24 alkenyl or unsubstituted C2-C24 alkynyl, preferably R4 is selected from C6-C12 alkyl, unsubstituted C6-C12 alkenyl or unsubstituted C6-C12 alkynyl, more preferably R4 is selected from C6-C12 alkyl, most preferably C6 alkyl.

[0011] Furthermore, the preparation method of the above hydrophobic auxiliary material is as follows: using amino acids and derivatives thereof protected by Fmoc groups and compounds containing hydroxyl groups or amino groups as raw materials, and carrying out esterification or amidation reaction under the action of a dehydrating agent and a catalyst to obtain a hydrophobic auxiliary material for emulsification; the compound containing hydroxyl groups or amino groups is Hm-X-R4; X is selected from O or N, when X=O, m=1, when X=N, m=1 or 2, that is, a hydroxyl-containing compound HO-R4, an amino-containing compound NH2-R4 or a compound containing A compound of secondary amino -NH- (which can be expressed as R5-NH-R6), R4, R5 and R6 are all selected from C1-C24 alkyl, unsubstituted C2-C24 alkenyl or unsubstituted C2-C24 alkynyl, and the total number of carbon atoms of R5 and R6 is equal to R4, preferably, R4 is selected from C6-C12 alkyl, unsubstituted C6-C12 alkenyl or unsubstituted C6-C12 alkynyl, more preferably, R4 is selected from C6-C12 alkyl, and most preferably, it is C6 alkyl.

[0012] Furthermore, the dehydrating agent is any one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N,N-dicyclohexylcarbodiimide, and N,N'-diisopropylcarbodiimide.

[0013] Furthermore, the catalyst is any one of pyridine, 4-dimethylaminopyridine (DMAP), triethylamine or solid phase DMAP.

[0014] The hydrophobic auxiliary material for aiding emulsification can be used as an auxiliary material for the preparation of hydrophobic drug protein nanoformulations.

[0015] Wherein, the hydrophobic drug is one or more of taxanes, macrolides, camptothecins, anthracyclines and other active ingredients, specifically: the taxanes include but are not limited to paclitaxel or docetaxel, cabazitaxel, docetaxel lipophilic derivatives; the macrolides include but are not limited to rapamycin and its derivatives, epothilone B and its derivatives, tanspiramycin and its derivatives; the camptothecins include but are not limited to 10-hydroxycamptothecin, SN38 and its derivatives; the anthracyclines include but are not limited to aclarubicin and pirarubicin; the other active ingredients include but are not limited to colchicine and its derivatives, thiocolchicine dimer, amiodarone, liothyronine, cyclosporine, exemestane, flutamide, fulvestrant, romidepsin, semustine, and ibuprofen.

[0016] The protein is selected from one or more combinations of albumin, transferrin, insulin, endostatin, hemoglobin, myoglobin, lysozyme, immunoglobulin, α-2-macroglobulin, fibronectin, laminin, collagen, gelatin, artificial peptides, and artificial proteins.

[0017] The present invention also provides a method for preparing a hydrophobic drug albumin nanoformulation, comprising the following steps:

[0018] 1) dissolving a hydrophobic drug and a hydrophobic auxiliary material for emulsification in an organic solvent;

[0019] 2) adding to an aqueous solution containing human serum albumin and shearing into colostrum;

[0020] 3) High pressure homogenization of colostrum into nanoparticles;

[0021] 4) removing the organic solvent by rotary evaporation and filtering to obtain a sterile aqueous solution of hydrophobic drug albumin nanoparticles;

[0022] 5) freeze-drying the nanoparticle aqueous solution to obtain a hydrophobic drug albumin nano freeze-dried powder preparation;

[0023] Wherein, the hydrophobic auxiliary material for aiding emulsification is any of the above-mentioned hydrophobic auxiliary materials for aiding emulsification or a hydrophobic auxiliary material prepared by any of the above-mentioned methods.

[0024] The hydrophobic drug albumin nanoformulation can be in the form of lyophilized powder, injection, spray, or dry powder.

[0025] Compared with the prior art, the present invention has at least the following beneficial effects:

[0026] The hydrophobic excipient for emulsification designed in the present invention has a structure in which the Fmoc-amino acid part at the head can be well combined with the hydrophobic drug, and the fatty acid chain part at the tail can be embedded in protein molecules such as albumin to form an auxiliary bridge, thereby facilitating the preparation of drugs such as docetaxel into stable albumin nanoformulations, thereby achieving the purpose of improving the safety and effectiveness of the preparations.

[0027] In the preparation process of the albumin nanoformulation, the emulsification-aiding hydrophobic excipient of the present invention and the hydrophobic drugs such as docetaxel are dissolved together in the organic phase, which can not only effectively improve the stability of the albumin nanoformulations such as docetaxel, but also make the prepared albumin nanoformulations more stable at room temperature, so as to realize the preparation of high-concentration products; and this type of albumin nanoformulation does not contain easily hemolytic excipients such as polysorbate 80 and ethanol, and there is no risk of hemolysis; experimental studies have proved that the in vivo efficacy of the albumin docetaxel nanoformulation prepared by the present invention is better than that of the docetaxel injection used clinically. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the NMR spectrum of the lysine methyl ester excipient;

[0029] Figure 2 This is the NMR spectrum of the auxiliary material lysine ethyl ester;

[0030] Figure 3 This is the NMR spectrum of the auxiliary material lysine n-hexyl ester;

[0031] Figure 4 This is the NMR spectrum of the auxiliary material of lysine n-dodecyl ester;

[0032] Figure 5 This is the NMR spectrum of the lysine oleyl ester auxiliary material;

[0033] Figure 6 The particle size distribution of albumin docetaxel nanoformulation with lysine methyl ester as excipient;

[0034] Figure 7 The particle size distribution and transmission electron microscopy image of albumin docetaxel nanoformulation with lysine ethyl ester as excipient;

[0035] Figure 8 The particle size distribution of albumin docetaxel nanoformulation with lysine n-hexyl ester as excipient;

[0036] Fig. 9 The particle size distribution of albumin docetaxel nanoformulation with lysine oleyl ester as excipient;

[0037] Fig.10 Figure 2 shows the hemolysis experiment of different preparations;

[0038] Fig.11In vivo anti-cervical cancer effect of albumin docetaxel nanoformulation. DETAILED DESCRIPTION

[0039] In order to make the technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with several embodiments and drawings. It should be noted that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0040] The present invention designs a hydrophobic auxiliary material for emulsification, and its general structural formula is:

[0041]

[0042] Wherein, R1 is a group formed by amino acids and their derivatives losing hydrogen on the amino group and hydroxyl on the carboxyl group, and the general formula is -NH-CHR3-CO-, R3 represents a variable group (i.e., a side chain group used to represent different amino acids), and the amino acids and their derivatives are selected from glycine, alanine, valine, leucine, isoleucine, methionine (methionine), proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine, BOC-valine, BOC-asparagine, BOC-glutamine, BOC- C-serine, BOC-threonine, BOC-arginine, BOC-lysine, preferably lysine, BOC-lysine, and most preferably BOC-lysine; wherein the R2 group is -XHn-R4; X is selected from O or N, n=0 when X=O, and n=1 or 0 when X=N; R4 is selected from C1-C24 alkyl, unsubstituted C2-C24 alkenyl or unsubstituted C2-C24 alkynyl, and preferably, R4 is selected from C6-C12 alkyl, unsubstituted C6-C12 alkenyl or unsubstituted C6-C12 alkynyl, and more preferably, R4 is selected from C6-C12 alkyl, and most preferably, C6 alkyl.

[0043] The following is a detailed description of BOC-lysine as an example of amino acids and their derivatives. When other amino acids and their derivatives are used, they also have the same or similar effects as BOC-lysine.

[0044] Example 1 Synthesis of Lysine Methyl Ester Excipient

[0045] Fmoc-Lys(BOC)-OH (9.374 g, 0.020 mmol), diisopropylcarbodiimide (DIC) (2.543 g, 0.022 mmol) and 4-dimethylaminopyridine (DMAP) (0.129 g, 0.001 mmol) were added to a 100 mL round-bottom flask in sequence and dissolved in 20 mL of anhydrous methanol. Stir at 30 °C for 18 h and observe the reaction using thin layer chromatography. When the reaction was basically completed, the unreacted methanol solvent was removed from the reaction solution under reduced pressure and the reactants were redissolved in dichloromethane and washed with 5% citric acid aqueous solution, saturated sodium bicarbonate, and saturated brine respectively; the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was collected and the solvent was removed under reduced pressure; the product 1 (7.170 g, yield 74.3%) was obtained after separation and purification by column chromatography (DCM: MeOH = 500: 1).

[0046] Lysine methyl ester excipient 1 H NMR data are as follows, the NMR spectrum is as follows Figure 1 shown.

[0047] 1 H NMR(400MHz,Chloroform-d)δ7.75(d,J=7.5Hz,2H),7.59(dd,J=7.5,2.7Hz,2H),7.38(t,J=7.5Hz,2H),7.33–7.26(m,2H),5.37(d,J=8.2Hz,1H) ,4.51(d,J=24.4Hz,1H),4.44–4.14(m,4H),3.73(s,3H),3.09(d,J=7.3 Hz,2H),2.00–1.73(m,2H),1.69(s,2H),1.41(s,9H),1.36–1.26(m,2H).

[0048] Example 2 Synthesis of Lysine Ethyl Ester Auxiliary Material

[0049] Fmoc-Lys(BOC)-OH (9.386 g, 0.020 mmoL), DIC (2.520 g, 0.022 mmol) and DMAP (0.127 g, 0.001 mmol) were added to a 100 mL round-bottom flask in sequence and dissolved in 20 mL of anhydrous ethanol. Stir at 30 °C for 16 h and observe the reaction using thin layer chromatography. When the reaction was almost complete, the reaction solution was decompressed to remove the unreacted ethanol solvent and then the reactants were redissolved in dichloromethane and washed with 5% citric acid aqueous solution, saturated sodium bicarbonate and saturated brine respectively; the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was collected and the solvent was removed under reduced pressure; the product 1 (8.059 g, yield 81.0%) was obtained after separation and purification by column chromatography (DCM: MeOH = 600: 1).

[0050] Lysine ethyl ester excipient 1 H NMR data are as follows, the NMR spectrum is as follows Figure 2 shown.

[0051] 1 H NMR(400MHz,Chloroform-d)δ7.74(d,J=7.6Hz,2H),7.58(dt,J=11.2,5.7Hz, 2H),7.38(t,J=7.5Hz,2H),7.29(t,J=7.3Hz,2H),6.057(s,11H),5.39(d,J=8 .2Hz,1H),4.63–4.22(m,4H),4.18(q,J=7.3,5.5Hz,2H),3.19–2.92(m,2H),1 .82(dt,J=10.5,5.6Hz,2H),1.49(s,2H),1.41(s,11H),1.26(t,J=7.2Hz,3H).

[0052] Example 3 Synthesis of lysine n-hexyl ester

[0053] Fmoc-Lys(BOC)-OH (9.699 g, 0.021 mmol), DIC (2.522 g, 0.022 mmol), DMAP (0.118 g, 0.001 mmol) and n-hexanol (2.038 g, 0.020 mmol) were added to a 100 mL round-bottom flask in sequence and dissolved in 50 mL of anhydrous dichloromethane. Stir at 30 °C for 18 h and observe the reaction using thin layer chromatography. When the reaction was almost complete, wash with 5% citric acid aqueous solution, saturated sodium bicarbonate, and saturated brine respectively; dry the organic phase with anhydrous sodium sulfate, filter, collect the filtrate and remove the solvent under reduced pressure; separate and purify by column chromatography (DCM: MeOH = 500: 1) to obtain product 1 (7.634 g, yield 69.1%).

[0054] Lysine n-hexyl ester excipient 1 H NMR data are as follows, the NMR spectrum is as follows Figure 3 shown.

[0055] 1H NMR(400MHz,Chloroform-d)δ7.75(d,J=7.5Hz,2H),7.59(d,J=6.8Hz,2H),7.38(t ,J=7.5Hz,2H),7.30(t,J=7.4Hz,2H),5.39(d,J=8.3Hz,1H),4.50(d,J=30.2Hz,1H) ,4.44–4.17(m,4H),4.12(t,J=6.8Hz,2H),3.09(s,2H),1.87–1.60(m,4H),1.49(q ,J=6.6Hz,2H),1.41(s,9H),1.27(td,J=13.8,12.6,8.7Hz,8H),0.89–0.83(m,3H).

[0056] Example 4 Synthesis of lysine n-dodecyl ester

[0057] Fmoc-Lys(BOC)-OH (9.342 g, 0.020 mmoL), DIC (2.529 g, 0.022 mmol), DMAP (0.120 g, 0.001 mmol) and n-dodecanol (3.717 g, 0.020 mmoL) were added to a 100 mL round-bottom flask in sequence and dissolved in 50 mL of anhydrous dichloromethane. Stir at 30 °C for 19 h and observe the reaction using thin layer chromatography. When the reaction was almost complete, wash with 5% citric acid aqueous solution, saturated sodium bicarbonate, and saturated brine respectively; dry the organic phase with anhydrous sodium sulfate, filter, collect the filtrate, and remove the solvent under reduced pressure; separate and purify by column chromatography (DCM: MeOH = 500: 1) to obtain product 1 (9.120 g, yield 71.8%).

[0058] Lysine n-dodecyl ester excipient 1 H NMR data are as follows, the NMR spectrum is as follows Figure 4 shown.

[0059] 1 H NMR (400MHz, Chloroform-d) δ7.74(d,J=7.5Hz,2H),7.59(d,J=7.5Hz,2H),7.38(t,J=7.5Hz,2H),7.30(t,J=7.4Hz,2H),5.38(d,J=8.2Hz,1H),4.50(d ,J=29.2Hz,1H),4.42–4.16(m,4H),4.11(t,J=6.8Hz,2H),3.09(s,2H),1.9 2–1.60(m,4H),1.49(q,J=6.7Hz,2H),1.23(s,20H),0.86(t,J=6.7Hz,3H).

[0060] Example 5 Synthesis of Lysine Oleic Ester

[0061] Fmoc-Lys(BOC)-OH (9.397 g, 0.020 mmoL), DIC (2.602 g, 0.022 mmol), DMAP (0.119 g, 0.001 mmol) and oleyl alcohol (5.357 g, 0.020 mmoL) were added to a 100 mL round-bottom flask in sequence and dissolved in 50 mL of anhydrous dichloromethane. Stir at 30 °C for 19 h and observe the reaction using thin layer chromatography. When the reaction was almost complete, wash with 5% citric acid aqueous solution, saturated sodium bicarbonate, and saturated brine respectively; dry the organic phase with anhydrous sodium sulfate, filter, collect the filtrate, and remove the solvent under reduced pressure; separate and purify by column chromatography (DCM: MeOH = 500: 1) to obtain product 1 (9.447 g, yield 65.9%).

[0062] Lysine ester excipient 1 H NMR data are as follows, the NMR spectrum is as follows Figure 5 shown.

[0063] 1 H NMR(400MHz,Chloroform-d)δ7.74(d,J=7.5Hz,2H),7.59(d,J=7.5Hz,2H),7.38(t,J=7.4 Hz,2H),7.29(t,J=7.4Hz,2H),5.43–5.35(m,1H),5.35–5.25(m,2H),4.50(d,J=30.3Hz,1 H),4.42–4.16(m,4H),4.11(t,J=6.8Hz,2H),3.09(s,2H),2.06–1.94(m,4H),1.89–1.61( m,4H),1.49(q,J=6.7,6.2Hz,2H),1.41(s,9H),1.30–1.22(m,24H),0.86(t,J=6.6Hz,3H).

[0064] Example 6 Preparation of albumin docetaxel nanoformulation using lysine methyl ester as excipient

[0065] Prepare albumin docetaxel nanoparticles with a final docetaxel concentration of 4 mg / mL. Weigh 200 mg of docetaxel and 800 mg of lysine methyl ester excipient, dissolve in 2.5 mL of chloroform solution, quickly add to 50 mL of aqueous solution containing 1.8 g of human serum albumin, rapidly shear at 13000 rpm for 10 minutes, then quickly transfer to microjet 26000 psi high pressure homogenization for 10 minutes, and then use rotary evaporation to remove the organic solvent to obtain albumin nanoparticles. The albumin nanoparticles can be lyophilized to obtain lyophilized powder. After filtration and sterilization, lyophilization is obtained to obtain lyophilized powder. After reconstitution with physiological saline, the concentration is determined by high performance liquid chromatography, and the nanoparticles are diluted to 4 mg / mL based on the drug concentration measured by high performance liquid chromatography. Its particle size distribution is as follows Figure 6 shown.

[0066] Example 7 Preparation of albumin docetaxel nanoformulation with lysine ethyl ester as excipient

[0067] Prepare albumin docetaxel nanoparticles with a final docetaxel concentration of 4 mg / mL. Weigh 200 mg of docetaxel and 800 mg of lysine ethyl ester excipient, dissolve in 2.5 mL of chloroform solution, quickly add to 50 mL of aqueous solution containing 1.8 g of human serum albumin, rapidly shear at 13000 rpm for 10 minutes, then quickly transfer to microjet 26000 psi high pressure homogenization for 10 minutes, and then use rotary evaporation to remove the organic solvent to obtain albumin nanoparticles. The albumin nanoparticles can be freeze-dried to obtain freeze-dried powder. After filtration and sterilization, freeze-drying is obtained to obtain freeze-dried powder. After reconstitution with physiological saline, the concentration is determined by high performance liquid chromatography, and the nanoparticles are diluted to 4 mg / mL based on the drug concentration measured by high performance liquid chromatography. Its particle size distribution and transmission electron microscopy are as follows Figure 7 shown.

[0068] Example 8 Preparation of albumin docetaxel nanoformulation using lysine n-hexyl ester as excipient

[0069] Prepare albumin docetaxel nanoparticles with a final docetaxel concentration of 4 mg / mL. Weigh 200 mg of docetaxel and 800 mg of lysine n-hexyl ester excipient, dissolve in 2.5 mL of chloroform solution and quickly add to 50 mL of aqueous solution containing 1.8 g of human serum albumin, rapidly shear at 13000 rpm for 10 minutes, then quickly transfer to microjet 26000 psi high pressure homogenization for 10 minutes, and then use rotary evaporation to remove the organic solvent to obtain albumin nanoparticles. The albumin nanoparticles can be freeze-dried to obtain freeze-dried powder. After filtration and sterilization, freeze-drying is obtained. After reconstitution with physiological saline, the concentration is determined by high performance liquid chromatography, and the nanoparticles are diluted to 4 mg / mL based on the drug concentration measured by high performance liquid chromatography. Its particle size distribution is as follows Figure 8 shown.

[0070] Example 9 Preparation of albumin docetaxel nanoformulation with lysine n-dodecyl ester as excipient

[0071] Prepare albumin docetaxel nanoparticles with a final docetaxel concentration of 4 mg / mL. Weigh 200 mg of docetaxel, 800 mg of lysine n-dodecyl ester excipient, dissolve in 2.5 mL of chloroform solution, quickly add to 50 mL of aqueous solution containing 1.8 g of human serum albumin, quickly shear at 13000 rpm for 10 min, then quickly transfer to microjet 26000 psi high pressure homogenization for 10 minutes, and then use rotary evaporation to remove the organic solvent to obtain albumin nanoparticles. The albumin nanoparticles can be lyophilized to obtain lyophilized powder. After filtration and sterilization, lyophilization is performed to obtain lyophilized powder. After reconstitution with physiological saline, the concentration is determined by high-performance liquid chromatography, and the nanoparticles are diluted to 4 mg / mL according to the drug concentration measured by high-performance liquid chromatography. The particle size is 35.5 nm.

[0072] Example 10 Preparation of albumin docetaxel nanoformulation with lysine oleyl ester as excipient

[0073] Prepare albumin docetaxel nanoparticles with a final docetaxel concentration of 4 mg / mL. Weigh 200 mg of docetaxel and 800 mg of lysine n-dodecyl ester excipient, dissolve in 2.5 mL of chloroform solution and quickly add to 50 mL of aqueous solution containing 1.8 g of human serum albumin, rapidly shear at 13000 rpm for 10 minutes, then quickly transfer to microjet 26000 psi high pressure homogenization for 10 minutes, and then use rotary evaporation to remove the organic solvent to obtain albumin nanoparticles. The albumin nanoparticles can be freeze-dried to obtain freeze-dried powder. After filtration and sterilization, freeze-drying is obtained. After reconstitution with physiological saline, the concentration is determined by high performance liquid chromatography, and the nanoparticles are diluted to 4 mg / mL based on the drug concentration measured by high performance liquid chromatography. Its particle size distribution is as follows Fig. 9 shown.

[0074] Example 11 Stability of Albumin Docetaxel Nanoformulations with Different Excipients

[0075] According to the above examples, albumin docetaxel nanoparticles containing different excipients were prepared, and the organic solvent was removed by rotary evaporation to obtain albumin nanoparticles. PBS was used to dilute the mixture to a suitable concentration, and then the particle size was measured and the stability was observed. The results are shown in Table 1. Without adding Fmoc excipients, the albumin docetaxel nanoparticles prepared using albumin alone had a large particle size and were easily precipitated and precipitated. The albumin docetaxel nanoparticles with Fmoc excipients had a smaller particle size and better stability.

[0076] Table 1 Properties and stability of albumin docetaxel nanoformulations with different excipients

[0077]

[0078] After learning that albumin docetaxel nanoparticles with Fmoc excipients have better stability, the in vitro stability of preparation 6 albumin docetaxel nanoparticles was further evaluated under an in vitro simulated environment. After the lyophilized powder was reconstituted with PBS to 5 mg / mL, it was diluted to 0.1 mg / mL with in vitro simulated solutions (PBS, 20% FBS), and parallel samples were set up and incubated in a 37°C water bath. 2 mL was taken out at different times to measure the particle size. As shown in Table 2, in PBS and 20% FBS solutions, preparation 6 albumin docetaxel nanoparticles had good particle size stability in an environment simulated by body fluids.

[0079] Table 2 Particle size stability of formulation 6 albumin docetaxel nanoformulation

[0080]

[0081]

[0082] Example 12 Drug hemolytic toxicity evaluation experiment

[0083] Take fresh ICR mouse blood in an anticoagulant tube, centrifuge at 1500rpm for 10min, and collect the red blood cells in the lower layer. Wash the red blood cells with normal saline several times until the supernatant no longer appears red, and disperse the red blood cells with normal saline into a 2% red blood cell suspension. Take 0.4mL of red blood cell suspension, add 0.4mL of normal saline, 1% Triton X-100, and albumin nanoparticles with the same docetaxel concentration (0.4mg / mL) and clinical injection form of docetaxel as the negative control group, positive control combination and test drug group. After incubation in a 37°C water bath for 1h, centrifuge at 1500rpm for 10min and take pictures. Fig.10 As shown, under this drug concentration condition, the clinical dosage form formula will cause more serious hemolysis, while all nanoparticle treatment groups have no obvious hemolysis. Because the Fmoc hydrophobic excipients of the present invention are not amphiphilic, the albumin docetaxel nanoparticles prepared by the method of the present invention all have similar anti-hemolysis effects.

[0084] Example 13 In vivo evaluation experiment of the drug's anti-cervical cancer effect

[0085] Cervical cancer cells HeLa were inoculated into the left abdominal wall of BALB / c nude mice, and the volume was about 50mm 3 Treatment was started at 1:00 p.m. on days 0, 3, and 6. Normal saline, clinical injection docetaxel injection, and albumin nanoparticle preparation (docetaxel equivalent dose: 10 mg / Kg for the low-dose group and 20 mg / Kg for the high-dose group) were injected into the tail vein of the mice, and then the tumor growth of the nude mice was monitored. Fig.11As shown, the therapeutic effect of albumin nanoparticle formulation was better than docetaxel injection at low and high doses.

[0086] The above-described embodiments are only some of the preferred solutions of the present invention, but they are not intended to limit the present invention. A person skilled in the relevant technical field may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. A hydrophobic auxiliary material for emulsification, characterized in that: It is composed of amino acids and their derivatives protected by Fmoc groups and fatty acid chains formed thereon, and serves as a hydrophobic auxiliary material to assist emulsification, wherein the amino acids and their derivatives protected by Fmoc groups are partially used to combine with hydrophobic active drugs, and the fatty acid chains are used to be embedded in the molecules that need to be assisted in emulsification.

2. The hydrophobic auxiliary material for emulsification according to claim 1, characterized in that: The general structural formula of the hydrophobic auxiliary material for emulsification is: Wherein, R1 is a group formed by amino acids and their derivatives losing hydrogen on the amino group and hydroxyl on the carboxyl group, with the general formula -NH-CHR3-CO-, R3 represents a variable group, and the amino acids and their derivatives are selected from glycine, alanine, valine, leucine, isoleucine, methionine (methionine), proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine, BOC-valine, BOC-asparagine, BOC-glutamine, BOC-serine, BOC-threonine, BOC-arginine, BOC-lysine, preferably lysine and BOC-lysine, and most preferably BOC-lysine; Wherein, the R2 group is -XHn-R4, X is selected from O or N, and when X=O, n=0, and when X=N, n=1 or 0; R4 is selected from C1-C24 alkyl, unsubstituted C2-C24 alkenyl or unsubstituted C2-C24 alkynyl, preferably, R4 is selected from C6-C12 alkyl, unsubstituted C6-C12 alkenyl or unsubstituted C6-C12 alkynyl, more preferably, R4 is selected from C6-C12 alkyl, and most preferably, it is C6 alkyl.

3. The method for synthesizing the hydrophobic auxiliary material for emulsification according to claim 1, characterized in that: The invention comprises the following steps: using amino acids and derivatives thereof protected by Fmoc groups and compounds containing hydroxyl or amino groups as raw materials, and carrying out esterification or amidation reaction under the action of a dehydrating agent and a catalyst to obtain a hydrophobic auxiliary material for emulsification; the compound containing hydroxyl or amino groups is Hm-X-R4; X is selected from O or N, m=1 when X=O, and m=1 or 2 when X=N, and R4 is selected from C1-C24 alkyl, unsubstituted C2-C24 alkenyl or unsubstituted C2-C24 alkynyl, preferably, R4 is selected from C6-C12 alkyl, unsubstituted C6-C12 alkenyl or unsubstituted C6-C12 alkynyl, more preferably, R4 is selected from C6-C12 alkyl, and most preferably, it is C6 alkyl.

4. The method for synthesizing the hydrophobic auxiliary material for emulsification according to claim 3, characterized in that: The dehydrating agent is any one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N,N-dicyclohexylcarbodiimide and N,N'-diisopropylcarbodiimide.

5. The method for synthesizing the hydrophobic auxiliary material for emulsification according to claim 3, characterized in that: The catalyst is any one of pyridine, 4-dimethylaminopyridine (DMAP), triethylamine or solid phase DMAP.

6. Use of the hydrophobic auxiliary material for emulsification according to claim 1 or 2 or the hydrophobic auxiliary material obtained by the method according to any one of claims 3 to 5, characterized in that: It is used as an excipient in the preparation of hydrophobic drug protein nanoformulations.

7. The use according to claim 6, characterized in that: The hydrophobic drug is one or more of taxanes, macrolides, camptothecins, anthracyclines and other active ingredients, wherein the taxanes include but are not limited to paclitaxel or docetaxel, cabazitaxel, and docetaxel lipophilic derivatives; the macrolides include but are not limited to rapamycin and its derivatives, epothilone B and its derivatives, tanspiramycin and its derivatives; the camptothecins include but are not limited to 10-hydroxycamptothecin, SN38 and its derivatives; the anthracyclines include but are not limited to aclarubicin and pirarubicin; the other active ingredients include but are not limited to colchicine and its derivatives, thiocolchicine dimer, amiodarone, liothyronine, cyclosporine, exemestane, flutamide, fulvestrant, romidepsin, semustine, and ibuprofen.

8. The use according to claim 6, characterized in that: The protein is selected from one or more combinations of albumin, transferrin, insulin, endostatin, hemoglobin, myoglobin, lysozyme, immunoglobulin, α-2-macroglobulin, fibronectin, laminin, collagen, gelatin, artificial peptides, and artificial proteins.

9. A method for preparing a hydrophobic drug albumin nanoformulation, characterized in that: These include: 1) dissolving a hydrophobic drug and a hydrophobic auxiliary material for emulsification in an organic solvent; 2) adding to an aqueous solution containing human serum albumin and shearing into colostrum; 3) High pressure homogenization of colostrum into nanoparticles; 4) removing the organic solvent by rotary evaporation and filtering to obtain a sterile aqueous solution of hydrophobic drug albumin nanoparticles; 5) freeze-drying the nanoparticle aqueous solution to obtain a hydrophobic drug albumin nano freeze-dried powder preparation; Wherein, the hydrophobic auxiliary material for aiding emulsification is the hydrophobic auxiliary material for aiding emulsification as described in claim 1 or 2, or the hydrophobic auxiliary material prepared by the method described in any one of claims 3-5.

10. A hydrophobic drug albumin nanoformulation, characterized in that: The preparation is prepared by the method as claimed in claim 9 and is in the form of freeze-dried powder, injection, spray, or dry powder.

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