Lymphatic-mediated transport of paclitaxel-like triglyceride prodrugs and lipid formulations

Abstract

The present invention belongs to the field of medical technology, and relates to a triglyceride prodrug based on lymphatic-mediated transport, and specifically to a paclitaxel triglyceride prodrug of different fatty acid chains for lymphatic-mediated transport and its lipid preparation and application in the preparation of an oral drug delivery system. The present invention also provides a lipid preparation of the paclitaxel triglyceride prodrug, comprising paclitaxel triglyceride prodrug and adjuvants, wherein the adjuvants include phospholipid emulsifiers, short-chain alcohols or ethers co-emulsifiers, and liquid oil phases, and by weight percentage, the liquid oil phase accounts for 30%-85% of the lipid preparation, the phospholipid emulsifier accounts for 10%-45% of the lipid preparation, and the short-chain alcohol or ethers co-emulsifier accounts for 5%-25% of the lipid preparation; paclitaxel triglyceride prodrug accounts for 1-10% of the total weight of the adjuvant. The lipid preparation preparation process of the present invention is simple, and is convenient for industrialization; it has good stability, is convenient for storage, and can significantly improve the bioavailability of paclitaxel.

Description

Technical field:

[0001] The present invention belongs to the field of medical technology and relates to triglyceride-like prodrugs based on lymphatic-mediated transport, and specifically to paclitaxel triglyceride prodrugs of different fatty acid chains for lymphatic-mediated transport and lipid preparations thereof and their application in preparing oral drug delivery systems. Background technology:

[0002] Cancer poses a serious threat to the health of all mankind. Chemotherapy is one of the most commonly used and effective strategies for cancer treatment, but most of the chemotherapy drugs currently used in clinical practice are cytotoxic drugs, which have disadvantages such as low solubility, poor stability, narrow therapeutic window and poor pharmacokinetic properties. Oral administration has always been the preferred route of administration in new drug development and clinical treatment because of its few adverse reactions, simple administration method and good patient compliance. However, since paclitaxel belongs to the Biopharmaceutics classification system IV (BCS IV) class of drugs, that is, it has poor water solubility, poor membrane permeability, and a short biological half-life in the body, there is an obvious first-pass metabolism effect in the liver, and it is easily excreted by P-glycoprotein (P-gp), the bioavailability of paclitaxel after oral administration is extremely low, and its clinical use is greatly limited. Therefore, the development of low-toxic and high-efficiency oral paclitaxel preparations remains a research hotspot. Paclitaxel oral self-microemulsification preparation developed by Daehwa Pharmaceutical of South Korea (DHP107) improves the oral absorption of paclitaxel by enhancing mucosal adhesion. However, this also brings a series of problems, including limited indications, which are currently only applicable to the treatment of gastric cancer. In addition, it also brings gastrointestinal side effects such as diarrhea and mucosal damage.

[0003] Lymphatic transport can avoid the first-pass effect and improve the oral bioavailability of drugs. In the gastrointestinal tract, highly lipid-soluble drugs (LogD>5 and solubility in long-chain triglycerides>50mg / g) can enter the lymphatic circulation by participating in lipoprotein assembly. The introduction of fatty acid chains can increase the lipophilicity of hydrophobic drugs to a certain extent. During the digestion of triglycerides in the intestine, pancreatic lipase specifically hydrolyzes fatty acids at positions 1 and 3, while fatty acids at positions 2 are almost not hydrolyzed. The fatty acid at position 2 is replaced with a drug, so that the prodrug simulates the digestion process of triglycerides in the intestine, enters the intestinal epithelial cells in the form of 2-monoglyceride prodrugs for re-esterification, participates in the assembly of lipoproteins, and then promotes lymphatic transport. Many researchers have made examples of triglyceride-like prodrugs. However, the structures of these triglyceride-like prodrugs are useless for the design of paclitaxel-triglyceride-like prodrugs. Since the parent drug cannot be effectively released, the efficacy of the prodrug is greatly affected. In Chinese patent CN106715456A, the inventor introduced a self-eliminating linker to promote the systemic release of the parent drug testosterone. However, for paclitaxel, a chemotherapy drug, a large amount of systemic exposure will bring serious side effects. Therefore, the linker is of no guiding significance for the design of an oral drug delivery system for paclitaxel.

[0004] In the prior art, triglyceride-like prodrugs are often prepared into nanoemulsions, but nanoemulsions have the disadvantages of unstable storage, easy stratification, cumbersome preparation, and poor formulation reproducibility. In addition, nanoemulsions generally contain a large amount of sodium deoxycholate as an emulsifier, which may cause serious gastrointestinal side effects and are not suitable for long-term multiple administration. Therefore, it is necessary to design a safe and simple oral dosage form of triglyceride-like prodrugs to improve the bioavailability of oral paclitaxel. Summary of the invention:

[0005] In order to overcome the defects of the prior art and improve the oral bioavailability of paclitaxel, the present invention selects 1,3 fatty acids with different chain lengths and saturations to synthesize paclitaxel-triglyceride prodrugs based on the lymphatic transport mechanism of natural triglycerides to promote the lymphatic transport of paclitaxel and improve its oral availability, and introduces reduction-sensitive connecting bonds to promote the oral absorption of anti-tumor drugs while enabling them to be specifically released at the target site, thereby improving the efficacy and reducing toxicity.

[0006] The present invention is achieved through the following technical solutions:

[0007] The first object of the present invention is to provide a paclitaxel-triglyceride-like prodrug represented by formula (I), (II) or (III) or its geometric isomers, pharmaceutically acceptable salts, hydrates, solvates:

[0008]

[0009]

[0010] The second object of the present invention is to provide a method for preparing the above compound. The specific preparation method is as follows:

[0011] (a) Synthesis of 1,3-diacerol: A certain amount of fatty acid (octanoic acid, stearic acid or oleic acid) is dissolved in anhydrous chloroform, and reacts with a certain amount of 1,3-dihydroxyacetone to form an ester under the catalysis of EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and DMAP (4-dimethylaminopyridine), and then hydrogenated with sodium borohydride to obtain 1,3-diacerol.

[0012] (b) 4,4'-dithiodibutyric acid is dissolved in an appropriate amount of acetic anhydride, stirred at room temperature to form the corresponding anhydride, reacted with 1,3-diacerol in the presence of EDCI and DMAP to obtain an intermediate product, and esterified with paclitaxel under the catalytic conditions of DMAP and EDCI to obtain prodrug (I), (II) or (III).

[0013]

[0014] The third object of the present invention is to provide a simple and safe pharmaceutical composition containing paclitaxel-triglyceride prodrug. The pharmaceutical composition is a lipid preparation of paclitaxel-triglyceride prodrug, comprising paclitaxel-triglyceride prodrug and excipients. The excipients include phospholipid emulsifiers, short-chain alcohols or ethers emulsifiers, and liquid oil phase. By weight percentage, the liquid oil phase accounts for 30%-85% of the lipid preparation, the phospholipid emulsifier accounts for 10%-45% of the lipid preparation, and the short-chain alcohol or ethers emulsifier accounts for 5%-25% of the lipid preparation; the paclitaxel-triglyceride prodrug accounts for 1-10% of the total weight of the excipients.

[0015] Furthermore, in the pharmaceutical composition of the present invention, by weight percentage, the liquid oil phase accounts for 55%-85% of the lipid preparation, the phospholipid emulsifier accounts for 10%-35% of the lipid preparation, and the short-chain alcohol or ether emulsifier accounts for 5%-10% of the lipid preparation.

[0016] Preferably, in the pharmaceutical composition of the present invention, by weight percentage, the liquid oil phase accounts for 55%-80% of the lipid preparation, the phospholipid emulsifier accounts for 15%-35% of the lipid preparation, and the short-chain alcohol or ether emulsifier accounts for 5%-10% of the lipid preparation.

[0017] Furthermore, the paclitaxel-triglyceride prodrug accounts for 2-5% of the total weight of the excipients.

[0018] Preferably, the paclitaxel-triglyceride prodrug accounts for 3-4% of the total weight of the excipients.

[0019] Wherein, the phospholipid is egg yolk lecithin, soybean lecithin, synthetic phospholipids such as 1,2-dioctanoyl-sn-glycero-3-phosphocholine, 1,2-didecanoyl-sn-glycero-3-phosphocholine, preferably egg yolk lecithin;

[0020] The liquid oil is selected from long-chain triglycerides, mixed long-chain glycerides, medium-chain triglycerides or a combination thereof, including: one or more of olive oil, almond oil, canola oil, castor oil, coconut oil, corn oil, cottonseed oil, fish oil, palm kernel oil, palm oil, peanut oil, rapeseed oil, safflower oil, sesame oil, shark liver oil, soybean oil, sunflower oil, hydrogenated coconut oil, hydrogenated cottonseed oil, hydrogenated palm oil, hydrogenated soybean oil, partially hydrogenated soybean oil, hydrogenated vegetable oil, Maisine, peceol, among which olive oil is preferred.

[0021] The short-chain alcohol or ether emulsifier is diethylene glycol monoethyl ether (Transcutol HP), ethanol, propylene glycol, etc., among which Transcutol HP is preferred.

[0022] Furthermore, the weight ratio of the short-chain alcohol or ether emulsifier, the phospholipid emulsifier, and the liquid oil phase is 1:2-4:5-20.

[0023] The lipid preparation of paclitaxel-triglyceride prodrug of the present invention is prepared by the following method:

[0024] The paclitaxel-triglyceride prodrug, short-chain alcohol or ether emulsifier, phospholipid emulsifier and liquid oil phase are mixed, and the auxiliary materials and the prodrug are uniformly mixed by ultrasound to obtain the lipid preparation of the prodrug.

[0025] Alternatively, the phospholipid emulsifier and the short-chain alcohol or ether emulsifier are dissolved in liquid oil, and ultrasonically mixed to obtain a uniform blank preparation. The paclitaxel-triglyceride prodrug is added to the blank preparation, and ultrasonically mixed to completely dissolve the prodrug therein, thereby obtaining a uniform lipid oral preparation.

[0026] When the phospholipid emulsifier is egg yolk lecithin, the short-chain alcohol or ether emulsifier is diethylene glycol monoethyl ether, the liquid oil phase is olive oil, and the liquid oil phase accounts for 55%-80% of the lipid preparation, the phospholipid emulsifier accounts for 15%-35% of the lipid preparation, and the short-chain alcohol or ether emulsifier accounts for 5%-10% of the lipid preparation, the obtained lipid preparation of paclitaxel-triglyceride prodrug has the best particle size and particle size distribution, has the best stability, and can significantly improve the oral bioavailability of paclitaxel.

[0027] The lipid preparation provided by the present invention uniformly mixes olive oil, phospholipids and diethylene glycol monoethyl ether for the first time to prepare a blank lipid preparation, and dissolves the synthesized paclitaxel-triglyceride-like prodrug into the above preparation. The preparation method is simple, does not require high-temperature heating, probe ultrasound and other processes, and only requires simple stirring or ultrasonic dissolution. The process is simple, easy to industrialize, and has strong repeatability. The obtained lipid preparation is in a uniform oil solution state. Compared with the thermodynamically unstable state of the nano preparation, the lipid preparation provided by the present invention is more stable and easy to store. The excipients used in the lipid preparation provided by the present invention are all FDA-approved pharmaceutical excipients, and the use of sodium deoxycholate is avoided. After oral administration, it will not cause gastrointestinal toxicity to the body, and the drug loading capacity of the preparation of the present invention is greatly improved compared with the prior art.

[0028] The advantages of the present invention are:

[0029] 1. Based on the absorption mechanism of natural triglycerides, the present invention designs paclitaxel-triglyceride prodrugs, promotes the lymphatic transport of paclitaxel, avoids the first-pass effect, and thus improves its oral bioavailability.

[0030] 2. The present invention designs three paclitaxel-triglyceride prodrugs substituted with different fatty acids, introduces reduction-sensitive disulfide bonds in the connecting bonds, and selects fatty acids at positions 1 and 3 with different carbon chain lengths and degrees of unsaturation. While improving lymphatic transport, paclitaxel can be released in the highly reducing environment of tumor cells, thereby improving the lymphatic transport and oral bioavailability of paclitaxel.

[0031] 3. The present invention connects the triglyceride backbone and paclitaxel with a reduction-sensitive disulfide bridge, which promotes the oral absorption of paclitaxel and enables the drug to be specifically released at the target site, thereby increasing efficacy and reducing toxicity.

[0032] 4. The present invention prepares a pharmaceutical composition of a lipid preparation based on the properties of paclitaxel-triglyceride prodrugs, which is highly suitable for the prepared paclitaxel-triglyceride prodrugs, has a simple preparation process, is easy to industrialize, is uniform and stable, and avoids the use of sodium deoxycholate compared to nanoemulsions, simplifies the preparation process, does not require a heating process or probe ultrasound, and is convenient for industrial production. Description of the drawings:

[0033] FIG1 is a high-resolution mass spectrum of paclitaxel-triglyceride-like prodrug;

[0034] (I) is PTX-SS-TG (OcA); (II) is PTX-SS-TG (OA); (III) is PTX-SS-TG (SA).

[0035] Figure 2 shows the paclitaxel-triglyceride prodrug 1 H-NMR spectrum;

[0036] (I) is PTX-SS-TG (OcA); (II) is PTX-SS-TG (OA); (III) is PTX-SS-TG (SA).

[0037] Figure 3 Gastrointestinal cytotoxicity of a lipid formulation of a paclitaxel-triglyceride-like prodrug.

[0038] FIG4 is a drug-time curve of paclitaxel-triglyceride prodrug lipid formulation in a rat pharmacokinetic experiment;

[0039] A: Free paclitaxel plasma concentration-time curves of paclitaxel triglyceride prodrugs, DHP107 and dopaclitaxel solution;

[0040] B: Blood concentration-time curve of the total amount of paclitaxel in the paclitaxel triglyceride prodrug;

[0041] C: Blood concentration-time curve of PTX-SS-TG (OcA);

[0042] D: Blood concentration-time curve of PTX-SS-TG(OA);

[0043] E: Blood concentration-time curve of PTX-SS-TG(SA).

[0044] FIG5 is a diagram showing an in vivo oral anti-tumor experiment of a paclitaxel-triglyceride prodrug lipid formulation;

[0045] A: Tumor growth curves of paclitaxel triglyceride prodrug, DHP107 and paclitaxel solution;

[0046] B: Body weight changes of paclitaxel triglyceride prodrug, DHP107, and paclitaxel solution;

[0047] C: Tumor-bearing rates of paclitaxel triglyceride prodrug, DHP107 and paclitaxel solution. Specific implementation method:

[0048] The present invention is further described by the following examples, but is not limited thereto.

[0049] Example 1 Preparation of Paclitaxel-Triglyceride Prodrug

[0050]

[0051] Dissolve 40mmol of octanoic acid or oleic acid or stearic acid in anhydrous chloroform, react with 20mmol of 1,3-dihydroxyacetone to form an ester under the catalysis of EDCI and DMAP, and then react with sodium borohydride to obtain 1,3-DG, which is separated by column chromatography to obtain a pure product (separation conditions are n-hexane: ethyl acetate = 30:1-15:1). Dissolve 8.8mmol of 4,4'-dithiodibutyric acid in an appropriate amount of acetic anhydride, react at room temperature for 2.5h, concentrate the solvent by rotary evaporation and redissolve it in an appropriate amount of anhydrous dichloromethane, and add 0.8mmol of 1,3-DG and DMAP. Vacuum nitrogen protection and react at room temperature for 24 hours. Remove the solvent by rotary evaporation, and obtain the intermediate product by column chromatography separation conditions (separation conditions are n-hexane: ethyl acetate = 12:1-8:1). 1 mmol of the intermediate product was dissolved in an appropriate amount of anhydrous chloroform, EDCI, DMAP, and 0.8 mmol of paclitaxel were added, and the mixture was reacted at room temperature for 24 hours under vacuum nitrogen protection. The solvent was removed by rotary evaporation, and the final product was obtained by preparative liquid phase separation (separation condition was pure acetonitrile).

[0052] The molecular weight of the prodrug in Example 1 was measured by high resolution mass spectrometry, and the results are shown in FIG1 :

[0053] Compound Ⅰ(PTX-SS-TG(OcA)):ESI-HRMS:Calcd.For C 74 H 91 NNaO 21 S 2 [MH + ]1399.5995found 1399.6012.

[0054] Compound II (PTX-SS-TG(OA)): ESI-HRMS: Calcd.For C 94 H 133 NNaO 21 S 2 [M-Na + ]1675.8812found 1675.8811.

[0055] Compound III (PTX-SS-TG (SA)): ESI-HRMS: Calcd.For C 94 H 137 NNaO 21 S 2 [M+Na + ]1679.9125found 1679.9154.

[0056] Nuclear magnetic resonance imaging 1 H-NMR spectrum was used to determine the structure of the prodrug in Example 1. The solvent used was CDCl 3, the results are shown in Figure 2.

[0057] 1 H NMR of compound I PTX-SS-TG (OcA) (600 MHz, CDCl 3 ,ppm)

[0058] δ8.14(d,J=7.6Hz,2H),7.74(d,J=7.5Hz,2H),7.61(t,J=7.4Hz,1H),7.51(d d,J=15.7,7.8Hz,3H),7.46–7.32(m,7H),6.92(d,1H),6.30(s,1H),6.26(t,1 H),5.98(dd,J=9.1,2.7Hz,1H),5.69(d,1H),5.51(d,1H),5.24(m,1H),4.98 (d,1H),4.45(dd,J=10.9,6.7Hz,1H),4.39–4.17(m,4H),4.13(dd,J=12.0,5. 8Hz,2H),3.82(d,1H),2.66(m,2H),2.64-2.49(m,4H),2.47(s,3H),2.44(t, 2H),2.38(dd,J=15.3,9.4Hz,1H),2.30(t,4H),2.23(s,3H),2.17(dd,J=15.3 ,8.9Hz,1H),2.00(m,4H),1.95(s,3H),1.89(t,1H),1.68(s,3H),1.62–1.57 (m,4H),1.30–1.25(m,18H),1.23(s,3H),1.14(s,3H),0.88(t,J=7.0Hz,6H).

[0059] 1 H NMR of compound II PTX-SS-TG(OA)(600MHz,CDCl 3 ,ppm)

[0060] δ8.15 (d, J = 7.4 Hz, 2H), 7.74 (d, J = 7.4 Hz, 2H), 7.61 (t, J = 7.3 Hz, 1H), 7.55–7.49 (m, 3H), 7.45–7.33 (m, 7H), 6.92 (d, 1H), 6.30 (s, 1H), 6.25 (t, 1H), 5.98 (dd, J = 9.2, 2.5 Hz, 1H), 5.69 (d, 1H), 5.52 (d, 1H), 5.29 (s, 4H), 5.24 (m, 1H), 4.98 (d, 1H), 4.45 (m, 1H), 4.34–4.19 (m, 4H), 4.13 (dd, J = 11.9, 5.7 Hz, 2H), 3.82 (d, 1H), 2.66 (m, 2H), 2.63–2.51 (m, 4H), 2.46 (s, 3H), 2.43 (t, 2H), 2.40–2.34 (m, 1H), 2.30 (t, 4H), 2.23 (s, 3H), 2.18–2.13 (m, 1H), 2.05–1.96 (m, 12H), 1.95 (s, 3H), 1.87 (t, 1H), 1.69 (s, 3H), 1.64 - 1.56 (m, 4H), 1.38 - 1.26 (m, 42H), 1.23 (s, 3H), 1.14 (s, 3H), 0.88 (t, 6H).

[0061] 1 1H NMR of Compound Ⅲ PTX - S - S - TG(SA) (600 MHz, CDCl 3 , ppm)

[0062] δ8.15(d,J=7.2Hz,2H)7.74(d,J=7.2Hz,2H),7.61(t,J=7.4Hz,1H),7.51(d d,J=12.8,7.5Hz,3H),7.47–7.32(m,7H),6.92(d,1H),6.30(s,1H),6.26(t, 1H),5.98(dd,J=9.3,3.0Hz,1H),5.69(d,1H),5.52(d,1H),5.28–5.15(m,1H ),4.98(d,1H),4.45(m,1H),4.34–4.19(m,4H),4.15(d,J=5.8Hz,1H),4.12( d,J=5.8Hz,1H),3.82(d,1H),2.67(m,2H),2.61–2.49(m,4H),2.46(s,3H), 2.44(t,2H),2.40–2.34(m,1H),2.30(t,4H),2.23(s,3H),2.17(dd,J=15.4, 9.0Hz,1H),2.00(m,4H),1.95(s,3H),1.93–1.84(m,1H),1.69(s,3H),1.63- 1.57(m,4H),1.25(m,58H),1.23(s,3H),1.14(s,3H),0.88(t,J=6.8Hz,6H).

[0063] The specific synthetic route of the above prodrug is as follows:

[0064]

[0065] Example 2 Prescription Screening of Paclitaxel-Triglyceride Prodrug

[0066] 12 mg of paclitaxel-triglyceride prodrug was dissolved in 400 mg of olive oil and preheated to 60°C. 120 mg of egg yolk lecithin and 40 mg of sodium deoxycholate were weighed and dissolved in 4 ml of deionized water and preheated to 60°C. The oil phase was slowly dripped into the stirred aqueous phase and stirred for 3 min to form colostrum. The colostrum was ultrasonicated with an ice bath probe for 10 min at an ultrasonic power of 500 W to prepare a paclitaxel triglyceride prodrug nanoemulsion. Taking PTX-SS-TG (OA) as an example, it was found that the emulsion particle size was stratified on the 5th day, exposing the defect of the emulsion's instability. The drug loading of the nanoemulsion was only 0.260%. The paclitaxel triglyceride prodrug was prepared into a nanoemulsion, which not only had a low drug loading but also had poor stability.

[0067] 2.1 Selection of oil phase

[0068] Taking PTX-SS-TG(OA) among the three prodrugs as an example, a prescription screening was carried out, and the solubility of PTX-SS-TG(OA) in several LCT-based vegetable oils was measured. The results are shown in Table 1.

[0069] Table 1. Solubility of PTX-SS-TG(OA) in several vegetable oils

[0070]

[0071] Based on the solubility results, olive oil was selected as the oil phase of the lipid formulation.

[0072] 2.2 Selection of emulsifier

[0073] Phospholipids can help improve the oral absorption of triglyceride-like prodrugs, so egg yolk lecithin was initially selected as an emulsifier to screen for co-emulsifiers. The emulsifier and co-emulsifier were dissolved in the oil phase, with the mass ratio of oil phase: emulsifier: co-emulsifier being 65:25:10, and the mixture was uniformly mixed by ultrasound to obtain a uniform blank preparation.

[0074] 200 mg of PTX-SS-TG(OA) prodrug was added to 1 g of the above blank preparation, and the equilibrium solubility of PTX-SS-TG(OA) prodrug in the blank preparation was measured.

[0075] Another 30 mg of PTX-SS-TG (OA) prodrug was added to 1 g of the blank preparation, and the prodrug was completely dissolved in it by ultrasound to obtain a uniform lipid oral preparation. PTX-SS-TG (OA) lipid preparations were prepared according to the above method, and 500 mg of each drug-containing preparation was added to 5 mL of simulated gastric fluid, stirred for 5 min to simulate gastric peristalsis, and the particle size and distribution were measured. The results of different emulsifiers are shown in Table 2.

[0076] Table 2. Properties of PTX-SS-TG(OA) lipid preparations prepared with different cosolvents

[0077]

[0078] The results showed that the performance of the prepared lipid preparations was different when different emulsifiers were added. When the emulsifier was Transcutol HP, the prepared PTX-SS-TG(OA) prodrug lipid preparation had the best drug loading capacity, particle size and distribution.

[0079] 2.3 Selection of phospholipids

[0080] Using olive oil as the oil phase and Transcutol HP as the emulsifier, the types of phospholipids were screened, and egg yolk lecithin, soybean lecithin, 1,2-dioctanoyl-sn-glycero-3-phosphocholine, and 1,2-didecanoyl-sn-glycero-3-phosphocholine were selected. PTX-SS-TG(OA) lipid preparations were prepared according to method 2.2, and the equilibrium solubility of PTX-SS-TG(OA) prodrug in the blank preparation was determined. 500 mg of each drug-containing preparation was added to 5 mL of simulated gastric fluid and stirred for 5 minutes to simulate gastric motility. The particle size and distribution were determined. There was no significant difference in the various indicators of lipid preparations prepared with different phospholipids, and egg yolk lecithin was relatively better.

[0081] Example 3 Preparation of PTX-SS-TG(OA) prodrug lipid formulation

[0082] PTX-SS-TG (OA) prodrug lipid preparations were prepared according to the method of 2.2 in Example 2 to obtain prodrug lipid preparations of different formulations, as shown in Table 3. 500 mg of the lipid preparation was added to 5 mL of simulated gastric fluid, stirred for 5 min to simulate gastric peristalsis, and the particle size and distribution were measured. Another 1 mL of the above lipid preparation and simulated gastric fluid mixture was mixed with 5 mL of pancreatic juice-bile, incubated for 2 h, centrifuged at low temperature (10000 rpm, 10 min), and the middle layer liquid was taken to measure the particle size after digestion, as well as the proportion of PTX-SS-TG (OA) in the middle layer. The results are shown in Table 4:

[0083] Table 3. PTX-SS-TG(OA) lipid preparations with different formulations

[0084]

[0085] Table 4. Properties of PTX-SS-TG(OA) lipid preparations of different formulations

[0086]

[0087]

[0088] As shown in Table 4, when the proportion of olive oil is less than 50% (formulations 1-3), the proportion of egg yolk lecithin and TranscutolHP is relatively high, and the solubility of PTX-SS-TG (OA) is high, so the drug loading is high. In addition, due to the strong surface activity of the emulsifier and the co-emulsifier, after mixing and stirring with simulated gastric juice, the particles obtained have a small and uniform particle size. However, after mixing with pancreatic juice-bile to simulate digestion, the particle size of the aqueous phase of the obtained intermediate layer is larger, and the proportion of PTX-SS-TG (OA) prodrug in the intermediate layer is less than 50%. For triglyceride-like lipid preparations, the initial in vitro particle size cannot accurately predict the oral absorption in vivo, and the intermediate aqueous phase after simulated digestion is equivalent to the dissolved phase of the intestinal fluid in vivo. The smaller the particle size, the greater the proportion of the prodrug in the intermediate aqueous phase, which may be beneficial to in vivo absorption. When the proportion of olive oil exceeds 80% and the proportion of egg yolk lecithin is less than 15%, although the results of in vivo simulation are still within an acceptable range, the initial simulated gastric fluid emulsification particle size is greater than 300nm, which may delay the subsequent intestinal digestion process and thus affect oral absorption; when TrancutolHP is less than 5%, olive oil and egg yolk lecithin are incompatible and cannot form a uniform lipid preparation.

[0089] When the content of olive oil is 30%-85%, the content of egg yolk lecithin is 10%-45% and the content of TrancutolHP is 5%-25%, a uniform and stable lipid preparation can be formed, and the particle size of the lipid preparation is less than 400nm.

[0090] When olive oil is at 55%-85%, egg yolk lecithin is at 10%-35%, and TrancutolHP is at 5%-10%, a uniform and stable lipid preparation can be formed, and the particle size is less than 400nm. PTX-SS-TG (OA) accounts for a large proportion in the middle layer, which is conducive to absorption in the body.

[0091] When the olive oil is 55%-80%, the egg yolk lecithin is 15%-35%, and the TrancutolHP is 5%-10%, a uniform and stable lipid preparation can be formed, the particle size of which is less than 300nm, and it is most conducive to in vivo absorption.

[0092] The lipid preparations of each formulation showed no stratification or precipitation at 15 days, indicating good stability.

[0093] Example 4 Preparation of Paclitaxel-Triglyceride Prodrug Lipid Formulation

[0094] Weigh 300 mg of egg yolk lecithin and dissolve it in 1 g of olive oil. Add 100 mg of diethylene glycol monoethyl ether and mix it evenly by ultrasound to obtain a uniform blank preparation. Accurately weigh 40 mg of the prodrug described in Example 1, add 1 g of the blank preparation, and ultrasound to completely dissolve the prodrug therein to prepare three uniform prodrug lipid preparations, namely PTX-SS-TG (SA), PTX-SS-TG (OcA), and PTX-SS-TG (OA). Accurately weigh an appropriate amount of paclitaxel mother drug and add it to the above blank preparation to obtain a paclitaxel lipid preparation.

[0095] Example 5 Gastrointestinal cytotoxicity of paclitaxel-triglyceride prodrug lipid formulation

[0096] The MTT method was used to investigate the cytotoxicity of paclitaxel-triglyceride prodrug lipid preparations on human cloned colon adenocarcinoma cells (CaCo-2). The cells were seeded into 96-well plates at a density of 1000 cells / well and incubated in an incubator for 24 hours to adhere to the wall. After the cells adhered to the wall, a series of concentrations of paclitaxel solution, commercially available paclitaxel self-microemulsion DHP107, and the paclitaxel-triglyceride prodrug lipid preparation prepared in Example 4 were added. After 12 hours of drug addition, the cell survival rate was determined by the MTT method.

[0097] The results are as follows Figure 3 As shown in the figure, the cytotoxicity of paclitaxel-triglyceride prodrug lipid preparations to human cloned colon adenocarcinoma cells is weaker than that of paclitaxel solution and commercially available paclitaxel self-microemulsion DHP107, indicating that the lipid preparations of the prodrug have no obvious damage to gastrointestinal cells and significantly reduce the gastrointestinal toxicity of paclitaxel. The effect of the lipid preparations of the prodrug in reducing toxicity is PTX-SS-TG(SA)>PTX-SS-TG(OcA)>PTX-SS-TG(OA).

[0098] Example 6 Pharmacokinetics of Paclitaxel-Triglyceride Prodrug Lipid Formulation

[0099] Using SD rats as a model, paclitaxel-triglyceride prodrug oil solution and paclitaxel oil solution were orally administered at a dose of 10 mg / kg. Blood was collected from the eye sockets at regular intervals to determine the concentration of paclitaxel parent drug in plasma. The drug-time curve was drawn and the corresponding pharmacokinetic parameters were calculated based on the measured concentrations (Table 2). In order to calculate the absolute bioavailability, paclitaxel solution was administered intravenously at a dose of 5 mg / kg, and the content of paclitaxel in plasma was determined. As shown in Figure 4 and Table 5, the AUC of the oral PTX-SS-TG (OA) prodrug lipid preparation group and the oral PTX-SS-TG (SA) prodrug lipid preparation group was 1.34 × 10.5 ... (0-12h)The relative bioavailability (F) of oral PTX-SS-TG (OcA) prodrug lipid preparation group, oral PTX-SS-TG (OA) prodrug lipid preparation group, and oral PTX-SS-TG (SA) prodrug lipid preparation group was 3.34 and 4.29 times higher than that of the oral PTX solution group, and 1.65 and 2.12 times higher than that of the commercially available DHP107 group. rel ) were 129.05%, 333.9% and 428.85% respectively. Compared with 4.95% of oral PTX solution, the oral absolute bioavailability (F ab ) were 6.39%, 16.52% and 21.22% respectively, and the oral bioavailability was improved. The oral absorption of PTX lipid preparation was similar to that of PTX-SS-TG (OcA) prodrug. ab % is 7.05%. The absolute bioavailability comparison results of the paclitaxel-triglyceride prodrug group are: PTX-SS-TG (SA)>PTX-SS-TG (OA)>PTX-SS-TG (OcA). The free paclitaxel content of paclitaxel-triglyceride prodrug measured in plasma is limited, so the total amount of paclitaxel contained in the prodrug is measured by appropriate methods as shown in Table 6. The paclitaxel-triglyceride prodrug substituted by stearic acid at positions 1 and 3 has the highest total amount of paclitaxel in plasma.

[0100] Table 5. Main pharmacokinetic parameters of paclitaxel-triglyceride prodrug

[0101]

[0102] Table 6. Total amount of paclitaxel in paclitaxel-triglyceride prodrugs

[0103]

[0104] Example 7 Pharmacodynamic behavior of paclitaxel triglyceride prodrug lipid formulation

[0105] In the 4T1 ectopic tumor-bearing Balb / c mouse model, three different oral paclitaxel triglyceride prodrug lipid preparations, paclitaxel lipid preparations, and paclitaxel solution were administered at a dose of 16 mg / kg (paclitaxel equivalent dose) every day; the paclitaxel solution intravenous injection group was set as the positive control, with a dose of 10 mg / kg, and the drug was given every two days; the PBS group was set as the blank control. The results are shown in Figure 5. The tumor volume comparison of the three triglyceride prodrug lipid preparation groups is: PTX-SS-TG (SA)>PTX-SS-TG (OA)>PTX-SS-TG (OcA). The paclitaxel-triglyceride prodrug substituted by stearic acid at 1,3 positions has a significant difference compared with the control group, and the mouse tumor-bearing rate is the lowest, while the body weight has no significant change, indicating that it has reliable safety and good anti-tumor effect.

Claims

1. A lipid formulation of paclitaxel-triglyceride prodrug, characterized in that: The invention comprises paclitaxel-triglyceride prodrug and excipients, wherein the excipients are composed of a phospholipid emulsifier, a short-chain alcohol or ether emulsifier and a liquid oil phase. By weight percentage, the liquid oil phase accounts for 55%-85% of the lipid preparation, the phospholipid emulsifier accounts for 10%-35% of the lipid preparation, and the short-chain alcohol or ether emulsifier accounts for 5%-10% of the lipid preparation; the paclitaxel-triglyceride prodrug accounts for 1%-10% of the total weight of the excipients; the short-chain alcohol or ether emulsifier is diethylene glycol monoethyl ether, and the liquid oil phase is a long-chain triglyceride; the phospholipid emulsion is egg yolk lecithin, soybean lecithin, 1,2-dioctanoyl-sn-glycero-3-phosphocholine or 1,2-didecanoyl-sn-glycero-3-phosphocholine; and the long-chain triglyceride is olive oil; The lipid preparation of paclitaxel-triglyceride prodrug is prepared by the following method: The paclitaxel-triglyceride prodrug, a short-chain alcohol or ether emulsifier, a phospholipid emulsifier and a liquid oil phase are mixed, and the auxiliary materials and the prodrug are mixed uniformly by ultrasonication to obtain the product; Alternatively, a phospholipid emulsifier and a short-chain alcohol or ether emulsifier are dissolved in a liquid oil phase, and ultrasonically mixed to obtain a uniform blank preparation; a paclitaxel-triglyceride prodrug is added to the blank preparation, and ultrasonically mixed to obtain a uniform blank preparation; The structure of the paclitaxel-triglyceride prodrug is one of (I)-(III): 。 2. The lipid formulation of paclitaxel-triglyceride prodrug according to claim 1, characterized in that Liquid oil phase accounts for 55%-80% of the lipid preparation, phospholipid emulsifiers account for 15%-35% of the lipid preparation, and short-chain alcohol or ether emulsifiers account for 5%-10% of the lipid preparation.

3. The lipid preparation of paclitaxel-triglyceride prodrug according to claim 1, characterized in that: The weight ratio of the short-chain alcohol or ether emulsifier, the phospholipid emulsifier and the liquid oil phase is 1:2-4:5-20.

4. The lipid formulation of paclitaxel-triglyceride prodrug according to any one of claims 1 to 3, characterized in that The paclitaxel-triglyceride prodrug accounts for 2%-5% of the total weight of the excipients.

5. The lipid preparation of paclitaxel-triglyceride prodrug according to claim 1, characterized in that The lipid preparation is composed of paclitaxel-triglyceride prodrug, egg yolk lecithin, olive oil and diethylene glycol monoethyl ether, wherein the olive oil accounts for 55%-80% of the lipid preparation, the egg yolk lecithin accounts for 15%-35% of the lipid preparation, the diethylene glycol monoethyl ether accounts for 5%-10% of the lipid preparation, and the paclitaxel triglyceride prodrug accounts for 3%-4% of the total weight of the excipients.

6. The lipid formulation of paclitaxel-triglyceride prodrug according to claim 1, characterized in that The paclitaxel-triglyceride prodrug is prepared by the following method: (a) Synthesis of 1,3-diglycerol: Caprylic acid, stearic acid or oleic acid is dissolved in anhydrous chloroform, and reacted with 1,3-dihydroxyacetone to form an ester under the catalysis of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine, and then hydrogenated with sodium borohydride to obtain 1,3-diglycerol; (b) Dissolve 4,4'-dithiodibutyric acid in an appropriate amount of acetic anhydride, stir at room temperature to form 4,4'-dithiodibutyric anhydride, react with 1,3-diacerol to obtain an intermediate product, and then react with paclitaxel to obtain the intermediate product.

7. Use of the lipid preparation of paclitaxel-triglyceride prodrug according to any one of claims 1 to 6 in the preparation of anti-tumor drugs.

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