Gefitinib liposome compound and preparation method thereof

Abstract

The invention relates to a gefitinib liposome compound and a preparation method thereof. The compound comprises gefitinib, a lipid material, a buffer solution and the like. The liposome complex comprises one or more lipid materials, and has a high ratio of active gefitinib to lipids. The encapsulation efficiency of the gefitinib liposome compound can be improved, and the gefitinib liposome compound has the characteristics of good stability, high targeting property, low toxic and side effects and the like. The preparation method disclosed by the invention is simple, strong in operability and easy to realize industrial production.

Description

[0001] This application is a divisional application of the invention patent application with application number 201610255645.9, application date April 22, 2016, and invention name “Gefitinib liposome complex and preparation method thereof”.

[0002] Gefitinib is a relatively effective anticancer agent used in systemic cancer treatment. This chemotherapy drug is very effective in the treatment of experimental animals and human tumor models, and is particularly suitable for the treatment of locally advanced or metastatic non-small cell lung cancer (NSCLC) that has been previously treated with chemotherapy or is not suitable for chemotherapy.

[0003] Like other cancer chemotherapy agents, gefitinib compounds are toxic. The main disadvantages of gefitinib compounds are frequent adverse reactions to the skin, gastrointestinal tract, abnormal liver function, etc. Among them, interstitial pneumonia is a rare but fatal adverse reaction of gefitinib, with a high mortality rate.

[0004] Methods for reducing the toxicity of gefitinib compounds include combined chemotherapy, synthetic analogs (Fahmy et al., 2016.2.11, Urol Int.; Xiaoqing Wu et al., 2010.6.1, Bioorg Med Chem.18(11):3812-3822), immunotherapy and liposome encapsulation (Maruyama S et al., 2003 Oct Gan To Kagaku Ryoho.30(11):1773-5.; Shao Yue, China Excellent Master's Thesis 2015). Compared with the free form dosage form, the anti-tumor drug including gefitinib encapsulated in liposomes has lower toxicity while maintaining anti-tumor activity (Shao Yue, China Excellent Master's Thesis 2015).

[0005] However, due to the low water solubility of bioactive agents, gefitinib is slightly soluble at pH = 1, but almost insoluble at pH > 7, and gefitinib has low lipophilicity, resulting in a low bioactive agent / lipid ratio, so it is difficult to effectively embed gefitinib in liposomes or lipid complexes.

[0006] Another problem with liposomes and lipid complexes containing gefitinib is the stability of the compound. In particular, the persistence of the efficacy of the bioactive agent and the retention of the bioactive agent in the liposome during storage are well-known problems (Freise et al., 1982; Gondal et al., 1993; Potkul et al., 1991 Am J Obstet Gynecol. 164(2):652-658; Steerenberg et al., 1988; Sweiss et al., 1993). SUMMARY OF THE INVENTION

[0008] The present invention describes a novel lipid-embedded gefitinib liposome complex and a method for preparing the same. The method described in the present invention is a novel method for forming the novel gefitinib liposome complex.

[0009] Among other things, the present invention provides a composition comprising a liposome or lipid complex and gefitinib, wherein the liposome comprises one or more lipids, wherein the weight ratio of gefitinib to lipid is 1:50-1:1, or 1:30-1:3, or 1:20-1:5. The one or more lipids may include, for example, 50-100 mol% DPPC and 0-50 mol% cholesterol, and for example 50-70 mol% DPPC and 10-30 mol% cholesterol.

[0010] At the same time, the present invention also provides a method for preparing a gefitinib liposome complex, comprising the following steps: (a) mixing a gefitinib homogeneous solution with a lipid solution; (b) injecting the mixed solution (a) into an aqueous medium for mixing; (c) forming a mixture of the two at a first temperature; and (d) subsequently forming a further mixture at a second temperature lower than the first temperature. Wherein steps (c) and (d) are effective for improving the encapsulation efficiency of gefitinib. Step (c) is generally achieved by heating, while step (d) is generally achieved by cooling. In an alternative embodiment, the cycle starts from the cooling step, switches to the heating step, and repeats these two steps. The method includes continuing to repeat steps (c) and (d) for two or three or more cycles. The gefitinib solution can be prepared by dissolving gefitinib in dimethyl sulfoxide (DMSO). Dipalmitoylphosphatidylcholine (DPPC) and cholesterol are dissolved in an organic solvent including anhydrous ethanol to form a lipid solution. After all steps (c) and (d) are completed, the method for preparing gefitinib liposome complexes may further include: (e) filtering through a membrane having molecular weight site-selective capability to remove unencapsulated gefitinib and retain the desired liposomes or lipid complexes.

[0011] The present invention further provides a gefitinib liposome complex prepared according to the method of the present invention and a pharmaceutical formulation of the complex of the present invention, wherein the formulation comprises a pharmaceutically acceptable carrier or diluent and is suitable for administration to a patient by inhalation or injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The anticancer cell activities of the gefitinib liposome complex according to the present invention and the gefitinib original drug in three lung cancer cells are shown. Summary of the invention

[0013] The present invention includes a novel lipid complex of gefitinib liposomes, wherein the ratio of gefitinib to lipid is very high. The weight ratio of gefitinib to lipid in the present invention is between 1:1 and 1:30. Most preferably, the weight ratio of gefitinib to lipid is between 1:5 and 1:10.

[0014] The method for preparing the gefitinib liposome complex comprises mixing gefitinib with a lipid solution and subjecting the mixture to one or more cycles at two separate temperatures. The gefitinib liposome complex can be formed by this method.

[0015] The preparation method of pulmonary administration inhalation type gefitinib liposomes described in the present invention includes the following features: dissolving gefitinib in DMSO, dissolving the lipid material in an organic solvent miscible with water such as anhydrous ethanol and methanol, mixing the two and injecting physiological saline, isotonic glucose solution or phosphate buffer solution (PBS) preheated to above the phase transition temperature of the lipid material under stirring to obtain a liposome solution; removing free gefitinib by ultrafiltration, dialysis or Sephadex column to obtain the gefitinib liposome solution.

[0016] As can be determined by routine experimentation, the appropriate temperature for use in the method may vary in some cases depending on the lipid mixture employed in the method.

[0017] The prepared gefitinib liposome complex has a high drug-to-lipid ratio and the formulation can be suitable for inhalation or injection.

[0018] The lipids used in the present invention can be synthetic, semi-synthetic or naturally occurring lipids, including phospholipids, vitamin E, sterols, fatty acids, glycolipids, anionic lipids, and cationic lipids. The phospholipids can include phosphatidylcholine (EPC), phosphatidylglycerol (EPG), phosphatidylinositol (EPI), phosphatidylserine (EPS), phosphatidylethanolamine (EPE), and phosphatidic acid (EPA): soybean counterparts, soybean phosphatidylcholine (SPC): SPG, SPS, SPI, SPE and SPA: hydrogenated egg and soybean counterparts (such as HEPC, HSPC), sterically modified phosphatidylcholine ethanolamine, cholesterol derivatives, carotenoids, other phosphatidylcholines formed by lipid bonds, the esters are composed of glycerol containing 12-26 carbon atom chains at the 2nd and 3rd positions and glycerol containing different main groups containing choline, glycerol, inositol, serine, ethanolamine at the 1st position, and the corresponding phosphatidic acid. These fatty acid chains can be saturated or unsaturated, and phospholipids can be made up of fatty acids of different lengths and saturation. In particular, the composition of the formula can include DPPC, the main component of naturally occurring lung surfactant. Other examples include myristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) and dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) and distearoylphosphatidylcholine (DSPC) and distearoylphosphatidylglycerol (DSPG), dioleoylphosphatidyl-choline (PSPC) and palmitoylstearoylglycerol (PSPG), triglycerides, seranide, diacylglycerol, sphingosine, sphingomyelin and the monoacylphospholipids such as mono-oleoyl-phosphatidylethanolamine (MOPE).

[0019] The test result clearly shows that gefitinib is carried out to entrapment in the liposome vesicle formation process. The result further shows that, because the gefitinib concentration is much higher than the solubility limit, the physical state of gefitinib is solid or combined with lipid. The result further shows that freezing mixture is not needed in the process, but the temperature cooled to above the freezing temperature can produce better effect. The result further shows that the embedding effect of 3-4 cooling and heating cycles is similar to the embedding effect of 6 cycles, which illustrates that the temperature treatment of 3-4 cycles is enough to realize the encapsulation of the preferred level.

[0020] The result further shows that while improving the efficiency of embedding gefitinib, this process can be amplified. Therefore, the present invention further provides a method for supplying 200mLS or more or 800LS or more (appropriate small amount of increase) suitable for whole administration. Under all other identical conditions, it can be believed that the effect of increasing is usually easily reached than small-scale production of larger volumes. If such volume is suitable for administration, it is possible to reduce volume to make it suitable for storage.

[0021] The results further showed that the gefitinib liposome complex prepared by the method of the present invention could remain entrapped for more than one year due to minimal leakage. Further confirmation of the uniqueness of the formulation showed that gefitinib was entrapped in the liposome structure and was not prone to leakage. Example

[0022] Example 1

[0023] 20 mg of gefitinib was dissolved in 1.0 ml of DMSO, 100 mg of dipalmitoylphosphatidylcholine (DPPC) and 40 mg of cholesterol were dissolved in 1.0 ml of anhydrous ethanol, preheated to 65°C, and injected into 20 ml of physiological saline solution preheated to 65°C; slowly cooled to 5°C, then heated to 65°C, repeated 3 times, and then cooled to room temperature to obtain gefitinib liposome complex. Free gefitinib was removed by dialysis to obtain gefitinib liposome complex solution. The encapsulation efficiency before removing free drug was 90%.

[0024] Example 2

[0025] 10 mg of gefitinib was dissolved in 1.0 ml of DMSO, 40 mg of hydrogenated soybean lecithin (HSPC) and 27 mg of cholesterol were dissolved in 1.0 ml of anhydrous ethanol, and the two were mixed and injected into 5 ml of 50 isotonic glucose solution under magnetic stirring; the temperature was lowered to 0, and then slowly heated to 50, and the temperature was repeated twice, and the liposome solution was obtained after it was lowered to room temperature. The gefitinib liposome solution was obtained by dialyzing the isotonic glucose solution to remove free gefitinib. The encapsulation efficiency was 82% before removing the free drug.

[0026] Example 3

[0027] 30 mg of gefitinib was dissolved in 1.0 ml of DMSO, 130 mg of egg yolk lecithin (Egg PC) and 50 mg of cholesterol were dissolved in 1.0 ml of ethanol, and the two were mixed and injected into 15 ml of 40 pH 6.8 PBS phosphate buffer solution under magnetic stirring; the temperature was cooled to 0, and then slowly heated to 40, and the temperature was repeated 4 times, and the liposome solution was obtained after cooling to room temperature. The gefitinib liposome solution was obtained by dialyzing in an isotonic glucose solution to remove free gefitinib. The encapsulation efficiency was 85% before removing the free drug.

[0028] Although the present invention has only focused on describing preferred embodiments, it is obvious to those skilled in the art that various preferred devices and methods may be used, and that attempts may be made to use methods different from those described herein. Therefore, the present invention includes all modifications within the spirit and scope of the following claims.

Claims

1. A gefitinib liposome complex, comprising a liposome or lipid complex and gefitinib embedded therein, wherein the liposome or lipid complex comprises one or more lipid materials, wherein the weight ratio of gefitinib to lipid is 1:50-1:1; preferably 1:30-1:3; more preferably 1:20-1:

5.

2. The composition according to claim 1, wherein the one or more lipids include egg yolk lecithin, soybean lecithin, hydrogenated soybean lecithin, dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoylphosphatidylcholine, dimyristoylphosphatidylglycerol, etc.; Preferably, the one or more lipids include dipalmitoylphosphatidylcholine (DPPC); Further preferably, the one or more lipids include 50-100 mol% DPPC and 0-50 mol% cholesterol; or 50-70 mol% DPPC and 10-30 mol% cholesterol.

3. The composition according to claim 1, wherein the lipids comprise: Vitamin E, cholesterol and cholesterol derivatives, etc.; preferably, the one or more lipids include cholesterol.

4. The composition according to claim 1, wherein the embedded drug is not limited to gefitinib, and also includes imatinib, erlotinib, dasatinib, sunitinib, sorafenib, nilotinib, afatinib, icotinib, canertinib, etc.

5. A method for preparing gefitinib liposome complexes, comprising the following main steps: (a) mixing a gefitinib homogeneous solution with a lipid solution; (b) injecting the mixed solution from (a) into an aqueous medium for mixing; (c) forming a mixture of the two at a first temperature; and (d) subsequently forming a further mixture at a second temperature lower than the first temperature, Among them, steps (c) and (d) are effective in improving the encapsulation efficiency of gefitinib.

6. The method according to claim 5, further comprising continuing to repeat steps (c) and (d) for a total of two or more cycles, or preferably a total of three or more cycles, to increase the encapsulation efficiency of gefitinib.

7. The method according to claim 5, wherein: In step (a), gefitinib is dissolved in one or more solvents including dimethyl sulfoxide (DMSO) to form a gefitinib homogeneous solution, wherein dipalmitoylphosphatidylcholine (DPPC) and cholesterol are dissolved in one or more organic solvents including anhydrous ethanol to form a lipid solution; Preferably, in step (b), the aqueous medium comprises one or more combinations of water, physiological saline, phosphate buffered saline (PBS), isotonic glucose solution; More preferably, after all steps (c) and (d) are completed, the method further comprises: (e) filtering through a membrane with molecular weight selective function to remove the unencapsulated gefitinib drug and retain the desired liposome complex.

8. The method according to claim 6, wherein: The temperature range of forming the mixture in step (c) includes 5 degrees Celsius to 70 degrees Celsius; or 15 degrees Celsius to 65 degrees Celsius; or 30 degrees Celsius to 65 degrees Celsius; Preferably, the temperature range of forming the mixture in step (d) includes between -20 degrees Celsius and 25 degrees Celsius; or between -10 degrees Celsius and 20 degrees Celsius; or between -5 degrees Celsius and 10 degrees Celsius; Further preferably, the temperature difference between steps (c) and (d) is 25 degrees Celsius or more.

9. A pharmaceutical formulation comprising the composition of claim 1 and a pharmaceutically acceptable carrier or diluent.

10. A pharmaceutical formulation comprising the composition of claim 1, suitable for inhalation or injection by a patient.

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