A hydrophobic auxiliary material for emulsification, and its synthesis method and application

Abstract

The present invention discloses an emulsification-aiding hydrophobic adjuvant, a synthesis method, and applications thereof. The adjuvant is composed of Fmoc-amino acids and their derivatives and fatty acid chains formed thereon. As the emulsification-aiding hydrophobic adjuvant, the Fmoc-amino acids and their derivatives are partially used to bind to hydrophobic active drugs, and the fatty acid chains are used to embed into the molecules to be emulsified. Utilizing this emulsification-aiding hydrophobic adjuvant can effectively improve the stability of albumin nanoformulations of drugs such as docetaxel, thereby facilitating the preparation of corresponding high-concentration products. Furthermore, such albumin nanoformulations do not contain hemolytic adjuvants such as polysorbate 80 and ethanol, eliminating the risk of hemolysis and enabling better drug effects.

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, a synthesis method and application thereof. Background Art

[0002] Docetaxel, a taxane drug with stronger microtubule-inhibiting activity than paclitaxel, is a widely used anti-tumor drug in clinical practice. Due to its strong hydrophobicity, its injection solution requires high concentrations of polysorbate 80 and ethanol as excipients for solubilization, which can lead to safety concerns such as unnecessary hemolysis. To address these issues, docetaxel can be formulated into an injectable nanoformulation. Existing docetaxel nanoformulations include docetaxel albumin and docetaxel micelles. Docetaxel albumin nanoformulations have attracted considerable attention due to their lack of excipient safety concerns, and are expected to become the next albumin nanoformulation after paclitaxel albumin. However, the preparation of docetaxel using the same, similar, or improved processes as paclitaxel albumin preparations is difficult. This is due to the structural characteristics and hydrophobicity of docetaxel, resulting in large particle size, poor stability, and increased coagulation, making it particularly difficult to prepare at high concentrations.

[0003] There are two existing methods for preparing albumin nanoparticles. The first involves dissolving the hydrophobic material in an organic solvent, preparing colostrum, and then shearing it with a high-shear instrument such as a high-pressure homogenizer to form an albumin nanoformulation. The organic solvent is then removed by rotary evaporation or filtration to obtain the final albumin nanoformulation. The second method involves disrupting the albumin structure using a reducing agent or heating, followed by prolonged mixing and stirring with a drug solution to form an albumin nanoformulation. The final product is obtained after filtration and concentration. Both of these methods are currently common methods for preparing docetaxel albumin, with the first method being more commonly used because it was the original method for developing paclitaxel albumin nanoformulations. However, docetaxel albumin prepared using these methods exhibits poor in vitro stability and is prone to coagulation. Therefore, researchers have added various excipients to stabilize docetaxel albumin nanoformulations based on existing methods. For example, CN113577305A discloses the stabilization of docetaxel albumin nanoformulations by adding cationic lipids. However, the addition of these excipients may actually alter the original starting point. Instead of fundamentally addressing the hemolytic risk of these excipients, they merely mitigate the risk by switching to excipients with lower hemolytic risk or reducing their dosage. The addition of safer excipients, such as poloxamer, requires high process stability and can only be applied to a single formulation or process. Consequently, the resulting docetaxel albumin nanoformulations exhibit unsatisfactory stability.

[0004] Based on this, the present invention designs a novel hydrophobic excipient that can assist in the emulsification of albumin. Unlike common ionic amphiphilic excipients that easily cause hemolysis risks, according to a specific embodiment of the present invention, the excipient is composed of an Fmoc-amino acid as a head and a fatty acid chain as a tail. The Fmoc-amino acid at the head can combine well with the hydrophobic drug, and the fatty acid chain at the tail can be embedded in the albumin molecule 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 efficacy of the preparation. Summary of the Invention

[0005] The purpose of the present invention is to address the deficiencies of the existing technology and provide an emulsification-aiding hydrophobic excipient, a synthesis method and application thereof. 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 solutions adopted in the present invention are as follows:

[0007] An emulsification-aiding hydrophobic excipient is composed of amino acids and derivatives protected by Fmoc groups and fatty acid chains formed thereon. As an emulsification-aiding hydrophobic excipient, the amino acids and derivatives 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 to be emulsified.

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

[0009]

[0010] Wherein, R1 is a group formed after an amino acid and its derivatives lose the hydrogen on the amino group and the 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- 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, the R4 is selected from C6-C12 alkyl, unsubstituted C6-C12 alkenyl or unsubstituted C6-C12 alkynyl, more preferably, the R4 is selected from C6-C12 alkyl, most preferably, a C6 alkyl.

[0011] Furthermore, the preparation method of the above-mentioned hydrophobic auxiliary material is as follows: using an amino acid and its derivatives protected by an Fmoc group and a compound containing a hydroxyl group or an amino group as raw materials, an esterification or amidation reaction occurs under the action of a dehydrating agent and a catalyst to obtain an emulsifying hydrophobic auxiliary material; the compound containing a hydroxyl group or an amino group 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 group-containing compound HO-R4, an amino group-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 emulsification-assisting hydrophobic excipient can be used as an excipient for the preparation of hydrophobic drug protein nanoformulations.

[0015] 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, and docetaxel lipophilic derivatives; the macrolides include but are not limited to rapamycin and its derivatives, epothilone B and its derivatives, and tanespiramycin 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 excipient 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 designed to aid emulsification in the present invention has a structure in which the Fmoc-amino acid portion at the head can be well combined with hydrophobic drugs, and the fatty acid chain portion at the tail can be embedded into protein molecules such as albumin, forming an auxiliary bridge. This is conducive to the preparation of drugs such as docetaxel into stable albumin nanoformulations, achieving the purpose of improving the safety and effectiveness of the preparations.

[0027] During the preparation of albumin nanoformulations, the emulsification-aiding hydrophobic excipient of the present invention is dissolved together with a hydrophobic drug such as docetaxel in an organic phase. This not only effectively improves the stability of albumin nanoformulations such as docetaxel, but also makes the prepared albumin nanoformulations more stable at room temperature, thereby enabling the preparation of high-concentration products. Moreover, such albumin nanoformulations do not contain easily hemolytic excipients such as polysorbate 80 and ethanol, thus omitting the risk of hemolysis. Experimental studies have demonstrated that the albumin docetaxel nanoformulations prepared using the present invention have superior in vivo efficacy to clinically used docetaxel injections. 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 lysine ethyl ester excipient;

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

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

[0032] Figure 5 This is the NMR spectrum of the lysine oleate excipient;

[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] Figure 9 The particle size distribution of albumin docetaxel nanoformulation with lysine oleate as excipient;

[0037] Figure 10 Figure 2 shows hemolysis experiments of different preparations;

[0038] Figure 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 intended to limit the present invention.

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

[0041]

[0042] Wherein, R1 is a group formed after an amino acid and its derivatives lose the hydrogen on the amino group and the 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, most preferably BOC-lysine; wherein the R2 group is -XHn-R4; X is selected from O or N, n=0 when X=O, 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, most preferably, C6 alkyl.

[0043] The following describes in detail 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 sequentially to a 100 mL round-bottom flask and dissolved in 20 mL of anhydrous methanol. The mixture was stirred at 30°C for 18 h, and the reaction was observed by thin-layer chromatography. When the reaction was nearly complete, the unreacted methanol solvent was removed under reduced pressure, and the reaction mixture was redissolved in dichloromethane and washed with 5% aqueous citric acid, 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 removed under reduced pressure. The product 1 was isolated and purified by column chromatography (DCM:MeOH = 500:1) to obtain product 1 (7.170 g, 74.3% yield).

[0046] Lysine methyl ester excipient 1 The H NMR data are 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 Excipient

[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 sequentially to a 100 mL round-bottom flask and dissolved in 20 mL of anhydrous ethanol. The mixture was stirred at 30°C for 16 h, and the reaction was observed by thin-layer chromatography. When the reaction was nearly complete, the unreacted ethanol solvent was removed under reduced pressure, and the reaction mixture was redissolved in dichloromethane and washed with 5% aqueous citric acid, 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 removed under reduced pressure. The product 1 was isolated and purified by column chromatography (DCM:MeOH = 600:1) to obtain product 1 (8.059 g, 81.0% yield).

[0050] Lysine ethyl ester excipient 1 The H NMR data are 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] To a 100 mL round-bottom flask, 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 sequentially and dissolved in 50 mL of anhydrous dichloromethane. The mixture was stirred at 30°C for 18 h, and the reaction was monitored by thin-layer chromatography. When the reaction was nearly complete, the mixture was washed with 5% aqueous citric acid, 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 removed under reduced pressure. The product 1 was isolated and purified by column chromatography (DCM:MeOH = 500:1) to obtain product 1 (7.634 g, 69.1% yield).

[0054] Lysine n-hexyl ester excipient 1 The H NMR data are 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] To a 100 mL round-bottom flask, 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 sequentially and dissolved in 50 mL of anhydrous dichloromethane. The mixture was stirred at 30°C for 19 h, and the reaction was monitored by thin-layer chromatography. When the reaction was nearly complete, the mixture was washed with 5% aqueous citric acid, 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 removed under reduced pressure. The product 1 was isolated and purified by column chromatography (DCM:MeOH = 500:1) to obtain product 1 (9.120 g, 71.8% yield).

[0058] Lysine n-dodecyl ester excipient 1 The H NMR data are as follows, and 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 Oleate

[0061] To a 100 mL round-bottom flask, 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 sequentially and dissolved in 50 mL of anhydrous dichloromethane. The mixture was stirred at 30°C for 19 h, and the reaction was monitored by thin-layer chromatography. When the reaction was nearly complete, the mixture was washed with 5% aqueous citric acid, 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 removed under reduced pressure. The product 1 was isolated and purified by column chromatography (DCM:MeOH = 500:1) to obtain product 1 (9.447 g, 65.9% yield).

[0062] Lysine oleate excipient 1 The H NMR data are 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 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 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 is as follows Figure 6 shown.

[0066] Example 7 Preparation of Albumin Docetaxel Nanoformulation Using 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 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 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 according to 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 Using Lysine n-Dodecyl Ester as Excipient

[0071] Albumin-docetaxel nanoparticles with a final docetaxel concentration of 4 mg / mL were prepared. 200 mg of docetaxel and 800 mg of the excipient, lysine n-dodecyl ester, were weighed and dissolved in 2.5 mL of chloroform solution. The mixture was rapidly added to a 50 mL aqueous solution containing 1.8 g of human serum albumin. The mixture was rapidly sheared at 13,000 rpm for 10 minutes. The mixture was then quickly transferred to a microfluidizer at 26,000 psi for 10 minutes. The organic solvent was then removed by rotary evaporation to produce the albumin nanoparticles. The albumin nanoparticles were lyophilized to obtain a lyophilized powder. After filtration and sterilization, the powder was lyophilized to obtain a lyophilized powder. After reconstitution with saline, the concentration was determined by HPLC. Based on the drug concentration determined by HPLC, the nanoparticles were diluted to 4 mg / mL. The particle size was 35.5 nm.

[0072] Example 10 Preparation of Albumin Docetaxel Nanoformulation Using Lysine Oleate 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 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 is as follows Figure 9 shown.

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

[0075] Albumin-docetaxel nanoformulations containing various excipients were prepared according to the above examples. The organic solvent was removed by rotary evaporation to obtain albumin nanoparticles. The nanoformulations were diluted with PBS to an appropriate concentration, and their particle size and stability were measured. The results are shown in Table 1. Albumin-docetaxel nanoformulations prepared solely with albumin without the addition of Fmoc excipients had large particle sizes and were prone to coagulation and precipitation. Albumin-docetaxel nanoformulations with the addition of Fmoc excipients exhibited smaller particle sizes and improved 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 Formulation 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 two 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, Formulation 6 albumin docetaxel nanoparticles had good particle size stability in the environment of simulated body fluids in PBS and 20% FBS solutions, respectively.

[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 10 minutes, 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 shows red color, and disperse the red blood cells with normal saline to form 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 formulation of docetaxel as negative control group, positive control combination and test drug group. After incubation in a 37℃ water bath for 1h, centrifuge at 1500rpm for 10min and take pictures. Figure 10 As shown, at this drug concentration, the clinical dosage form formulation resulted in severe hemolysis, while no significant hemolysis was observed in any of the nanoparticle-treated groups. Because the Fmoc-based hydrophobic excipients of the present invention are not amphiphilic, the albumin-docetaxel nanoparticles prepared using the present invention exhibited similar anti-hemolytic 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 14:00. On days 0, 3, and 6, normal saline, clinical injection of docetaxel, 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. Figure 11As shown in the results, the albumin nanoparticle formulation had a better therapeutic effect than docetaxel injection at low and high doses.

[0086] The embodiments described above are merely some preferred embodiments of the present invention and are not intended to limit the present invention. Persons skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, any technical solution obtained by equivalent substitution or equivalent transformation falls within the scope of protection of the present invention.

Claims

1. A method for preparing a hydrophobic drug albumin nanoformulation, characterized in that: These include: 1) A hydrophobic drug and a hydrophobic excipient to aid emulsification are dissolved in an organic solvent; the hydrophobic drug is a taxane drug, and the hydrophobic excipient to aid emulsification is one or more of lysine methyl ester, lysine ethyl ester, lysine n-hexyl ester, lysine n-dodecyl ester, and lysine oleyl ester, wherein the structural formulas of lysine methyl ester, lysine ethyl ester, lysine n-hexyl ester, lysine n-dodecyl ester, and lysine oleyl ester are: 、 、 、 、 ； 2) Add to an aqueous solution containing human serum albumin and shear 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.

2. A hydrophobic drug albumin nanoformulation, characterized in that: Prepared by the method as claimed in claim 1.

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