WO2017063559A1 - 一种考布他汀a4衍生物及其制剂 - Google Patents

一种考布他汀a4衍生物及其制剂 Download PDF

Info

Publication number
WO2017063559A1
WO2017063559A1 PCT/CN2016/101907 CN2016101907W WO2017063559A1 WO 2017063559 A1 WO2017063559 A1 WO 2017063559A1 CN 2016101907 W CN2016101907 W CN 2016101907W WO 2017063559 A1 WO2017063559 A1 WO 2017063559A1
Authority
WO
WIPO (PCT)
Prior art keywords
cobstatin
stearate
preparation
injection
nano
Prior art date
Application number
PCT/CN2016/101907
Other languages
English (en)
French (fr)
Inventor
周琴琴
单彬
Original Assignee
上海天氏利医药科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 上海天氏利医药科技有限公司 filed Critical 上海天氏利医药科技有限公司
Publication of WO2017063559A1 publication Critical patent/WO2017063559A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
    • A61K31/23—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin of acids having a carboxyl group bound to a chain of seven or more carbon atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C67/00—Preparation of carboxylic acid esters
    • C07C67/08—Preparation of carboxylic acid esters by reacting carboxylic acids or symmetrical anhydrides with the hydroxy or O-metal group of organic compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
    • C07C69/02—Esters of acyclic saturated monocarboxylic acids having the carboxyl group bound to an acyclic carbon atom or to hydrogen
    • C07C69/22—Esters of acyclic saturated monocarboxylic acids having the carboxyl group bound to an acyclic carbon atom or to hydrogen having three or more carbon atoms in the acid moiety
    • C07C69/24—Esters of acyclic saturated monocarboxylic acids having the carboxyl group bound to an acyclic carbon atom or to hydrogen having three or more carbon atoms in the acid moiety esterified with monohydroxylic compounds

Definitions

  • the invention relates to the technical field of medicine, in particular to a testosterone A4 derivative and a preparation thereof.
  • Combretastatin A4 (CA4, abbreviated as CA) is a natural potent small molecule anti-mitotic agent isolated from the bark of South African shrub. Its action site is the same as that of colchicine. The polymerization of tubulin can inhibit the depolymerization of tubulin and the accumulation of mitotic proteins in cells (Shang Hai, et al. Progress in the study of tubulin inhibitors [J]. Acta Pharmaceutica Sinica, 20l0, 45(9): 1078 -1088). In 1991, G.R. Pettit et al. determined its chemical structure and applied for a US patent (US 4,996,237).
  • cobstatin A4 can selectively inhibit the binding of tumor tubulin and change the skeleton structure and morphology of endothelial cells by utilizing the physiological differences between tumor tissue and normal tissue endothelial cells. Its vascular permeability disturbs blood flow, causing apoptosis of tumor vascular endothelial cells, leading to secondary tumor cell death.
  • Cobstatin A4 is a natural vascular targeted drug with high anti-tumor activity and is effective for breast cancer, colon cancer, medullary thyroid carcinoma, non-small cell lung cancer, lymphoma, etc., and has great clinical application prospects. .
  • CA4P cobstatin A4 disodium phosphate
  • CA4P is currently being developed by OXIGENE and has been in clinical phase II/III (Li Hongxue, et al. Cobstatin A-4 phosphate prodrug: CA4P [J]. Chinese Journal of New Drugs and Clinical Medicine, 2010, 29(11) :816-821).
  • CA4P has gradually deepened its research on safety, effectiveness and chemical stability. It has comprehensively evaluated it from various angles and found that CA4P also has many shortcomings. It needs to be further resolved, mainly in the following aspects:
  • 2CA4P has certain toxic and side effects on cardiovascular, which may lead to high blood pressure, tachycardia, bradycardia, atrial fibrillation and myocardial infarction, as well as pain, vomiting, headache and fatigue at the tumor site.
  • the series is uncomfortable, resulting in poor compliance of clinical medications (SubbiahlM. et al. Cardiovascular toxicity profiles of vascular disrupting agents [J].
  • 3CA4P can change the blood of normal organs Flow, such as the brain, heart, spleen, kidney, etc., changes in blood flow in the heart and brain can even endanger the patient's life (Murata, R., J. et al. Comprehensive effects of combretastatin A-4disodium phosphate and 5, 6-dimethylxanthenone-4-acetic acid on blood perfusion in a murine tumour and normal tissues [J]. International journal of radiation biology. 2001, 77: 195-204).
  • 1CA4P has a fast elimination rate and a wide volume distribution, which weakens its anti-tumor effect to a certain extent (Li Ma, et al. Targeted treatment of choroidal neovascularization using integrin-mediated sterically stabilized liposomes loaded with combretastatin A4 [J].Journal of ocular pharmacology and therapeutics,2009,25(3):195-200;Zhang Xiaolan,Wang Zhiqiang, et al.Comparative study of pharmacokinetics of Combretastatin A4 and its phosphate disodium in rats[J].
  • 1CA4P is unstable to light, prone to cis-trans isomerization, and produces inactive trans isomers (Mo Yi, et al. Photolysis kinetics of Combretastatin A4phosphate solution [J]. Chinese Journal of Pharmaceutical Sciences, 2007, 42(7): 516-519); 2CA4P aqueous solution is more unstable than solid powder, easily decomposes to form cobstatin A4, precipitates and precipitates.
  • CA4P is a water-soluble drug, so it is not suitable for preparing CA4P into nano-formulation.
  • CA4P is a prodrug of combretastatin A4. It is cobstatin A4 that actually exerts antitumor effect in vivo, while cobstatin A4 is insoluble in water. If it can prepare capbutastat A4 into nanometer preparation, it is anti-tumor.
  • EP2896412A1 also mentions the modification of poorly soluble drugs to give fat-soluble derivatives, including combretastatin A4, which is modified by the use of saturated or unsaturated fatty chains to modify the above-mentioned poorly soluble drugs, but the main The purpose is to coat or encapsulate various drug-modified derivatives on the surface or inside of an implanted stent made of a polymer material, which is a local implant preparation for treating human physiological channel stenosis, and The substantial effects produced by the invention are completely different.
  • the main purpose is to solve the problem of the nano-formulation medicinal properties, so we successfully synthesized the C2 ⁇ C18 different carbon chain length of the cobstatin A4 fatty acid ester.
  • the results showed that only the cobstatin A4 palmitate (C16) and the cobstatin A4 stearate (C18) were in the form of a white powder, and the rest were in the form of a waxy or oily liquid (see Example 1).
  • impurities in the oily compound and the organic solvent are not easily removed, which is disadvantageous for quality control, and the solid powder can be removed by recrystallization or the like, and a high quality compound can be easily obtained, and safety can be ensured.
  • the cobstatin A4 fatty acid esters with different carbon chain lengths from C2 to C18 can basically prepare good nano-preparation, and no essential difference is found.
  • CA4P original drug
  • the cobstatin A4 fatty acid ester can indeed solve the defect that the cobstatin A4 is difficult to prepare directly into the nano-preparation, but surprisingly the fatty acid carbon chain only has stearic acid in the range of C2 ⁇ C18 (C18 The ester exhibits an extraordinary antitumor effect, and it has been unexpectedly found that the cobstatin A4 stearate having a substantial effect is a core technical feature of the present invention.
  • a nano-formulation of cobstatin A4 was successfully developed based on the physicochemical properties unique to the cobstatin A4 stearate.
  • the invention provides a safe, efficient and stable quality of the cobstatin A4 stearate and its nano preparation injection, which lays a solid foundation for the research and application of the cobstatin A4 in China.
  • the present invention provides a cobstatin A4 stearate obtained by reacting a phenolic hydroxyl group in the chemical structure of the cobstatin A4 with stearoyl chloride.
  • the fatty acid carbon chain of cobstatin A4 stearate is C18, which is included in the protection range of the international patent (WO 2007059118A1) with the C2-C21 alkyl substituent and the phenolic hydroxyl group of the cobstatin A4.
  • the patent has not been studied differently, and its practical significance is unknown. We have repeatedly found that the antitumor effect of cobstatin A4 stearate is the most prominent, through multiple parallel experiments in animal efficacy.
  • the tumor inhibition rate is as high as 90%, while the anti-tumor rate of other C2-C21 cobstatin A4 fatty acid esters and the original drug (CA4P) is maintained at about 60%. This unexpected result is still unknown. The underlying mechanism is still under investigation (see Example 1). Judging from the results of many in vivo effects, cobstatin A4 stearate has substantial advantages over other fatty acid esters. In fact, the actual value of cobstatin A4 and fatty acid ester-forming schemes has been discovered. The cobstatin A4 stearate having a substantial effect is a core technical feature of the present invention.
  • the invention provides a cobstatin A4 stearate nano preparation injection, which is prepared by encapsulating the cobstatin A4 stearate in a lipid material by a special prescription process.
  • a preparation process it was found that the nano-formulations that met the requirements could not be obtained by the conventional prescription process, and a series of problems such as large particle size, uneven particle size distribution, turbidity, and drug precipitation occurred.
  • the nano-formulation encapsulating the fat-soluble drug can basically obtain a good preparation through the adjustment of several prescription processes, but it is completely beyond our expectation for the cobstatin A4 fatty acid ester.
  • a high-pressure homogenization method for fusion a high-pressure homogenization method for a film
  • a high-pressure homogenization method for injecting ethanol or diethyl ether a high-pressure homogenization method for injecting ethanol or diethyl ether
  • the ideal drug-loaded nanoformulation was not obtained (see Example 2).
  • the high-pressure homogenization step did not receive the intentional effect of the ethanol injection alone, and the obtained nano-preparation was semi-transparent, and the particle size was about 100 nm, and the distribution was very uniform. .
  • cobstatin A4 fatty acid esters were verified, and basically a good nano preparation was obtained.
  • a good nano-formulation can be prepared by the injection stirring method, which is purely unexpected.
  • the preparation method is a matching process for the cobstatin A4 fatty acid ester (see Example 3).
  • Another invention of the present invention is to systematically match the dosage of the preparation auxiliary materials in combination with the inherent characteristics of the cobstatin A4 stearate, and to prepare a safe, efficient and stable quality test in the true sense. Butatin A4 stearate and its nanoformulation injections (see Examples 4-5).
  • a cobstatin A4 derivative which is a cobstatin A4 saturated fatty acid ester and is a nano preparation.
  • the cobstatin A4 derivative is cobstatin A4 stearate obtained by esterification of stearyl chloride with cobstatin A4, and the structural formula is as follows;
  • the organic solvent in the step (A) is selected from the group consisting of anhydrous chloroform, anhydrous dichloromethane, N,N-dimethylacetamide, and anhydrous N,N-dimethylformamide. Or more than one; preferably dichloromethane.
  • the acid binding agent in the step (A) is selected from one or more of N,N-diisopropylethylamine, triethylamine, 4-dimethylaminopyridine and pyridine; preferably N,N- Diisopropylethylamine.
  • a cobstatin A4 stearate nano preparation which is an injection, which is an injection or a lyophilized powder for injection; the injection is made from combretastatin A4 stearin
  • the acid ester and phospholipid composition preferably, it is composed of combretastatin A4 stearate, phospholipid, DSPE-PEG2000.
  • the cobstatin A4 stearate nanoformulation is formulated from the following formulations (the percentages below refer to the total volume of the injection):
  • the cobstatin A4 stearate nanoformulation is formulated from the following formula (the following percentages refer to Relative to the total volume of the injection):
  • the cobstatin A4 stearate nanoformulation is formulated from the following formula (the percentages below refer to the total volume of the injection):
  • the phospholipid in the above formula is selected from the group consisting of hydrogenated soybean phospholipid, hydrogenated egg yolk phospholipid, dipalmitoylphosphatidylcholine, phosphatidylcholine, egg yolk phospholipid, soybean phospholipid, phosphatidylserine, dimyristoyl phosphatidylcholine, One or more of stearylphosphatidylcholine, phosphatidylethanolamine, and sphingomyelin; preferably hydrogenated soybean phospholipid, egg yolk phospholipid.
  • the pH adjusting agent in the above formula is selected from the group consisting of citric acid, hydrochloric acid, phosphoric acid, disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, disodium citrate, trisodium citrate, hydrogen.
  • One or more of sodium oxide adjusting the pH to 3-7, preferably adjusting the pH to 3.5-6; more preferably adjusting the pH to 4-6.
  • the lyoprotectant in the above formulation is selected from one or more of trehalose, sucrose, lactose, mannitol, dextran 40, xylitol, sorbitol; preferably trehalose, sucrose, mannitol.
  • a method for preparing a cobstatin A4 stearate nanoformulation comprising the steps of:
  • the organic solvent is selected from one or more of anhydrous ethanol, tert-butanol and propylene glycol in an amount of from 1 to 10% by volume; preferably anhydrous ethanol or propylene glycol, preferably in a total volume. 2-6%.
  • the aqueous phase of a certain temperature has a temperature of 40-80 ° C, preferably 50-70 ° C.
  • the present invention provides an antitumor pharmaceutical preparation comprising cobstatin A4 stearate as the sole active ingredient.
  • the anti-tumor pharmaceutical preparation is a nano preparation, and any of the above formulas may be selected.
  • Cobstatin A4 stearate significantly increased the antitumor effect of cobstatin A4.
  • the anti-tumor effect of cobstatin A4 stearate is the most prominent in many of the cobstatin A4 fatty acid esters and the original drug (CA4P), and the tumor inhibition rate is over 90%. . It not only solves the problem of poor fat solubility of cobstatin A4, but also significantly improves the efficacy.
  • Stearic acid is a saturated fatty acid with good chemical stability, thus providing a guarantee for the stability of the cobstatin A4 stearate.
  • the combecitastat A4 stearate nano preparation injection of the invention belongs to the nano-encapsulated preparation, has obvious slow-release effect, greatly slows the elimination rate of the drug in the body, and the drug concentration targeting the tumor site will be more high.
  • the combecitastat A4 stearate nano preparation injection of the present invention encapsulates the drug in the lipid material, thereby avoiding direct contact of the drug with water and light, and further improving the chemical stability of the drug.
  • Example 1 Comparative evaluation of a series of different cobstatin A4 fatty acid esters in vitro and in vivo
  • Nano formulations of different cobstatin A4 fatty acid esters were prepared under the same formulation conditions.
  • cobstatin A4 fatty acid ester about 300mg according to testosterone A4), hydrogenated soybean phospholipid (HSPC) 0.3g, DSPE-PEG2000 0.3g, dissolved in 2mL absolute ethanol to obtain the organic phase; weighed 3g of trehalose and 7g of mannitol, placed in an appropriate amount of water for injection, stirred and dissolved to obtain an aqueous phase; the organic phase was injected under stirring Heat to 65 ° C in the aqueous phase, mix well, and dilute to 100 ml with water for injection. Adjust the pH to about 5.0 with citric acid or sodium hydroxide; then sterilize the solution through 0.22 ⁇ m; dispense, freeze-dry, and seal to obtain the cobstatin A4 fatty acid ester nano preparation.
  • HSPC hydrogenated soybean phospholipid
  • cobstatin A4 fatty acid ester nano preparations were reconstituted with water for injection to a concentration of about 3 mg/ml (calculated as cobstatin A4).
  • the appearance and average particle size of the complex solution were examined at room temperature, and the state of the complex solution was observed after standing for 5 hours at room temperature. The results are shown in the following table:
  • the test showed that the 10 cobstatin A4 fatty acid esters were designed to be successfully prepared into nano-preparation, and after reconstitution, it was a translucent emulsion, and the particle size was basically distributed around 100 nm.
  • the nano solution of the cobstatin A4 oleate changed from the initial translucent emulsion to the turbid emulsion, and the particle size increased significantly, possibly oleic acid unsaturated fatty acid.
  • the other nine fatty acid esters have good stability, so the use of the cobstatin A4 saturated fatty acid ester may be more medicinal and also demonstrate the superiority of the present invention. Therefore, only the remaining 9 saturated fatty acid esters were compared in the next in vivo pharmacodynamic evaluation.
  • S180 cells were cultured in DMEM containing 10% serum and 1% double antibody at a temperature of 37 ° C and 5% CO 2 .
  • the activity was good, and the cells were collected, and the cell concentration was 1 ⁇ 10 7 /ml diluted with serum-free DMEM to obtain a tumor cell suspension.
  • Each mouse was inoculated with 0.2 ml of cell suspension by intraperitoneal injection, and a total of 6 Kunming mice were inoculated. Eight days after the inoculation, ascites was taken and passed to the third generation.
  • the third-generation ascites cells were extracted and diluted with physiological saline to a concentration of 1 ⁇ 10 6 /ml to obtain a dilution of ascites cells. 0.2 ml of the ascites cell dilution was taken and injected subcutaneously into the right forelimb of Kunming mice to obtain a mouse S180 tumor model.
  • the physiological saline group, the original test drug cobstatin A4 phosphate disodium salt solution positive control group, the cobstatin A4 fatty acid ester nano preparations test group, and 8 S180 tumor model mice in each group were respectively set.
  • the original drug is to dissolve the drug substance of cobstatin A4 disodium phosphate directly into the water for injection to make 3mg/ml (calculated as combretastatin A4) as a positive control preparation; and prepare according to the first (3) of the first embodiment.
  • a series of cobstatin A4 fatty acid ester lyophilized nano preparations were dissolved in water for injection to 3 mg/ml (calculated as combretastatin A4) as a test preparation.
  • mice were administered by tail vein injection at a dose of 15 mg/kg each time (calculated as cobstatin A4), once a day for 6 days, and the mice were sacrificed the next day after drug withdrawal, and the tumor was removed. Weigh and calculate the tumor inhibition rate. Since the number of samples of the designed testosterone A4 fatty acid ester nano-preparation was as high as 9, the above test protocol was divided into two experiments, which were respectively recorded as Comparative Test 1 and Comparative Test 2.
  • Tumor inhibition rate (normal weight of saline group - tumor weight of administration group) / tumor weight of physiological saline group ⁇ 100%
  • the antitumor effects of the cobstatin A4 acetate, hexanoate, phthalate, myristate, and stearate nanoformulations were compared in the same dosage form and drug loading. Unexpectedly, only the anti-tumor effect of the cobstatin A4 stearate nano-preparation was the most prominent, the tumor inhibition rate was as high as 91.09%, while the other tumor inhibition rates of the cobstatin A4 fatty acid ester and the original drug (CA4P) were both. Maintaining at about 60%, the remarkable anti-tumor effect of combretastatin A4 stearate is a core technical feature of the present invention.
  • Tumor inhibition rate Saline 1.98 ⁇ 0.66 / Cobstatin A4 disodium phosphate solution 0.68 ⁇ 0.33 65.66% Cobstatin A4 Butyrate Nanoformer 0.71 ⁇ 0.21 64.14% Cobstatin A4 octanoate nanoformulation 0.75 ⁇ 0.31 62.12% Cobstatin A4 laurate nano preparation 0.78 ⁇ 0.39 60.61% Cobstatin A4 palmitate nanoformulation 0.63 ⁇ 0.19 68.18% Cobstatin A4 stearate nanoformulation 0.14 ⁇ 0.11 92.93%
  • the antitumor effects of the cobstatin A4 butyrate, caprylate, laurate, palmitate, and stearate nanoformulations were compared in the same dosage form and drug loading.
  • the results showed that only the anti-tumor effect of the cobstatin A4 stearate nano-preparation was the best, the tumor inhibition rate was as high as 92.93%, while the anti-tumor rate of other cobstatin A4 fatty acid esters and the original drug (CA4P) remained. Maintaining at around 60% further demonstrates that the antitumor activity of cobstatin A4 stearate is substantially different from that of other fatty acid esters.
  • the preparation of a series of combretastatin A4 fatty acid esters after the preparation of the cobstatin A4 can indeed solve the problem of poor fat solubility of the cobstatin A4, and the nano preparation prepared by the saturated combecitastat A4 fatty acid ester.
  • Example 2 Comparative study on the preparation process of cobstatin A4 stearate nano preparation injection
  • the molten high pressure homogenization method is to mix a drug or a drug with a lipid material, and to make it melt or semi-molten at a certain temperature, emulsify it in water under the action of a surfactant, and then further reduce it by a high-pressure homogenizer.
  • the particle size gives a nano-formulation. According to its principle, we used this method to treat the cobstatin A4 stearin with the most conservative drug loading of 0.5mg/ml.
  • the acid ester nanoform preparation was prepared and evaluated for quality.
  • the prepared capbutastatin A4 stearate nano preparation has a large particle size and distribution value (PDI); the particle size is 311.2 nm, the distribution is extremely uneven, and more importantly, it cannot be removed.
  • the bacteria were filtered, and the drug loading was only 0.5 mg/ml. To this end, we have done a corresponding study on the ratio of different lipid materials, and the results are basically similar. It is impossible to see any possibility of preparing the combretastatin A4 stearate nanoformulation by the melt high pressure homogenization method.
  • the film high-pressure homogenization method dissolves the drug and the lipid material in a volatile organic solvent, removes the organic solvent under vacuum, forms a lipid film, adds water to hydrate, and obtains a crude nanoparticle solution; The solution was reduced in particle size by a high pressure homogenizer to obtain a nano preparation. According to its principle, we used this method to prepare the cobstatin A4 stearate nanoformulation with the most conservative drug loading of 0.5 mg/ml, and evaluated the quality.
  • the membrane high pressure homogenization method was used.
  • the drug loading was only 0.5 mg/ml
  • the prepared capbutastatin A4 stearate nano preparation was improved compared with the melt high pressure homogenization method, but the particle size and distribution value (PDI) ) is still very large, the particle size is 227.1nm, the distribution is extremely uneven, and more importantly, it is impossible to carry out sterilization filtration.
  • PDI particle size and distribution value
  • the drug and the lipid material are dissolved together in a water-soluble organic solvent to form a lipid solution; the lipid solution is injected into water to form a crude nanoparticle solution; and the crude nanoparticle solution is passed through a high pressure
  • the mass machine reduces the particle size to obtain a nano preparation. According to its principle, we used this method to prepare the cobstatin A4 stearate nanoformulation with the most conservative drug loading of 0.5 mg/ml, and evaluated the quality.
  • the prepared capbutastatin A4 stearate nano preparation has a significant improvement compared with the melt high pressure homogenization method and the film high pressure homogenization method. Dropped to 200nm, but the distribution is still uneven, the particle size is large and small, still can not successfully complete the sterilization filtration. Similarly, we have also done a corresponding study on the ratio of different lipid materials, and finally did not obtain a nano-formulation with uniform particle size and relatively stable. Therefore, it is considered that the high-pressure homogenization method is not applicable to the cobstatin A4 stearin. Preparation of an acid ester nanoformulation.
  • the commonly used nano-preparation preparation method for the cobstatin A4 stearate, can not obtain the nano-formulation that meets the requirements of intravenous injection, which may be related to the special physical and chemical properties of the cobstatin A4 stearate. related. Therefore, it is important to find a preparation method that matches it, which will directly affect the application prospect of cobstatin A4 stearate.
  • Example 3 Study on the preparation process of cobstatin A4 stearate nano preparation injection
  • Prescription 1 Prescription 2
  • Prescription 3 Prescription 4
  • Prescription 5 Cobstatin A4 stearate 0.05g 0.3g 0.3g 0.3g 0.3g DSPE-PEG2000 0.5g 0.5g 0.5g 0.5g Hydrogenated soybean phospholipid / / 0.5g / / Dipalmitoylphosphatidylcholine / / / 0.5g / Phosphatidylcholine / / / / 0.5g Egg yolk phospholipid 0.5g 0.5g / / / Absolute ethanol 3ml 3ml 3ml 3ml 3ml 3ml Water for Injection To 100ml To 100ml To 100ml To 100ml To 100ml To 100ml To 100ml To 100ml To 100ml
  • cobstatin A4 stearate, DSPE-PEG2000 and different kinds of phospholipids were weighed and dissolved in 3 ml of absolute ethanol to obtain an organic phase; the formulation amount of water for injection was weighed into an aqueous phase; The mixture was poured into an aqueous phase at 65 ° C under stirring, and then filtered and sterilized to obtain a cobstatin A 4 stearate nanoformulation solution, which was designated as a nanoparticle solution.
  • the cobstatin A4 stearate nano preparation with smaller particle size, uniform distribution and smooth filtration can be prepared under different drug loadings and different phospholipid types.
  • the obtained nanoparticles have a non-uniform particle size distribution, which causes damage to the nanoparticles, resulting in failure to smoothly filter and filter. Therefore, the preparation of the cobstatin A4 stearate nano-formulation is necessary by the injection stirring method, otherwise the drug-forming problem will not be solved.
  • Example 4 The role of DSPE-PEG2000 in combretastatin A4 stearate nanoformulation
  • Prescription 1 Prescription 2
  • Prescription 3 Prescription 4
  • Prescription 5 Cobstatin A4 stearate 0.3g 0.3g 0.3g 0.3g 0.3g Hydrogenated soybean phospholipid 0.3g 0.3g 0.3g 0.3g 0.3g DSPE-PEG2000 0g 0.05g 0.1g 0.5g 1.0g Absolute ethanol 3ml 3ml 3ml 3ml 3ml Water for Injection To 100ml To 100ml To 100ml To 100ml To 100ml To 100ml To 100ml To 100ml To 100ml To 100ml
  • cobstatin A4 stearate, hydrogenated soybean phospholipid, DSPE-PEG2000 was weighed and dissolved in 3 ml of absolute ethanol to obtain an organic phase; the formulation amount of water for injection was weighed as an aqueous phase; The mixture was poured into an aqueous phase at 65 ° C under stirring, and then filtered and sterilized to obtain a cobstatin A 4 stearate nanoformulation solution.
  • DSPE-PEG2000 has a great influence on the quality of the cobstatin A4 stearate nano preparation.
  • the prepared nanoparticles have a white emulsion appearance, a large particle size, a wide distribution, and even precipitation, and it is impossible to carry out sterilization filtration, which is for the sterile preparation.
  • the nano-formulation has been fundamentally improved. Good nano-particles can be obtained in the range of 0.05-1.0%.
  • the particle size is obviously reduced. However, when the dosage is 1%, the particle size distribution becomes wider, and more small particles appear.
  • the particle size distribution is more uniform in the range of 0.1-0.5%. Therefore, the effect of DSPE-PEG2000 on the quality of the cobstatin A4 stearate nanoformulation is fundamental, so the dosage is controlled in the range of 0.05-1.0%, preferably 0.1-0.5%.
  • the nanoparticle of the invention may also be a freeze-dried powder injection, so it is particularly important in the freeze-drying protection section, otherwise it may cause an increase in particle size or a drug. Leakage problem.
  • Prescription 1 Prescription 2
  • Prescription 3 Prescription 4
  • Prescription 5 Cobstatin A4 stearate 0.3g 0.3g 0.3g 0.3g 0.3g Hydrogenated soybean phospholipid 0.3g 0.3g 0.3g 0.3g 0.3g DSPE-PEG2000 0.3g 0.3g 0.3g 0.3g 0.3g Absolute ethanol 3ml 3ml 3ml 3ml Trehalose 10g / / / / sucrose / 10g / / / lactose / / 10g / / Dextran 40 / / / 10g / Mannitol / / / / 10g Water for Injection To 100ml To 100ml To 100ml To 100ml To 100ml To 100ml To 100ml To 100ml To 100ml To 100ml
  • the formula amount of cobstatin A4 stearate, hydrogenated soybean phospholipid, DSPE-PEG2000 was weighed and dissolved in 3 ml of absolute ethanol to obtain an organic phase; the formulation amount of the freeze-dried protective agent was weighed, and the formulation amount of water for injection was placed. Medium, stirring and dissolving to obtain an aqueous phase; the organic phase is poured into an aqueous phase at 65 ° C with stirring, and mixed, and the pH is adjusted to about 4.5 with citric acid; The solution was sterilized by 0.22 ⁇ m; sub-packaged, lyophilized, and sealed, that is, the cobstatin A4 stearate nano-preparation lyophilized powder needle was obtained.
  • the cobstatin A4 stearate nanopreparation lyophilized powder needle containing different kinds of lyoprotectants was reconstituted into about 3 mg/ml with water for injection, and the appearance of the powder and the complex solution were observed respectively, and the particles of the complex solution were measured.
  • the path is as follows:
  • Trehalose has a significant effect on the lyophilization of the cobstatin A4 stearate nanoformulation.
  • the particle size changes little before and after lyophilization, and the particle size distribution is uniform, followed by sucrose, and other commonly used protective agents against cobstatin.
  • the A4 stearate nanoformulation has little effect, so trehalose is preferred. It has been proved by a large number of experiments that trehalose is more effective in combination with other lyoprotectants, especially in combination with mannitol.
  • the dosage is better in the range of 5-15%.
  • the formula amount of scopolstatin A4 stearate 0.3 g, hydrogenated soybean phospholipid 0.1 g, 0.2 g DSPE-PEG2000 was weighed and dissolved in 2 ml of absolute ethanol to obtain an organic phase; trehalose 5 g and mannitol 5 g were weighed.
  • Example 17 Preparation of combretastatin A4 stearate nanoformulation
  • cobstatin A4 stearate 0.5 g of egg yolk phospholipid, 0.5 g of DSPE-PEG2000, dissolved in 3 ml of absolute ethanol to obtain an organic phase; weighed 4 g of sucrose, 1 g of lactose, 10 g of mannitol, to 75 ml Injecting water, stirring and dissolving, heating to 50 ° C, to obtain an aqueous phase; the organic phase is poured into the aqueous phase under stirring, and mixed, and the volume is adjusted to 100 ml by using water; the pH is adjusted to 3.50 with hydrochloric acid and disodium hydrogen phosphate. Filtration with 1.2 ⁇ m filter membrane, 0.22 ⁇ m filter membrane sterilization; sub-packaging, lyophilization, sealing, that is, the cobstatin A4 stearate nano-preparation freeze-dried powder needle.
  • cobstatin A4 stearate 0.05 g of dipalmitoylphosphatidylcholine, 0.05 g of phosphatidylserine, 0.05 g of DSPE-PEG2000, dissolved in 1 ml of absolute ethanol to obtain an organic phase; 8g of sugar, 2.0g of sorbitol, 2g of dextran, 8g of mannitol, to 75ml of water for injection, stirred and dissolved, heated to 70 ° C to obtain an aqueous phase; the organic phase is poured into the aqueous phase under stirring, mixed, and water for injection Make up to 100ml; adjust the pH value to 3.00 with hydrochloric acid; filter with 0.45 ⁇ m filter, 0.22 ⁇ m filter; remove, freeze-dry, seal, then get the broth statin A4 stearate nano-preparation lyophilized powder needle.
  • cobstatin A4 stearate 1.0 g of hydrogenated soybean phospholipid, 0.5 g of DSPE-PEG2000, add 3 ml of propylene glycol, 2 ml of absolute ethanol to dissolve, and obtain an organic phase; measure 90 ml for injection and heat to 50 ° C
  • the aqueous phase is obtained; the organic phase is poured into the aqueous phase under stirring, mixed, and the volume is adjusted to 100 ml with water for injection; the pH is adjusted to 4.30 with citric acid; filtered with a 0.45 ⁇ m filter and sterilized with a 0.22 ⁇ m filter. ; Packing, sealing, that is, cobstatin A4 stearate nano preparation injection.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Engineering & Computer Science (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Emergency Medicine (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Preparation (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

本发明属医药技术领域，具体涉及一种考布他汀A4衍生物制剂，其中该衍生物是在取代反应条件和缚酸剂存在的条件下由考布他汀A4与硬脂酰氯发生反应得到，其结构式如（Ｉ）。本发明还提供了一种该衍生物的纳米制剂。该制剂具有提高药物抗肿瘤效果、增加药物稳定性、降低药物毒副作用等诸多优点。

Description

一种考布他汀A4衍生物及其制剂 技术领域
本发明涉及医药技术领域，具体涉及一种考布他汀A4衍生物及其制剂。
背景技术
考布他汀A4(Combretastatin A4，简称CA4，结构式如式A所示)是从南非灌木柳树皮中分离得到的天然强效小分子抗有丝分裂剂，其作用位点与秋水仙碱相同，能够抑制微管蛋白的聚合，同时能够抑制细胞中微管蛋白的解聚和有丝分裂物的累积(尚海，等.微管蛋白抑制剂的研究进展[J].药学学报,20l0,45(9):1078-1088)。1991年G.R.Pettit等测定了它的化学结构并申请了美国专利(US4,996,237)。一系列的构效关系研究表明，考布他汀A4通过利用肿瘤组织与正常组织内皮细胞的生理差异，可有选择性地抑制肿瘤微管蛋白的结合，改变其内皮细胞的骨架结构与形态，增强其血管渗透性，扰乱血流，从而引起肿瘤血管内皮细胞凋亡，导致次级肿瘤细胞死亡。考布他汀A4是一种天然血管靶向药物，具有极高的抗肿瘤活性，对乳腺癌、结肠癌、甲状腺髓样癌、非小细胞肺癌、淋巴癌等都有效，具有巨大的临床应用前景。
Figure PCTCN2016101907-appb-000001
由于考布他汀A4水溶性差，难以经血管给药(许勤龙，等.微管蛋白抑制剂Combretastatin A-4类似物的研究进展[J].中国新药杂志,2013,22(3):301-318)，极大地阻碍了临床药物制剂的开发与应用。直至20世纪90年代中期，人们将考布他汀A4与磷酸进行酯化成盐反应，得到考布他汀A4磷酸二钠(Combretastatin A4磷酸盐，简称：CA4P)(结构式如式B所示)，极易溶于水，属考布他汀A4的水溶性前药，在体内经非特异性磷酸酶迅速水解成考布他汀A4，故在不影响原药构效关系的前提下，CA4P很好的解决了考布他汀A4水溶性差的问题，为考布他汀A4的研究与应用奠定了坚实的基础。CA4P目前由OXIGENE公司进行开发，已进行到临床II/III期(李洪雪，等.考布他丁A-4磷酸盐前药：CA4P[J].中国新药与临床杂志,2010,29(11):816-821)。
Figure PCTCN2016101907-appb-000002
近些年，由于CA4P具有优越的抗肿瘤活性，故人们对其安全性、有效性以及化学稳定性方面的研究逐渐深入，从多个角度对其进行全面的评价，发现CA4P亦存在诸多的不足，有待进一步予以解决，主要表现在如下几个方面：
安全性方面：①2003年，英国进行了CA4P的Ⅰ期临床试验，选择不同肿瘤患者，通过静脉注射单一剂量的CA4P，多数病人的最大耐受剂量仅为52-68mg/m2，而小鼠最大耐受剂量高达1000mg/kg，故Ⅰ期临床试验发现CA4P对人类产生的毒副作用要远远大于动物模型。因此，认为仍需进一步研究CA4P的作用机制并降低其毒副作用(沈媛媛，刘成霞.Combretastatin A4及类似物在大肠肿瘤治疗中的研究进展[J].滨州医学院学报，2010,33(1):57-59)；②CA4P对心血管具有一定的毒副作用，易引发高血压、心动过速、心动过缓、心房纤颤和心肌梗塞等反应，以及出现肿瘤部位疼痛、呕吐、头痛和疲劳等一系列不适表现，致使临床用药的顺从性较差(SubbiahlM.et al.Cardiovascular toxicity profiles of vascular disrupting agents[J].Oncologist,2011,16(8):1120-1130)；③CA4P可改变正常器官的血流量，如脑、心脏、脾、肾等，心脏和脑部位的血流改变甚至会危及到患者的生命安全(Murata,R.,J.et al.Comparative effects of combretastatin A-4disodium phosphate and5,6-dimethylxanthenone-4-acetic acid on blood perfusion in a murine tumour and normal tissues[J].International journal of radiation biology.2001,77:195-204)。
有效性方面：①CA4P体内消除速率快，体积分布广，在一定程度上削弱了其应有的抗肿瘤效果(Li Ma,et al.Targeted treatment of choroidal neovascularization using integrin-mediated sterically stabilized liposomes loaded with combretastatin A4[J].Journal of ocular pharmacology and therapeutics,2009,25(3):195-200；张小兰,王志强,等.Combretastatin A4及其磷酸酯二钠在大鼠体内的药代动力学比较研究[J].中国临床药理学与治疗学,2013，18(11):1205-1210)；②CA4P对肿瘤的增殖抑制作用只限于肿瘤内部，肿瘤边缘较窄区域内的肿瘤细胞继续增殖，致使单用CA4P停药后，肿瘤易复发，故往往需引入一些常规的细胞毒类抗肿瘤化疗药物与其联合使用，给临床用药带来了一定的安全性风险(任萱，林莉萍，等.Combretastatin A4的抗肿瘤作用研究进展[J].中国新药杂志,2007,16(17):1336-1339)。
化学稳定性方面：①CA4P对光极不稳定，易发生顺反异构化，生成无效的反式异构体(莫毅，等.Combretastatin A4phosphate溶液光解动力学研究[J].中国药学杂志,2007,42(7):516-519)；②CA4P水溶液比固体粉末更不稳定，易分解生成考布他汀A4而析出沉淀，并变色。
为了更好的发挥CA4P独特的抗肿瘤活性，针对CA4P所存在的缺陷，我们考虑将其制备成纳米制剂，其优点：①将药物包裹在纳米粒中，特别是长循环纳米粒，具有明显的 缓控释作用，大大减缓了药物在体内的消除速率，体积分布更广，使其有足够的时间将药物靶向在肿瘤部位，减少在正常组织中的分布，提高了临床用药的安全性；②纳米粒具有显著的肿瘤靶向性，在保证有效性的基础上进一步提高疗效；③药物通过包裹后，避免药物与水、光的直接接触，可提高药物的化学稳定性。纳米制剂的上述优点正是CA4P迫切需要达到的理想效果，故对CA4P而言，将其开发成纳米制剂将是正确的研究方向。
我们知道，一般纳米粒不适用于水溶性药物的包裹，存在包封率低、药物易渗漏在水相中的缺点，而恰恰CA4P属水溶性药物，故不适合将CA4P制备成纳米制剂。但CA4P是考布他汀A4的前药，真正在体内发挥抗肿瘤作用的是考布他汀A4，而考布他汀A4则不溶于水，如果能将考布他汀A4制备成纳米制剂，则抗肿瘤性将更加直接，无需经体内磷酸酶水解CA4P释放考布他汀A4才能起效的过程。不巧的是，考布他汀A4根本无法制备成符合静脉注射要求的纳米制剂。文献(Li Ma,et al.Targeted treatment of choroidal neovascularization using integrin-mediated sterically stabilized liposomes loaded with combretastatin A4[J].Journal of ocular pharmacology and therapeutics,2009,25(3):195-200)报道将考布他汀A4制成脂质体，其包封率很差，只有73.6％。文献(Wenbing Dai,et al.Spatiotemporally Controlled Co-delivery of Anti-vasculatureAgent and Cytotoxic Drug by Octreotide-Modified Stealth Liposomes[J].Pharmaceutical research,2012,29:2902-2911)报道将考布他汀A4与阿霉素共同包进脂质体中，但考布他汀A4的包封率也只有70％～80％，并且发现考布他汀A4释放很快。陈柔等研制的考布他汀A4壳聚糖基多功能纳米粒包封率也只有62.8％(陈柔,杨金荣,等.壳聚糖基多功能纳米药物载体的体外研究[J].高等学校化学学报，2012,33(7):1586-1590)。因此，将考布他汀A4制成纳米制剂普遍存在包封率低都低于80％、药物释放快等问题。而中国药典规定微囊、微球、脂质体药物包封率不得低于80％(中国药典2部2010版附录XIX E微囊、微球与脂质体制剂指导原则)。
我们结合文献报道，并根据实际情况也对考布他汀A4纳米制剂进行了大量的研究。结果显示，无论选用什么样的处方组成与用量，均不能达到预期的效果，所制备的纳米制剂甚至在几分钟内就有考布他汀A4析出，根本看不出能够直接将考布他汀A4开发成纳米制剂的任何可能性。分析原因，可能是考布他汀A4的脂溶性较差，导致和脂质材料的相容性不匹配所致。为此，我们拟通过结构改造来提高考布他汀A4的脂溶性，即用脂肪酸与考布他汀A4结构中唯一的一个羟基进行酯化，得到脂溶性较好的考布他汀A4脂肪酸酯，从而将其制备成纳米制剂，使其成为考布他汀A4的脂溶性前药。
通过文献检索，国外也有将脂肪酸与考布他汀A4结构中的羟基进行酯化，得到考布他汀A4脂溶性前药的报道。文献(N.S.Sitnikov et al，Antitumor liposomes bearing a prodrug of  combretastatin A_4and a tetrasaccharide ligand of selectins[J].Russian Chemical Bulletin,International Edition,2010,59(12):2290-2296)与(E.V.Moiseeva et al，Liposome formulations of Combretastatin A4and its 4-arylcoumarin analogue prodrugs:the antitumor effect in the mouse model of breast cancer[J].Biochemistry(Moscow)Supplement Series B:Biomedical Chemistry,2011,5(3):276-283)合成了考布他汀A4棕榈酸酯与考布他汀A4油酸酯，但并没有进行系统深入的研究。另外，油酸本身存在双键，导致其易氧化，不稳定(王炜,张伟敏.单不饱和脂肪酸的功能特性[J].中国食物与营养,2005,4:44-46)，从而不利于制剂储存。在文献(N.S.Sitnikov et al，Antitumor liposomes bearing a prodrug of combretastatin A_4and a tetrasaccharide ligand of selectins[J].Russian Chemical Bulletin,International Edition,2010,59(12):2290-2296)中关于考布他汀A4棕榈酸酯脂质体只是有提到，并没有深入研究，而且所采用的配方工艺与本发明中的配方工艺都不同。国际专利(WO 2007059118A1)，该专利选取C2～21烷基、取代的烷基、烃基、取代的烃基、芳环、芳杂环、芳烷基、取代的芳环、取代的芳杂环、取代的芳烷基等各种不同的取代基将考布他汀A4的酚羟基酯化修饰得到各种脂溶性衍生物，该专利包含了上百个衍生化合物，但均未明确记载相应实质性效果。欧洲专利(EP2896412A1)也有提到对难溶性药物的修饰得到脂溶性衍生物，其中包括考布他汀A4，该专利通过用饱和或者不饱和脂肪链对上述难溶性药物进行修饰，但该专利的主要目的是将各种药物修饰后的衍生物涂布在或被包裹在由聚合物材料构成的植入支架的表面或内部，是一种局部植入制剂，用于治疗人体生理通道狭窄，与本发明所产生的实质效果完全不同。由此可见，我们拟通过脂肪酸与考布他汀A4结构中的羟基进行酯化反应的实施方案虽然已有公开，但已公开的文献报道中未对其进行差异化研究，均未体现出任何真正的、实质性的效果。
我们拟设计的考布他汀A4脂肪酸酯，主要目的是解决纳米制剂成药性的问题，故我们随即成功合成了C2～C18不同碳链长短的考布他汀A4脂肪酸酯。结果显示，只有考布他汀A4棕榈酸酯(C16)与考布他汀A4硬脂酸酯(C18)呈白色粉末状，其余均呈蜡状或油状液体(见实施例1)。通常油状化合物中的杂质以及有机溶剂不易去除，不利于质量控制，而固体粉末则可以通过重结晶等方式除去杂质，较易得到高质量的化合物，安全性可得到保障。故以考布他汀A4硬脂酸酯为模型药物，拟初步建立考布他汀A4脂肪酸酯纳米制剂的制备方法，从而对系列不同碳链长短的考布他汀A4脂肪酸酯进行进一步的评价。但在纳米制剂制备的过程中发现，采用常规的处方工艺根本无法得到符合要求的纳米制剂，出现粒径较大、粒径分布不均匀、浑浊、药物析出等一系列问题。一般来说，纳米制剂包裹脂溶性药物，通过若干次的处方工艺的调整基本能够得到良好的制剂，但对考布他汀A4脂肪 酸酯来讲，完全超出了我们的预料。通常注射用纳米制剂的制备方法有三种，即熔融高压均质法、薄膜高压均质法与注入高压均质法，采用这三种方法无论选用什么样的处方组成与配比，均无法得到理想的载药纳米制剂(见实施例2)。但在一次偶然的试验中发现，不经过高压均质环节，而单用乙醇注入搅拌法反而收到了意向不到的效果，得到的纳米制剂呈半透明状，粒径在100nm左右，分布非常均匀(见实施例3)。采用该制备方法，对其他系列不同碳链长短的考布他汀A4脂肪酸酯进行了验证，基本上均能得到良好的纳米制剂(见实施例1)。
从纳米制剂成药性角度来看，C2～C18不同碳链长短的考布他汀A4脂肪酸酯基本均能制备出良好的纳米制剂，未见有本质的区别。为此，我们在相同的制剂类型下(纳米制剂)对其进行体内药效学的平行对比，结果让人意想不到的是，只有考布他汀A4硬脂酸酯的抗肿瘤效果最为突出，抑瘤率高达90％以上，而其他的考布他汀A4脂肪酸酯与原研药(CA4P)的抑瘤率均维持在60％左右。在相同的制剂类型下(纳米制剂)，为什么单单考布他汀A4硬脂酸酯会体现出如此显著的抗肿瘤效果，我们目前还不得而知，纯属意外发现，相关机理性研究我们正在开展中。
综上所述，我们起初只是拟开发一种考布他汀A4的纳米制剂，利用纳米制剂固有的优势来解决原研药(CA4P)存在不足。为了保证纳米制剂的成药性，我们制备了系列不同碳链长短的脂溶性考布他汀A4脂肪酸酯。事实上，考布他汀A4脂肪酸酯的确能很好地解决了考布他汀A4难以直接制备成纳米制剂的缺陷，但惊奇的是脂肪酸碳链在C2～C18的范围内只有硬脂酸(C18)酯表现出了非凡的抗肿瘤效果，故意外发现具有实质性效果的考布他汀A4硬脂酸酯是本发明的核心技术特征。另外，根据考布他汀A4硬脂酸酯特有的理化性质，成功开发一种考布他汀A4的纳米制剂。本发明真正意义上提供了一种安全、高效、质量稳定的考布他汀A4硬脂酸酯及其纳米制剂注射剂，为我国对考布他汀A4的研究与应用奠定了坚实的基础。
发明内容
本发明的目的在于提供一种安全、高效、质量稳定的考布他汀A4硬脂酸酯及其纳米制剂。
本发明提供的一种考布他汀A4硬脂酸酯，是将考布他汀A4化学结构中的酚羟基与硬脂酰氯反应而得到的。考布他汀A4硬脂酸酯的脂肪酸碳链为C18，这恰恰包含在国际专利(WO 2007059118A1)用C2-C21烷基取代基与考布他汀A4的酚羟基进行酯化反应的保护范围之内，但该专利并未进行差异化研究，也不得而知其现实意义所在。我们通过多次动物体内药效平行对比试验，惊奇的发现只有考布他汀A4硬脂酸酯的抗肿瘤效果最为突出， 抑瘤率高达90％以上，而其他C2-C21的考布他汀A4脂肪酸酯与原研药(CA4P)的抑瘤率均维持在60％左右，这种意料之外的结果目前还不得而知其内在的机理，我们仍在研究之中(见实施例1)。从诸多体内药效结果来看，考布他汀A4硬脂酸酯与其他脂肪酸酯有着实质性的优势，真正意义上挖掘出了考布他汀A4与脂肪酸成酯方案的实际价值，故意外发现具有实质性效果的考布他汀A4硬脂酸酯是本发明的核心技术特征。
本发明提供一种考布他汀A4硬脂酸酯纳米制剂注射剂，是将考布他汀A4硬脂酸酯通过特需的处方工艺将其包裹在脂质材料中制备而成。在制备过程中发现，采用常规的处方工艺根本无法得到符合要求的纳米制剂，出现粒径较大、粒径分布不均匀、浑浊、药物析出等一系列问题。一般来说，纳米制剂包裹脂溶性药物，通过若干次的处方工艺的调整基本能够得到良好的制剂，但对考布他汀A4脂肪酸酯来讲，完全超出了我们的预料。通常注射用纳米制剂的制备方法有三种，即熔融高压均质法、薄膜高压均质法与乙醇或乙醚注入高压均质法，采用这三种方法无论选用什么样的处方组成与配比，均无法得到理想的载药纳米制剂(见实施例2)。但在一次偶然的试验中发现，不经过高压均质环节，而单用乙醇注入搅拌法反而收到了意向不到的效果，得到的纳米制剂呈半透明状，粒径在100nm左右，分布非常均匀。同时对其他系列不同碳链长短的考布他汀A4脂肪酸酯进行了验证，基本上均能得到良好的纳米制剂。对考布他汀A4脂肪酸酯而言，采用注入搅拌法能够制备出良好的纳米制剂，纯属预料之外。分析原因，可能是考布他汀A4脂肪酸酯性质比较特殊，该制备方法对考布他汀A4脂肪酸酯而言，属匹配性工艺(见实施例3)。本发明的另一发明点是，结合考布他汀A4硬脂酸酯固有的特性，对制剂辅料用量进行了系统性的匹配性研究，真正意义上制备了一种安全、高效、质量稳定的考布他汀A4硬脂酸酯及其纳米制剂注射剂(见实施例4-5)。
本发明的第一方面，提供了一种考布他汀A4衍生物制剂，所述的考布他汀A4衍生物是考布他汀A4饱和脂肪酸酯，是一种纳米制剂。
较优的，所述的考布他汀A4衍生物是考布他汀A4硬脂酸酯，是通过硬脂酰氯与考布他汀A4进行酯化反应获得，其结构式如下所示；
Figure PCTCN2016101907-appb-000003
本发明的第二方面，提供了一种考布他汀A4硬脂酸酯，其合成步骤如下：
Figure PCTCN2016101907-appb-000004
(A)在反应容器中加入考布他汀A4，用适量的有机溶剂溶解，然后加入相当于1.2-1.5当量的考布他汀A4的缚酸剂，在冰浴条件下搅拌5～30分钟；
(B)冰浴条件下，在上述反应液中缓慢滴加相当于1.2-1.5当量的考布他汀A4的硬脂酰氯，室温下反应；
(D)反应结束后去除溶剂，得到考布他汀A4硬脂酸酯粗品；
(D)将考布他汀A4硬脂酸酯粗品重结晶，得考布他汀A4硬脂酸酯。
所述的步骤(A)中的有机溶剂选自无水三氯甲烷、无水二氯甲烷、N,N-二甲基乙酰胺、无水N,N-二甲基甲酰胺中的一种或一种以上；优选二氯甲烷。
所述的步骤(A)中缚酸剂选自N,N-二异丙基乙胺、三乙胺、4-二甲氨基吡啶、吡啶中的一种或一种以上；优选N,N-二异丙基乙胺。
本发明的第三方面，提供一种考布他汀A4硬脂酸酯纳米制剂，为注射剂，所述的注射剂是注射液或注射用冻干粉针剂；所述的注射剂由考布他汀A4硬脂酸酯、磷脂组成；较优的，由考布他汀A4硬脂酸酯、磷脂、DSPE-PEG2000组成。
所述的考布他汀A4硬脂酸酯纳米制剂由下列配方配制而成(以下百分比是指相对于注射液总体积而言的)：
Figure PCTCN2016101907-appb-000005
较优的，所述的考布他汀A4硬脂酸酯纳米制剂由下列配方配制而成(以下百分比是指 相对于注射液总体积而言的)：
Figure PCTCN2016101907-appb-000006
较优的，所述的考布他汀A4硬脂酸酯纳米制剂由下列配方配制而成(以下百分比是指相对于注射液总体积而言的)：
Figure PCTCN2016101907-appb-000007
其中，上述配方中的磷脂选自氢化大豆磷脂、氢化蛋黄磷脂、二棕榈酰磷脂酰胆碱、磷脂酰胆碱、蛋黄磷脂、大豆磷脂、磷脂酰丝氨酸、二肉豆蔻酰磷脂酰胆碱、二硬脂酰磷脂酰胆碱、磷脂酰乙醇胺、鞘磷脂中的一种或一种以上；优选氢化大豆磷脂、蛋黄磷脂。
上述配方中的pH调节剂选自枸橼酸、盐酸、磷酸、磷酸氢二钠、磷酸二氢钠、磷酸氢二钾、磷酸二氢钾、枸橼酸二钠、枸橼酸三钠、氢氧化钠中的一种或一种以上；调节pH值至3-7，优选调节pH值至3.5-6；更优选调节pH值至4-6。
上述配方中的冻干保护剂选自海藻糖、蔗糖、乳糖、甘露醇、右旋糖酐40、木糖醇、山梨醇中的一种或一种以上；优选海藻糖、蔗糖、甘露醇。
本发明的第四方面，提供一种考布他汀A4硬脂酸酯纳米制剂的制备方法，包括如下步骤：
称取配方量的考布他汀A4硬脂酸酯、磷脂、DSPE-PEG2000、胆固醇，加适量有机溶媒使溶解，得有机相；称取配方量的冻干保护剂，置于适量注射用水中，搅拌溶解，加热至一定的温度，得水相；将有机相在搅拌条件下注入水相中，混匀，注射用水定容；用pH调节剂调节pH值；过滤、除菌；分装、封口，即得考布他汀A4硬脂酸酯纳米制剂注射液； 或者冻干制备成考布他汀A4硬脂酸酯纳米制剂冻干粉针。
其中，所述的有机溶媒选自无水乙醇、叔丁醇、丙二醇中的一种或一种以上，用量为总体积的1-10％；优选无水乙醇、丙二醇，用量优选为总体积的2-6％。所述的一定温度的水相，其温度是40-80℃，优选50-70℃。
本发明的第五方面，提供上述考布他汀A4衍生物制剂在制备抗肿瘤药物中的应用。
进一步地，本发明提供了一种抗肿瘤药物制剂，是以考布他汀A4硬脂酸酯作为唯一活性成份。
较优的，所述的抗肿瘤药物制剂为纳米制剂，可选用以上任一配方。
本发明优点在于：
(1)考布他汀A4硬脂酸酯显著地提高了考布他汀A4的抗肿瘤效果。通过动物体内药效平行对比试验得出，在众多考布他汀A4脂肪酸酯以及原研药(CA4P)中，考布他汀A4硬脂酸酯的抗肿瘤效果最为突出，抑瘤率高达90％以上。不仅解决了考布他汀A4脂溶性差的问题，同时显著地提高了疗效。
(2)硬脂酸属饱和脂肪酸，化学稳定性良好，故对考布他汀A4硬脂酸酯的稳定性提供了保障。
(3)本发明的考布他汀A4硬脂酸酯纳米制剂注射剂，属纳米包裹制剂，有明显的缓控释作用，大大减缓了药物在体内的消除速率，靶向肿瘤部位的药物浓度将更高。
(4)本发明的考布他汀A4硬脂酸酯纳米制剂注射剂，是将药物包裹在脂质材料中，故避免药物与水、光的直接接触，进一步提高了药物的化学稳定性。
附图说明
图1.考布他汀A4乙酸酯、己酸酯、癸酸酯、肉豆蔻酸酯、硬脂酸酯纳米制剂抗小鼠S180实体瘤的药效学比较结果；
图2.考布他汀A4丁酸酯、辛酸酯、月桂酸酯、棕榈酸酯、硬脂酸酯纳米制剂抗小鼠S180实体瘤的药效学比较结果。
具体实施方式
下面结合实施例对本发明提供的具体实施方式作详细说明。
实施例1：对系列不同考布他汀A4脂肪酸酯进行体内外的对比评价
国际专利(WO 2007059118A1)，所记载的脂肪酸碳链长短在C2～C21的范围内，基本上涵盖了所有常见的脂肪酸，但未明确记载相应的实质性效果。为此，在平行对比试验中，我们在该范围内随机设计合成10个不同脂肪酸碳链的考布他汀A4酯，即考布他汀A4乙酸酯、丁酸酯、己酸酯、辛酸酯、癸酸酯、月桂酸酯、肉豆蔻酸酯、棕榈酸酯、硬脂酸酯、 油酸酯，其中包括饱和脂肪酸与不饱和脂肪酸的考布他汀A4酯，对其进行体内外对比评价。
(一)系列不同考布他汀A4脂肪酸酯的制备
1、考布他汀A4乙酸酯的制备
在反应容器中加250mg的考布他汀A4，加入10ml无水三氯甲烷溶解，再加入122.56μl的N,N-二异丙基乙胺，在冰浴条件下搅拌5分钟，缓慢滴加74.44μl的乙酰氯，冰浴条件下继续反应10分钟，再在室温下反应3小时，纯化反应物，得到淡黄色油状液体考布他汀A4乙酸酯278.64mg，收率98.35％。
该物质的1HNMR和MS(ESI)数据如下：
1H NMR(600MHz,CDCl3):δ7.10(dd,1H,J＝8.5,2.0Hz,Ar-H),6.97(d,1H,J＝2.5Hz,Ar-H),6.83(d,1H,J＝8.6Hz,Ar-H),6.51(s,2H,Ar-H),6.46(d,1H,J＝12.28Hz,C＝CH),6.44(d,1H,J＝12.28Hz,CH＝C),3.84(s,3H,OCH3),3.77(s,3H,OCH3),3.68(s,6H,2×OCH3),0.87(s,3H,J＝8.6Hz,CH3).
MS(ESI-)m/z:359(M+H+)
化学结构式如下：
Figure PCTCN2016101907-appb-000008
2、考布他汀A4丁酸酯的制备
在反应容器中加250mg的考布他汀A4，加入20ml无水二氯甲烷溶解，再加入95.96μl的三乙胺，在冰浴条件下搅拌15分钟，缓慢滴加101.15μl的丁酰氯，冰浴条件下继续反应15分钟，再在室温下反应2小时，纯化反应物，得到淡黄色油状液体考布他汀A4丁酸酯301.50mg，收率98.73％。
该物质的1HNMR和MS(ESI)数据如下：
1H NMR(600MHz,CDCl3):δ7.09(dd,1H,J＝8.6,2.2Hz,Ar-H),6.99(d,1H,J＝2.5Hz,Ar-H),6.81(d,1H,J＝8.6Hz,Ar-H),6.49(s,2H,Ar-H),6.45(d,1H,J＝12.28Hz,C＝CH),6.43(d,1H,J＝12.28Hz,CH＝C),3.82(s,3H,OCH3),3.79(s,3H,OCH3),3.69(s,6H,2×OCH3),1.70-1.74(m,2H,CH2),1.30-1.36(m,2H,CH2),0.87(t,3H,J＝8.6Hz,CH3).
MS(ESI-)m/z:404(M+NH4 +)
化学结构式如下：
Figure PCTCN2016101907-appb-000009
3、考布他汀A4己酸酯的制备
在反应容器中加250mg的考布他汀A4，加入20ml无水二氯甲烷溶解，再加入95.96μl的三乙胺，在冰浴条件下搅拌15分钟，缓慢滴加127.65μl的己酰氯，冰浴条件下继续反应15分钟，再在室温下反应4小时，纯化反应物，得到淡黄色油状液体考布他汀A4己酸酯320.60mg，收率97.88％。
该物质的1HNMR和MS(ESI)数据如下：
1H NMR(600MHz,CDCl3):δ7.12(dd,1H,J＝8.5,2.2Hz,Ar-H),6.98(d,1H,J＝2.4Hz,Ar-H),6.83(d,1H,J＝8.5Hz,Ar-H),6.49(s,2H,Ar-H),6.45(d,1H,J＝12.30Hz,C＝CH),6.43(d,1H,J＝12.30Hz,CH＝C),3.82(s,3H,OCH3),3.79(s,3H,OCH3),3.69(s,6H,2×OCH3),2.01-1.87(m,2H,CH2),1.05-1.46(m,6H,3×CH2),0.87(t,3H,J＝8.6Hz,CH3).
MS(ESI-)m/z:432(M+NH4 +)
化学结构式如下：
Figure PCTCN2016101907-appb-000010
4、考布他汀A4辛酸酯的制备
在反应容器中加250mg的考布他汀A4，加入30ml的无水二氯甲烷与N,N-二甲基乙酰胺的混合溶剂溶解，加入的122.56μlN,N-二异丙基乙胺，在冰浴条件下搅拌20分钟，缓慢滴加154.33μl的辛酰氯，冰浴条件下继续反应30分钟，再在室温下反应10小时。纯化反应物，得到淡黄色油状液体考布他汀A4辛酸酯332.78mg，收率97.74％。
该物质的1HNMR和MS(ESI)数据如下：
1H NMR(600MHz,CDCl3):δ7.15(dd,1H,J＝8.5,2.0Hz,Ar-H),7.01(d,1H,J＝2.5Hz,Ar-H),6.89(d,1H,J＝8.6Hz,Ar-H),6.55(s,2H,Ar-H),6.51(d,1H,J＝12.30Hz,C＝CH),6.43(d,1H,J＝12.30Hz,CH＝C),3.82(s,3H,OCH3),3.79(s,3H,OCH3),3.69(s,6H,2×OCH3),1.69-1.74(m,2H,CH2),1.26-1.30(m,10H,5×CH2),0.87(t,3H,J＝8.6Hz,CH3).
MS(ESI-)m/z:423(M+H+)
化学结构式如下：
Figure PCTCN2016101907-appb-000011
5、考布他汀A4癸酸酯的制备
在反应容器中加250mg的考布他汀A4，加入20ml无水N,N-二甲基甲酰胺溶解，再加入365.69mg的4-二甲氨基吡啶，在冰浴条件下搅拌15分钟，缓慢滴加226.07μl癸酰氯，冰浴条件下反应20分钟，再在室温下反应6小时，纯化反应物，得到淡黄色油状液体考布他汀A4癸酸酯358.74mg，收率99.42％。
该物质的1HNMR和MS(ESI)数据如下：
1H NMR(600MHz,CDCl3):δ7.07(dd,1H,J＝8.3,2.2Hz,Ar-H),6.90(d,1H,J＝2.6Hz,Ar-H),6.83(d,1H,J＝8.6Hz,Ar-H),6.46(s,2H,Ar-H),6.40(d,1H,J＝12.29Hz,C＝CH),6.38(d,1H,J＝12.29Hz,CH＝C),3.82(s,3H,OCH3),3.80(s,3H,OCH3),3.72(s,6H,2×OCH3),1.65-1.76(m,2H,CH2),1.26-1.30(m,14H,7×CH2),0.87(t,3H,J＝8.6Hz,CH3).
MS(ESI-)m/z:471(M+H+)
化学结构式如下：
Figure PCTCN2016101907-appb-000012
6、考布他汀A4月桂酸酯的制备
在反应容器中加250mg的考布他汀A4，加入15ml无水二氯甲烷溶解，再加入93.73μl的吡啶，在冰浴条件下搅拌20分钟，缓慢滴加207.48μl的月桂酰氯，冰浴条件下继续反应30分钟，再在室温下反应6小时，纯化反应物，得到淡黄色考布他汀A4月桂酸酯378.62mg，收率98.70％。
该物质的1HNMR和MS(ESI)数据如下：
1H NMR(600MHz,CDCl3):δ7.10(dd,1H,J＝8.5,2.0Hz,Ar-H),6.99(d,1H,J＝2.5Hz,Ar-H),6.83(d,1H,J＝8.6Hz,Ar-H),6.49(s,2H,Ar-H),6.45(d,1H,J＝12.30Hz,C＝CH),6.43(d,1H,J＝12.30Hz,CH＝C),3.82(s,3H,OCH3),3.79(s,3H,OCH3),3.69(s,6H,2×OCH3),1.69-1.74(m,2H,CH2),1.26-1.30(m,18H,9×CH2),0.87(t,3H,J＝8.6Hz,CH3).
MS(ESI-)m/z:499(M+H+)
化学结构式如下：
Figure PCTCN2016101907-appb-000013
7、考布他汀A4肉豆蔻酸酯的制备
在反应容器中加250mg的考布他汀A4，加入30ml的无水二氯甲烷与N,N-二甲基乙酰胺的混合溶剂溶解，加入的122.56μl N,N-二异丙基乙胺，在冰浴条件下搅拌20分钟，缓慢滴加234.06μl的肉豆蔻酰氯，冰浴条件下继续反应30分钟，再在室温下反应12小时。反应物在石油醚中重结晶，分离得到白色蜡状固体考布他汀A4肉豆蔻酸酯398.72mg，收率98.41％。
该物质的1HNMR和MS(ESI)数据如下：
1H NMR(600MHz,CDCl3):δ7.10(dd,1H,J＝8.5,2.1Hz,Ar-H),6.97(d,1H,J＝2.5Hz,Ar-H),6.83(d,1H,J＝8.6Hz,Ar-H),6.49(s,2H,Ar-H),6.45(d,1H,J＝12.30Hz,C＝CH),6.43(d,1H,J＝12.30Hz,CH＝C),3.82(s,3H,OCH3),3.79(s,3H,OCH3),3.69(s,6H,2×OCH3),1.69-1.74(m,2H,CH2),1.26-1.30(m,22H,11×CH2),0.87(t,3H,J＝8.6Hz,CH3).
MS(ESI-)m/z:527(M+H+)
化学结构式如下：
Figure PCTCN2016101907-appb-000014
8、考布他汀A4棕榈酸酯的制备
在反应容器中加500mg的考布他汀A4，加入15ml无水二氯甲烷溶解，再加入187.47μl的吡啶，在冰浴条件下搅拌20分钟，缓慢滴加651.66μl的棕榈酰氯，冰浴条件下继续反应30分钟，再在室温下反应12小时，反应物在石油醚中重结晶，分离得到白色粉状固体考布他汀A4棕榈酸酯854.64mg，收率99.31％。
该物质的1HNMR和MS(ESI)数据如下：
1H NMR(600MHz,CDCl3):δ7.14(dd,1H,J＝8.5,2.0Hz,Ar-H),6.95(d,1H,J＝2.5Hz,Ar-H),6.83(d,1H,J＝8.6Hz,Ar-H),6.47(s,2H,Ar-H),6.43(d,1H,J＝12.30Hz,C＝CH),6.40 (d,1H,J＝12.30Hz,CH＝C),3.82(s,3H,OCH3),3.79(s,3H,OCH3),3.69(s,6H,2×OCH3),1.60-1.75(m,2H,CH2),1.32-1.40(m,26H,13×CH2),0.88(t,3H,J＝8.6Hz,CH3).
MS(ESI-)m/z:555(M+H+)
化学结构式如下：
Figure PCTCN2016101907-appb-000015
9、考布他汀A4硬脂酸酯的制备
在反应容器中加500mg的考布他汀A4，加入20ml无水二氯甲烷溶解，再加入306.41μl的N,N-二异丙基乙胺，在冰浴条件下搅拌20分钟，缓慢滴加718.17μl的硬脂酰氯，冰浴条件下继续反应15分钟，再在室温下反应3小时，反应物在环己烷中重结晶，分离得到白色粉状固体考布他汀A4硬脂酸酯880.98mg，收率97.96％。
该物质的1HNMR和MS(ESI)数据如下：
1H NMR(600MHz,CDCl3):δ7.10(dd,1H,J＝8.6,2.0Hz,Ar-H),6.99(d,1H,J＝2.4Hz,Ar-H),6.83(d,1H,J＝8.6Hz,Ar-H),6.49(s,2H,Ar-H),6.45(d,1H,J＝12.29Hz,C＝CH),6.43(d,1H,J＝12.30Hz,CH＝C),3.82(s,3H,OCH3),3.79(s,3H,OCH3),3.69(s,6H,2×OCH3),1.69-1.74(m,2H,CH2),1.26-1.30(m,30H,15×CH2),0.87(t,3H,J＝8.6Hz,CH3)
MS(ESI-)m/z:583(M+H+)
化学结构式如下：
Figure PCTCN2016101907-appb-000016
10、考布他汀A4油酸酯的制备
在反应容器中加250mg的考布他汀A4，加入20ml无水二氯甲烷溶解，再加入122.56μl的N,N-二异丙基乙胺，在冰浴条件下搅拌20分钟，缓慢滴加356.70μl的油酸酰氯，冰浴条件下继续反应15分钟，再在室温下反应10小时，纯化反应物，得到淡黄色油状液体考布他汀A4油酸酯778.43mg，收率98.50％。
该物质的1HNMR数据如下：
1H NMR(600MHz,CDCl3):δ7.17(dd,1H,J＝8.6,2.2Hz,Ar-H),7.03(d,1H,J＝2.2Hz, Ar-H),6.98(d,1H,J＝8.6Hz,Ar-H),6.75(s,2H,Ar-H),6.46(d,1H,J＝12.28Hz,C＝CH),6.44(d,1H,J＝12.28Hz,CH＝C),5.56-5.87(m,2H,CH＝CH),3.83(s,3H,OCH3),3.71(s,3H,OCH3),3.68(s,6H,2×OCH3),1.84-1.96(m,6H,3×CH2),1.33-1.52(m,18H,9×CH2),0.87(s,3H,J＝8.6Hz,CH3).
化学结构式如下：
Figure PCTCN2016101907-appb-000017
(二)系列不同考布他汀A4脂肪酸酯的性状对比
将上述合成的不同脂肪酸考布他汀A4酯置于室温下观察其性状，结果如下表所示：
化合物名称 碳链长短 脂肪酸饱和度 性状
考布他汀A4乙酸酯 C2 饱和脂肪酸 淡黄色油状液体
考布他汀A4丁酸酯 C4 饱和脂肪酸 淡黄色油状液体
考布他汀A4己酸酯 C6 饱和脂肪酸 淡黄色油状液体
考布他汀A4辛酸酯 C8 饱和脂肪酸 淡黄色油状液体
考布他汀A4癸酸酯 C10 饱和脂肪酸 淡黄色油状液体
考布他汀A4月桂酸酯 C12 饱和脂肪酸 淡黄色油状液体
考布他汀A4肉豆蔻酸酯 C14 饱和脂肪酸 白色蜡状固体
考布他汀A4棕榈酸酯 C16 饱和脂肪酸 白色固体粉末
考布他汀A4硬脂酸酯 C18 饱和脂肪酸 白色固体粉末
考布他汀A4油酸酯 C18 不饱和脂肪酸 淡黄色油状液体
结果分析：
试验表明，只有考布他汀A4棕榈酸酯(C16)与考布他汀A4硬脂酸酯(C18)呈白色粉末状，其余均呈蜡状或油状液体。通常油状化合物中的杂质以及有机溶剂不易去除，不利于质量控制，而固体粉末则可以通过重结晶等方式除去杂质，较易得到高质量的化合物，安全性可得到保障。
(三)系列不同考布他汀A4脂肪酸酯纳米制剂的成药性对比
1、纳米制剂的制备方法
在相同的处方工艺条件下，制备不同考布他汀A4脂肪酸酯的纳米制剂。
称取考布他汀A4脂肪酸酯适量(按考布他汀A4计约300mg)、氢化大豆磷脂(HSPC)0.3g、 DSPE-PEG2000 0.3g，用2mL无水乙醇使溶解，得有机相；称取海藻糖3g、甘露醇7g，置于适量注射用水中，搅拌溶解，得水相；将有机相在搅拌条件下注入加热至65℃的水相中，混匀，用注射用水定容至100ml。用枸橼酸或氢氧化钠调节pH值至5.0左右；然后将料液过0.22μm除菌；分装、冻干、封口，即得考布他汀A4脂肪酸酯纳米制剂。
2、纳米制剂的成药性对比
取上述系列考布他汀A4脂肪酸酯纳米制剂，分别用注射用水复溶，使成约3mg/ml(按考布他汀A4计)。在室温下考察复溶液的外观、平均粒径，并在室温环境下放置5小时后观察复溶液的状态，结果如下表所示：
名称 外观 平均粒径 5小时放置外观
考布他汀A4乙酸酯纳米制剂 呈半透明乳浊液 90.53nm 未见明显变化
考布他汀A4丁酸酯纳米制剂 呈半透明乳浊液 110.3nm 未见明显变化
考布他汀A4己酸酯纳米制剂 呈半透明乳浊液 91.4nm 未见明显变化
考布他汀A4辛酸酯纳米制剂 呈半透明乳浊液 89.2nm 未见明显变化
考布他汀A4癸酸酯纳米制剂 呈半透明乳浊液 96.3nm 未见明显变化
考布他汀A4月桂酸酯纳米制剂 呈半透明乳浊液 110.7nm 未见明显变化
考布他汀A4肉豆蔻酸酯纳米制剂 呈半透明乳浊液 97.6nm 未见明显变化
考布他汀A4棕榈酸酯纳米制剂 呈半透明乳浊液 100.6nm 未见明显变化
考布他汀A4硬脂酸酯纳米制剂 呈半透明乳浊液 98.5nm 未见明显变化
考布他汀A4油酸酯纳米制剂 呈半透明乳浊液 103.1nm 呈浑浊的乳浊液
结果分析：
试验表明，所设计的10个考布他汀A4脂肪酸酯，均能将其成功制备成纳米制剂，且复溶后呈半透明乳浊液，粒径基本上分布在100nm左右。室温下放置5小时时，发现只有考布他汀A4油酸酯的纳米溶液由起初的半透明乳浊液变为浑浊的乳浊液，粒径明显增大，可能是油酸属不饱和脂肪酸的原因所致。其他9个脂肪酸酯则稳定性良好，故选用考布他汀A4饱和脂肪酸酯时成药性可能会更大些，也体现了本发明的优越性。故在接下来的体内药效评价时只对剩余的9个饱和脂肪酸酯进行比较。
(四)系列不同考布他汀A4脂肪酸酯对S180荷瘤小鼠肿瘤抑制作用的平行对比
取上述制备的考布他汀A4脂肪酸酯纳米制剂，在相同的制剂类型条件下(纳米制剂)分别对拟设计的9个考布他汀A4脂肪酸酯进行动物体内药效学的对比评价，同时与原研药考布他汀A4磷酸二钠注射剂进行比较。试验方案与结果如下所示：
1、小鼠S180肿瘤模型的建立与给药方案的设计
在温度为37℃与5％CO2环境下，用含有10％血清、1％双抗的DMEM中培养S180细胞。当细胞培养至对数生长期时，活性较好，收集细胞，并用不含血清的DMEM稀释细胞浓度为1×107个/ml，得肿瘤细胞悬液。每只小鼠以腹腔注射的形式接种0.2ml细胞悬液，共接种6只昆明小鼠。接种8天后，抽取腹水，传代至第三代。抽取第三代腹水细胞，用生理盐水稀释到浓度为1×106个/ml，得腹水细胞稀释液。取腹水细胞稀释液0.2ml，分别注射在昆明小鼠的右前肢皮下，得小鼠S180肿瘤模型。
分别设定生理盐水组、原研药考布他汀A4磷酸二钠盐溶液阳性对照组、考布他汀A4脂肪酸酯纳米制剂供试组，每组8只S180肿瘤模型小鼠。其中原研药是将考布他汀A4磷酸二钠原料药直接溶于注射用水中使成3mg/ml(按考布他汀A4计)，作为阳性对照制剂；取实施例1第(三)项下制备的系列考布他汀A4脂肪酸酯冻干纳米制剂，用注射用水溶解成3mg/ml(按考布他汀A4计)，作为供试制剂。采用小鼠尾静脉注射方式给药，剂量为每次15mg/kg(按考布他汀A4计)，每天给药一次，连续给药6天，停药后隔天处死小鼠，剥取肿瘤并称重，计算抑瘤率。由于设计的考布他汀A4脂肪酸酯纳米制剂样品数多达9个，故按上述试验方案，将其分为两次试验予以完成，分别记作对比试验一与对比试验二。
抑瘤率＝(生理盐水组瘤重-给药组瘤重)/生理盐水组瘤重×100％
2、对比试验一抗肿瘤效果
对小鼠S180实体瘤的药效学比较结果如下表所示，肿瘤照片见说明书附图中的图1。
组别 瘤重(g) 抑瘤率
生理盐水 2.47±0.52 /
考布他汀A4磷酸二钠盐溶液 0.98±0.41 60.32％
考布他汀A4乙酸酯纳米制剂 0.91±0.35 63.16％
考布他汀A4己酸酯纳米制剂 0.82±0.29 66.80％
考布他汀A4癸酸酯纳米制剂 0.88±0.31 64.37％
考布他汀A4肉豆蔻酸酯纳米制剂 0.84±0.42 65.99％
考布他汀A4硬脂酸酯纳米制剂 0.22±0.19 91.09％
结果分析：
在相同的剂型以及载药量情况下，对考布他汀A4乙酸酯、己酸酯、癸酸酯、肉豆蔻酸酯、硬脂酸酯纳米制剂的抗肿瘤效果进行比较。意外的发现，只有考布他汀A4硬脂酸酯纳米制剂的抗肿瘤效果最为突出，抑瘤率高达91.09％，而其他的考布他汀A4脂肪酸酯与原研药(CA4P)的抑瘤率均维持在60％左右，故考布他汀A4硬脂酸酯如此显著的抗肿瘤效果是本发明的核心技术特征。
3、对比试验二抗肿瘤效果
对小鼠S180实体瘤的药效学比较结果如下表所示，肿瘤照片见说明书附图中的图2。
组别 瘤重(g) 抑瘤率
生理盐水 1.98±0.66 /
考布他汀A4磷酸二钠盐溶液 0.68±0.33 65.66％
考布他汀A4丁酸酯纳米制剂 0.71±0.21 64.14％
考布他汀A4辛酸酯纳米制剂 0.75±0.31 62.12％
考布他汀A4月桂酸酯纳米制剂 0.78±0.39 60.61％
考布他汀A4棕榈酸酯纳米制剂 0.63±0.19 68.18％
考布他汀A4硬脂酸酯纳米制剂 0.14±0.11 92.93％
结果分析：
在相同的剂型以及载药量情况下，对考布他汀A4丁酸酯、辛酸酯、月桂酸酯、棕榈酸酯、硬脂酸酯纳米制剂的抗肿瘤效果进行比较。结果显示，依然只有考布他汀A4硬脂酸酯纳米制剂的抗肿瘤效果最好，抑瘤率高达92.93％，而其他的考布他汀A4脂肪酸酯与原研药(CA4P)的抑瘤率依然维持在60％左右，进一步说明考布他汀A4硬脂酸酯的抗肿瘤活性与与其他脂肪酸酯相比，具有实质性的不同。
终上所述，将考布他汀A4制备成系列考布他汀A4脂肪酸酯后，确实能够解决考布他汀A4脂溶性差的问题，且饱和的考布他汀A4脂肪酸酯所制备的纳米制剂将更加稳定；在国际专利(WO 2007059118A1)所记载的用C2-C21烷基取代基与考布他汀A4的酚羟基进行酯化反应的范围内，通过同剂型、同载药量、同剂量下对小鼠体内抗肿瘤性进行平行比较，惊奇的发现只有考布他汀A4硬脂酸酯的抗肿瘤活性最为显著，抑瘤率高达90％以上，基本上接近治愈的水平。另外，考布他汀A4硬脂酸酯呈白色固体粉末，质量更易控制。发现考布他汀A4硬脂酸酯独特的抗肿瘤效果，真正意义上挖掘出了考布他汀A4与脂肪酸成酯方案的实际价值，是构成本发明的核心技术特征。
实施例2：考布他汀A4硬脂酸酯纳米制剂注射剂制备工艺的对比研究
通常注射用纳米制剂的制备方法有三种，即熔融高压均质法、薄膜高压均质法与注入高压均质法，故以0.5mg/ml最保守的载药量分别对这三种制备方法进行可行性研究。
(一)熔融高压均质法
熔融高压均质法，是将药物或药物与脂质材料混合，在一定温度下使其呈熔融或半熔融状态，在表面活性剂作用下将其乳化在水中，然后通过高压均质机进一步降低粒径，得到纳米制剂。根据其原理，我们用该方法以0.5mg/ml最保守的载药量对考布他汀A4硬脂 酸酯纳米制剂进行制备，并进行质量评价。
1、熔融高压均质法制备考布他汀A4硬脂酸酯纳米制剂
称取考布他汀A4硬脂酸酯0.05g，加热熔融，将熔融液加入在70℃的含蛋黄磷脂0.5g、0.5g DSPE-PEG2000的注射用水，剪切乳化，用注射用水定容至100ml，得初乳；将初乳置于高压均质机中，在20000psi压力下高压均质3遍，即得考布他汀A4硬脂酸酯纳米制剂。
2、质量评价
外观 呈乳浊液状
粒径 311.2nm
粒径分布值(PDI) 0.428
0.22μm滤膜过滤通透性 几乎无法进行除菌过滤
结果分析：
采用熔融高压均质法，所制备考布他汀A4硬脂酸酯纳米制剂，粒径及分布值(PDI)均很大；粒径为311.2nm，分布极不均匀，更重要的是无法进行除菌过滤，况且载药量则仅为0.5mg/ml。为此，我们又对不同脂质材料的配比做了相应的研究，得到的结果基本类似，根本无法看出采用熔融高压均质法制备考布他汀A4硬脂酸酯纳米制剂的任何可能性。
(二)薄膜高压均质法
薄膜高压均质法，是将药物与脂质材料共同溶解在易挥发的有机溶剂中，在真空下将有机溶剂去除，形成脂质薄膜，加水水化，得到纳米粒粗品溶液；将纳米粒粗品溶液通过高压均质机降低粒径，得到纳米制剂。根据其原理，我们用该方法同样以0.5mg/ml最保守的载药量对考布他汀A4硬脂酸酯纳米制剂进行制备，并进行质量评价。
1、薄膜高压均质法制备考布他汀A4硬脂酸酯纳米制剂
称取考布他汀A4硬脂酸酯0.05g、蛋黄磷脂0.5g、0.5g DSPE-PEG2000，用5ml氯仿将其溶解，在40℃下减压去除氯仿，形成脂质薄膜，加水水化，用注射用水定容至100ml，得到纳米粒粗品溶液；将纳米粒粗品溶液置于高压均质机中，在20000psi压力下高压均质3遍，即得考布他汀A4硬脂酸酯纳米制剂。
2、质量评价
外观 呈乳浊液状
粒径 227.1nm
粒径分布值(PDI) 0.401
0.22μm滤膜过滤通透性 阻力相当大，过滤量极少
结果分析：
采用薄膜高压均质法，载药量仅为0.5mg/ml时，所制备考布他汀A4硬脂酸酯纳米制剂，虽然较熔融高压均质法有所改观，但粒径及分布值(PDI)依然很大，粒径为227.1nm，分布极不均匀，更重要的是无法进行除菌过滤。同样，我们也对不同脂质材料的配比也做了相应的研究，也未见有实质性的改善，故认为薄膜高压均质法根本不适用于考布他汀A4硬脂酸酯纳米制剂的制备。
(三)注入高压均质法
注入高压均质法，是将药物与脂质材料共同溶解在水溶性的有机溶剂中，形成脂质溶液；将脂质溶液注入在水中，形成纳米粒粗品溶液；将纳米粒粗品溶液通过高压均质机降低粒径，得到纳米制剂。根据其原理，我们用该方法同样以0.5mg/ml最保守的载药量对考布他汀A4硬脂酸酯纳米制剂进行制备，并进行质量评价。
1、注入高压均质法制备考布他汀A4硬脂酸酯纳米制剂
称取考布他汀A4硬脂酸酯0.05g、蛋黄磷脂0.5g、0.5g DSPE-PEG2000，用3ml无水乙醇将其溶解，得到脂质溶液；将脂质溶液注入在65℃的注射用水中，用注射用水定容至100ml，形成纳米粒粗品溶液；将纳米粒粗品溶液置于高压均质机中，在20000psi压力下高压均质3遍，即得考布他汀A4硬脂酸酯纳米制剂。
2、质量评价
外观 呈乳浊状溶液
粒径 151.6nm
粒径分布值(PDI) 0.422
0.22μm滤膜过滤通透性 能过滤，但阻力较大
结果分析：
采用注入高压均质法，载药量仅为0.5mg/ml时，所制备考布他汀A4硬脂酸酯纳米制剂，较熔融高压均质法与薄膜高压均质法有所明显改观，粒径降至200nm内，但分布依然不均匀，粒径有大有小，依然不能顺利完成除菌过滤。同样，我们也对不同脂质材料的配比也做了相应的研究，最终也未得到粒径均匀、相对稳定的纳米制剂，故认为注入高压均质法也不适用于考布他汀A4硬脂酸酯纳米制剂的制备。
综上所述，常用的纳米制剂制备方法，对考布他汀A4硬脂酸酯而言，均不能得到符合静脉注射要求的纳米制剂，这可能与考布他汀A4硬脂酸酯的特殊理化性质有关。故寻求一种与其相匹配的制备方法至关重要，将直接影响考布他汀A4硬脂酸酯的应用前景。
实施例3：考布他汀A4硬脂酸酯纳米制剂注射剂制备工艺的研究
大量试验表明，常用的纳米制剂制备方法，不适用于考布他汀A4硬脂酸酯纳米制剂 的制备。但相比而言，注入高压均质法制备的纳米粒相对最好。为了究其原因，我们反复对注入高压均质法进行分解性试验，意外地发现，在脂质溶液注入注射用水的瞬间就已经形成了相对较好的纳米制剂，而再通过常规的思路将其进行高压均质反而使纳米粒分布不均匀，甚至对纳米粒造成破坏。关于该结论，我们进行了多次对比试验予以了证实，现举例予以说明，如下所示：
1、用不同制备方法制备考布他汀A4硬脂酸酯纳米制剂
1.1处方
组分 处方1 处方2 处方3 处方4 处方5
考布他汀A4硬脂酸酯 0.05g 0.3g 0.3g 0.3g 0.3g
DSPE-PEG2000 0.5g 0.5g 0.5g 0.5g 0.5g
氢化大豆磷脂 / / 0.5g / /
二棕榈酰磷脂酰胆碱 / / / 0.5g /
磷脂酰胆碱 / / / / 0.5g
蛋黄磷脂 0.5g 0.5g / / /
无水乙醇 3ml 3ml 3ml 3ml 3ml
注射用水 至100ml 至100ml 至100ml 至100ml 至100ml
1.2样品制备
分别称取处方量的考布他汀A4硬脂酸酯、DSPE-PEG2000以及不同种类的磷脂，用3ml无水乙醇溶解，得有机相；称取配方量的注射用水，为水相；将有机相在搅拌条件下注入65℃的水相中，然后过滤、除菌，即得考布他汀A4硬脂酸酯纳米制剂溶液，记作纳米粒溶液。
取适量上述考布他汀A4硬脂酸酯纳米制剂溶液，将其置于高压均质机中，在20000psi压力下高压均质3遍，即得考布他汀A4硬脂酸酯纳米制剂均质溶液，记作纳米粒均质溶液。
2、制备工艺对纳米粒质量的影响
Figure PCTCN2016101907-appb-000018
Figure PCTCN2016101907-appb-000019
结果分析：
采用注入搅拌法，在不同载药量、不同磷脂种类下，均能制备出粒径较小、分布均匀、过滤顺畅的考布他汀A4硬脂酸酯纳米制剂。而结合高压均质后，所得到的纳米粒反而粒径分布不均匀，对纳米粒造成破坏，导致无法顺利除菌过滤。故采用注入搅拌法对考布他汀A4硬脂酸酯纳米制剂的制备是必要条件，否则将无法解决成药性问题。
实施例4：DSPE-PEG2000在考布他汀A4硬脂酸酯纳米制剂中的作用
在同样的处方及制备工艺下，对DSPE-PEG2000在考布他汀A4硬脂酸酯纳米制剂中的作用进行对比研究。
1、处方
组分 处方1 处方2 处方3 处方4 处方5
考布他汀A4硬脂酸酯 0.3g 0.3g 0.3g 0.3g 0.3g
氢化大豆磷脂 0.3g 0.3g 0.3g 0.3g 0.3g
DSPE-PEG2000 0g 0.05g 0.1g 0.5g 1.0g
无水乙醇 3ml 3ml 3ml 3ml 3ml
注射用水 至100ml 至100ml 至100ml 至100ml 至100ml
2、样品制备
分别称取处方量的考布他汀A4硬脂酸酯、氢化大豆磷脂、DSPE-PEG2000，用3ml无水乙醇溶解，得有机相；称取配方量的注射用水，为水相；将有机相在搅拌条件下注入65℃的水相中，然后过滤、除菌，即得考布他汀A4硬脂酸酯纳米制剂溶液。
3、不同DSPE-PEG2000用量对考布他汀A4硬脂酸酯纳米制剂的影响
组别 外观 粒径 粒径分布值(PDI) 0.22μm滤膜过滤顺畅性
处方1 白色乳浊状溶液 357.8nm 0.538 无法过滤，出现沉淀
处方2 半透明均一溶液 133.5nm 0.235 过滤较顺利，但略有阻力
处方3 半透明均一溶液 111.2nm 0.112 过滤极顺畅
处方4 半透明均一溶液 91.7nm 0.108 过滤极其顺畅
处方5 半透明均一溶液 63.9nm 0.292 过滤极其顺畅
结果分析：
在同样的处方及制备工艺下，DSPE-PEG2000对考布他汀A4硬脂酸酯纳米制剂的质量影响较大。首先，在不含DSPE-PEG2000时，所制备的纳米粒外观呈白色乳浊液，粒径很大，且分布很宽，甚至出现沉淀，更无法进行除菌过滤，这对无菌制剂来讲尤为重要；其次，添加DSPE-PEG2000后，纳米制剂得到了根本性的改观。用量在0.05-1.0％的范围内均能得到良好的纳米粒，随着用量的增加，粒径明显减少，但用量在1％时粒径分布反而变宽，出现较多的小粒子；当用量在0.1-0.5％的范围内粒径分布更加均一。故DSPE-PEG2000对考布他汀A4硬脂酸酯纳米制剂的质量影响是根本性的，故将用量控制在0.05-1.0％的范围内，优选用量为0.1-0.5％。
实施例5：海藻糖作为冻干保护剂的重要性研究
为了进一步保证考布他汀A4硬脂酸酯纳米制剂的稳定性，本发明的纳米粒也可以是一种冻干粉针剂，故在冻干保护环节尤为重要，否则会造成粒径增大或药物渗漏的问题。为此，我们结合考布他汀A4硬脂酸酯纳米制剂的固有特性，进行了大量保护剂匹配性研究，以保证冻干后粉针剂的质量。
1、处方
组分 处方1 处方2 处方3 处方4 处方5
考布他汀A4硬脂酸酯 0.3g 0.3g 0.3g 0.3g 0.3g
氢化大豆磷脂 0.3g 0.3g 0.3g 0.3g 0.3g
DSPE-PEG2000 0.3g 0.3g 0.3g 0.3g 0.3g
无水乙醇 3ml 3ml 3ml 3ml 3ml
海藻糖 10g / / / /
蔗糖 / 10g / / /
乳糖 / / 10g / /
右旋糖酐40 / / / 10g /
甘露醇 / / / / 10g
注射用水 至100ml 至100ml 至100ml 至100ml 至100ml
2、样品制备
称取配方量的考布他汀A4硬脂酸酯、氢化大豆磷脂、DSPE-PEG2000，用3ml无水乙醇溶解，得有机相；称取配方量的冻干保护剂，置于配方量的注射用水中，搅拌溶解，得水相；搅拌下将有机相注入65℃的水相中，混匀，用枸橼酸调节pH值至4.5左右；然后将 料液过0.22μm除菌；分装、冻干、封口，即得考布他汀A4硬脂酸酯纳米制剂冻干粉针。
3、不同冻干保护剂对考布他汀A4硬脂酸酯纳米制剂质量的影响
取含不同种类冻干保护剂的考布他汀A4硬脂酸酯纳米制剂冻干粉针，用注射用水复溶成约3mg/ml，分别观察粉体以及复溶液的外观，测定复溶液的粒径，结果如下所示：
组别 粉体外观 复溶液外观 粒径
处方1 饱满，但不细腻 半透明均一溶液 99.2nm
处方2 粉体严重萎缩 白色乳浊状均一溶液 135.3nm
处方3 饱满，但不细腻 白色乳浊状溶液，不透明 376.0nm
处方4 饱满，但不细腻 糊状乳浊液，有药物沉淀挂壁 532.4nm
处方5 粉体饱满细腻 白色乳浊状溶液，有药物沉淀挂壁 588.1nm
结果分析：
海藻糖对考布他汀A4硬脂酸酯纳米制剂的冻干保护作用尤为显著，冻干前后粒径变化不大，且粒径分布均一，蔗糖次之，其他的常用保护剂则对考布他汀A4硬脂酸酯纳米制剂作用不大，故优选海藻糖。通过大量实验验证，海藻糖与其他冻干保护剂合用则效果更佳，特别是与甘露醇合用收到了最佳的效果。用量在5-15％范围内效果更佳。
实施例6：考布他汀A4硬脂酸酯的制备
在反应容器中加500mg的考布他汀A4，加入20ml无水二氯甲烷溶解，再加入187.47μl的吡啶，在冰浴条件下搅拌30分钟，缓慢滴加718.17μl的硬脂酰氯，冰浴条件下继续反应15分钟，再在室温下反应2小时，反应物在石油醚重结晶，分离得到白色粉状固体考布他汀A4硬脂酸酯892.47mg，收率99.23％。
实施例7：考布他汀A4硬脂酸酯的制备
在反应容器中加250mg的考布他汀A4，加入20ml无水三氯甲烷溶解，再加入85.96μl的三乙胺，在冰浴条件下搅拌15分钟，缓慢滴加356.09μl的硬脂酰氯，冰浴条件下继续反应15分钟，再在室温下反应3小时，反应物在环己烷中重结晶，分离得到白色粉状固体考布他汀A4硬脂酸酯445.53mg，收率99.01％。
实施例8：考布他汀A4硬脂酸酯纳米制剂的制备
称取考布他汀A4硬脂酸酯0.3g、氢化大豆磷脂0.3g、0.3g DSPE-PEG2000，加3ml无水乙醇使溶解，得有机相；称取海藻糖3g、甘露醇7g，至85ml注射用水中，搅拌溶解，加热至65℃，得水相；将有机相在搅拌条件下注入水相中，混匀，注射用水定容至100ml；用枸橼酸调节pH值为4.53；过0.22μm滤膜除菌；分装、冻干、封口，即得考布他汀A4硬脂酸酯纳米制剂冻干粉针。
实施例9：考布他汀A4硬脂酸酯纳米制剂的制备
称取配方量的考布他汀A4硬脂酸酯0.3g、氢化大豆磷脂0.1g、0.2g DSPE-PEG2000，加2ml无水乙醇使溶解，得有机相；称取海藻糖5g、甘露醇5g，至85ml注射用水中，搅拌溶解，加热至60℃，得水相；将有机相在搅拌条件下注入水相中，混匀，注射用水定容至100ml；用盐酸调节pH值为4.12；过0.22μm滤膜除菌；分装、冻干、封口，即得考布他汀A4硬脂酸酯纳米制剂冻干粉针。
实施例10：考布他汀A4硬脂酸酯纳米制剂的制备
称取考布他汀A4硬脂酸酯0.3g、氢化大豆磷脂0.5g、0.5g DSPE-PEG2000，加4ml无水乙醇使溶解，得有机相；称取海藻糖5g、甘露醇10g，至75ml注射用水中，搅拌溶解，加热至70℃，得水相；将有机相在搅拌条件下注入水相中，混匀，注射用水定容至100ml；用枸橼酸调节pH值为3.54；过0.22μm滤膜除菌；分装、冻干、封口，即得考布他汀A4硬脂酸酯纳米制剂冻干粉针。
实施例11：考布他汀A4硬脂酸酯纳米制剂的制备
称取考布他汀A4硬脂酸酯0.5g、蛋黄磷脂1.0g、0.5g DSPE-PEG2000，加3ml无水乙醇使溶解，得有机相；称取海藻糖10g、甘露醇5g，至75ml注射用水中，搅拌溶解，加热至55℃，得水相；将有机相在搅拌条件下注入水相中，混匀，注射用水定容至100ml；用磷酸调节pH值为4.05；过0.22μm滤膜除菌；分装、冻干、封口，即得考布他汀A4硬脂酸酯纳米制剂冻干粉针。
实施例12：考布他汀A4硬脂酸酯纳米制剂的制备
称取考布他汀A4硬脂酸酯0.3g、蛋黄磷脂0.3g、0.3g DSPE-PEG2000，加3ml无水乙醇使溶解，得有机相；称取海藻糖3g、甘露醇7g，至80ml注射用水中，搅拌溶解，加热至65℃，得水相；将有机相在搅拌条件下注入水相中，混匀，注射用水定容至100ml；用枸橼酸调节pH值为4.56；过0.22μm滤膜除菌；分装、冻干、封口，即得考布他汀A4硬脂酸酯纳米制剂冻干粉针。
实施例13：考布他汀A4硬脂酸酯纳米制剂的制备
称取考布他汀A4硬脂酸酯0.1g、蛋黄磷脂0.3g、0.3g DSPE-PEG2000，加2ml无水乙醇使溶解，得有机相；称取海藻糖2g、甘露醇6g，至85ml注射用水中，搅拌溶解，加热至55℃，得水相；将有机相在搅拌条件下注入水相中，混匀，注射用水定容至100ml；用氢氧化钠调节pH值为6.00；过0.22μm滤膜除菌；分装、冻干、封口，即得考布他汀A4硬脂酸酯纳米制剂冻干粉针。
实施例14：考布他汀A4硬脂酸酯纳米制剂的制备
称取考布他汀A4硬脂酸酯0.2g、氢化大豆磷脂0.3g、0.3g DSPE-PEG2000，加3ml无水乙醇使溶解，得有机相；称取海藻糖3g、甘露醇7g，至85ml注射用水中，搅拌溶解，加热至55℃，得水相；将有机相在搅拌条件下注入水相中，混匀，注射用水定容至100ml；用氢氧化钠调节pH值为5.53；过0.22μm滤膜除菌；分装、冻干、封口，即得考布他汀A4硬脂酸酯纳米制剂冻干粉针。
实施例15：考布他汀A4硬脂酸酯纳米制剂的制备
称取考布他汀A4硬脂酸酯0.2g、氢化大豆磷脂0.3g、0.3g DSPE-PEG2000、胆固醇0.1g，加3ml无水乙醇使溶解，得有机相；称取海藻糖2g、蔗糖2g、甘露醇8g，至75ml注射用水中，搅拌溶解，加热至70℃，得水相；将有机相在搅拌条件下注入水相中，混匀，注射用水定容至100ml；用枸橼酸与枸橼酸三钠调节pH值为5.52；用0.8μm滤膜过滤、0.22μm滤膜除菌；分装、冻干、封口，即得考布他汀A4硬脂酸酯纳米制剂冻干粉针。
实施例16：考布他汀A4硬脂酸酯纳米制剂的制备
称取考布他汀A4硬脂酸酯0.5g、氢化大豆磷脂1.0g、0.7g DSPE-PEG2000，加5ml无水乙醇使溶解，得有机相；称取海藻糖6g、甘露醇9g，至75ml注射用水中，搅拌溶解，加热至65℃，得水相；将有机相在搅拌条件下注入水相中，混匀，注射用水定容至100ml；用枸橼酸与枸橼酸二钠调节pH值为4.66；用0.45μm滤膜过滤、0.22μm滤膜除菌；分装、冻干、封口，即得考布他汀A4硬脂酸酯纳米制剂冻干粉针。
实施例17：考布他汀A4硬脂酸酯纳米制剂的制备
称取考布他汀A4硬脂酸酯0.4g、蛋黄磷脂0.5g、0.5g DSPE-PEG2000，加3ml无水乙醇使溶解，得有机相；称取蔗糖4g、乳糖1g、甘露醇10g，至75ml注射用水中，搅拌溶解，加热至50℃，得水相；将有机相在搅拌条件下注入水相中，混匀，注射用水定容至100ml；用盐酸与磷酸氢二钠调节pH值为3.50；用1.2μm滤膜过滤、0.22μm滤膜除菌；分装、冻干、封口，即得考布他汀A4硬脂酸酯纳米制剂冻干粉针。
实施例18：考布他汀A4硬脂酸酯纳米制剂的制备
称取考布他汀A4硬脂酸酯0.3g、蛋黄磷脂0.3g、0.3g DSPE-PEG2000，加3ml无水乙醇使溶解，得有机相；量取注射用95ml，加热至50℃，得水相；将有机相在搅拌条件下注入水相中，混匀，注射用水定容至100ml；用磷酸二氢钠调节pH值为4.12；用0.45μm滤膜过滤、0.22μm滤膜除菌；分装、封口，即得考布他汀A4硬脂酸酯纳米制剂注射液。
实施例19：考布他汀A4硬脂酸酯纳米制剂的制备
称取考布他汀A4硬脂酸酯0.3g、氢化大豆磷脂0.3g、0.3g DSPE-PEG2000，加2ml无水乙醇溶解，得有机相；量取注射用95ml，加热至60℃，得水相；将有机相在搅拌条件 下注入水相中，混匀，注射用水定容至100ml；用磷酸氢二钠调节pH值为5.53；用0.45μm滤膜过滤、0.22μm滤膜除菌；分装、封口，即得考布他汀A4硬脂酸酯纳米制剂注射液。
实施例20：考布他汀A4硬脂酸酯纳米制剂的制备
称取考布他汀A4硬脂酸酯0.5g、磷脂酰胆碱1.0g、0.5g DSPE-PEG2000、胆固醇0.3g，加5ml无水乙醇使溶解，得有机相；量取注射用90ml，加热至70℃，得水相；将有机相在搅拌条件下注入水相中，混匀，注射用水定容至100ml；用磷酸与磷酸氢二钾调节pH值为5.76；用0.45μm滤膜过滤、0.22μm滤膜除菌；分装、封口，即得考布他汀A4硬脂酸酯纳米制剂注射液。
实施例21 考布他汀A4硬脂酸酯纳米制剂的制备
称取考布他汀A4硬脂酸酯0.1g、氢化大豆磷脂0.1g、0.1g DSPE-PEG2000，加1ml无水乙醇使溶解，得有机相；称取海藻糖3g、甘露醇7g，至85ml注射用水中，搅拌溶解，加热至65℃，得水相；将有机相在搅拌条件下注入水相中，混匀，注射用水定容至100ml；用氢氧化钠调节pH值为5.50；用0.45μm滤膜过滤、0.22μm滤膜除菌；分装、冻干、封口，即得考布他汀A4硬脂酸酯纳米制剂冻干粉针。
实施例22 考布他汀A4硬脂酸酯纳米制剂的制备
称取考布他汀A4硬脂酸酯2.0g、蛋黄磷脂3.0g、1.0g DSPE-PEG2000、胆固醇1.0g，加6ml无水乙醇使溶解，得有机相；量取注射用85ml，加热至65℃，得水相；将有机相在搅拌条件下注入水相中，混匀，注射用水定容至100ml；用磷酸氢二钾与磷酸二氢钾调节pH值为5.16；用0.45μm滤膜过滤、0.22μm滤膜除菌；分装、封口，即得考布他汀A4硬脂酸酯纳米制剂注射液。
实施例23 考布他汀A4硬脂酸酯纳米制剂的制备
称取考布他汀A4硬脂酸酯0.3g、氢化大豆磷脂0.3g、0.3g DSPE-PEG2000，加5ml叔丁醇使溶解，得有机相；称取海藻糖3g、甘露醇7g，至80ml注射用水中，搅拌溶解，加热至60℃，得水相；将有机相在搅拌条件下注入水相中，混匀，注射用水定容至100ml；用氢氧化钠调节pH值为5.50；用0.45μm滤膜过滤、0.22μm滤膜除菌；分装、冻干、封口，即得考布他汀A4硬脂酸酯纳米制剂冻干粉针。
实施例24 考布他汀A4硬脂酸酯纳米制剂的制备
称取考布他汀A4硬脂酸酯2.0g、氢化蛋黄磷脂5.0g、1.0g DSPE-PEG2000、胆固醇2g，加7ml叔丁醇、3ml无水乙醇使溶解，得有机相；称取海藻糖15g、乳糖3.0g、木糖醇2.0g、甘露醇10g，至45ml注射用水中，搅拌溶解，加热至70℃，得水相；将有机相在搅拌条件下注入水相中，混匀，注射用水定容至100ml；用氢氧化钠调节pH值为7.00；用1.2 μm滤膜过滤、0.22μm滤膜除菌；分装、冻干、封口，即得考布他汀A4硬脂酸酯纳米制剂冻干粉针。
实施例25 考布他汀A4硬脂酸酯纳米制剂的制备
称取考布他汀A4硬脂酸酯0.1g、二棕榈酰磷脂酰胆碱0.05g、磷脂酰丝氨酸0.05g、0.05g DSPE-PEG2000，加1ml无水乙醇使溶解，得有机相；称取海藻糖8g、山梨醇2.0g、右旋糖酐2g、甘露醇8g，至75ml注射用水中，搅拌溶解，加热至70℃，得水相；将有机相在搅拌条件下注入水相中，混匀，注射用水定容至100ml；用盐酸调节pH值为3.00；用0.45μm滤膜过滤、0.22μm滤膜除菌；分装、冻干、封口，即得考布他汀A4硬脂酸酯纳米制剂冻干粉针。
实施例26 考布他汀A4硬脂酸酯纳米制剂的制备
称取考布他汀A4硬脂酸酯0.3g、大豆磷脂0.1g、0.5g DSPE-PEG2000，加3ml丙二醇使溶解，得有机相；量取注射用90ml，加热至40℃，得水相；将有机相在搅拌条件下注入水相中，混匀，注射用水定容至100ml；用枸橼酸调节pH值为4.00；用0.45μm滤膜过滤、0.22μm滤膜除菌；分装、封口，即得考布他汀A4硬脂酸酯纳米制剂注射液。
实施例27 考布他汀A4硬脂酸酯纳米制剂的制备
称取考布他汀A4硬脂酸酯0.2g、二肉豆蔻酰磷脂酰胆碱0.5g、鞘磷脂0.1g、0.5g DSPE-PEG2000，加4ml无水乙醇使溶解，得有机相；称取海藻糖15g，至75ml注射用水中，搅拌溶解，加热至50℃，得水相；将有机相在搅拌条件下注入水相中，混匀，注射用水定容至100ml；用枸橼酸三钠调节pH值为6.50；用0.45μm滤膜过滤、0.22μm滤膜除菌；分装、冻干、封口，即得考布他汀A4硬脂酸酯纳米制剂冻干粉针。
实施例28 考布他汀A4硬脂酸酯纳米制剂的制备
称取考布他汀A4硬脂酸酯0.3g、蛋黄磷脂0.3g、0.3g DSPE-PEG2000，加2ml丙二醇使溶解，得有机相；称取海藻糖3g、甘露醇7g，至85ml注射用水中，搅拌溶解，加热至60℃，得水相；将有机相在搅拌条件下注入水相中，混匀，注射用水定容至100ml；用氢氧化钠调节pH值为6.00；用0.45μm滤膜过滤、0.22μm滤膜除菌；分装、冻干、封口，即得考布他汀A4硬脂酸酯冻干粉针。
实施例29 考布他汀A4硬脂酸酯纳米制剂的制备
称取考布他汀A4硬脂酸酯0.2g、蛋黄磷脂0.2g、0.5g DSPE-PEG2000，加2ml丙二醇、2ml无水乙醇使溶解，得有机相；称取海藻糖3g、甘露醇7g，至80ml注射用水中，搅拌溶解，加热至55℃，得水相；将有机相在搅拌条件下注入水相中，混匀，注射用水定容至100ml；用氢氧化钠调节pH值为5.68；用0.45μm滤膜过滤、0.22μm滤膜除菌；分装、冻干、封 口，即得考布他汀A4硬脂酸酯纳米制剂冻干粉针。
实施例30 考布他汀A4硬脂酸酯纳米制剂的制备
称取考布他汀A4硬脂酸酯1.5g、氢化大豆磷脂1.0g、0.5g DSPE-PEG2000，加3ml丙二醇、2ml无水乙醇使溶解，得有机相；量取注射用90ml，加热至50℃，得水相；将有机相在搅拌条件下注入水相中，混匀，注射用水定容至100ml；用枸橼酸调节pH值为4.30；用0.45μm滤膜过滤、0.22μm滤膜除菌；分装、封口，即得考布他汀A4硬脂酸酯纳米制剂注射液。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员，在不脱离本发明方法的前提下，还可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。

Claims (18)

  1. 一种考布他汀A4衍生物制剂，其特征在于，所述的考布他汀A4衍生物是考布他汀A4硬脂酸酯，其结构式如下所示：
    Figure PCTCN2016101907-appb-100001
  2. 根据权利要求1所述的考布他汀A4衍生物制剂，其特征在于，所述的考布他汀A4衍生物制剂是一种纳米制剂。
  3. 根据权利要求2所述的考布他汀A4衍生物制剂，其特征在于，所述的考布他汀A4硬脂酸酯是通过硬脂酰氯与考布他汀A4进行酯化反应获得。
  4. 根据权利要求2所述的考布他汀A4衍生物制剂，其特征在于，所述的考布他汀A4硬脂酸酯的合成步骤如下：
    Figure PCTCN2016101907-appb-100002
    (A)在反应容器中加入考布他汀A4，用适量的有机溶剂溶解，然后加入相当于1.2-1.5当量的考布他汀A4的缚酸剂，在冰浴条件下搅拌5～30分钟；
    (B)冰浴条件下，在上述反应液中缓慢滴加相当于1.2-1.5当量的考布他汀A4的硬脂酰氯，室温下反应；
    (C)反应结束后去除溶剂，得到考布他汀A4硬脂酸酯粗品；
    (D)将考布他汀A4硬脂酸酯粗品重结晶，得考布他汀A4硬脂酸酯。
  5. 根据权利要求1-4任一所述的考布他汀A4衍生物制剂，其特征在于，所述的考布他汀A4衍生物制剂是考布他汀A4硬脂酸酯纳米制剂，所述的纳米制剂是注射剂，所述的注射剂是注射液或注射用冻干粉针剂。
  6. 根据权利要求5所述的考布他汀A4衍生物制剂，其特征在于，所述的考布他汀A4 硬脂酸酯纳米制剂由考布他汀A4硬脂酸酯、磷脂组成。
  7. 根据权利要求5所述的考布他汀A4衍生物制剂，其特征在于，所述的考布他汀A4硬脂酸酯纳米制剂由考布他汀A4硬脂酸酯、磷脂、DSPE-PEG2000组成。
  8. 根据权利要求5所述的考布他汀A4衍生物制剂，其特征在于，所述的考布他汀A4硬脂酸酯纳米制剂由下列配方配制而成：
    Figure PCTCN2016101907-appb-100003
  9. 根据权利要求5所述的考布他汀A4衍生物制剂，其特征在于，所述的考布他汀A4硬脂酸酯纳米制剂由下列配方配制而成：
    Figure PCTCN2016101907-appb-100004
  10. 根据权利要求5所述的考布他汀A4衍生物制剂，其特征在于，所述的考布他汀A4硬脂酸酯纳米制剂由下列配方配制而成：
    Figure PCTCN2016101907-appb-100005
  11. 根据权利要求6-10任一所述的考布他汀A4衍生物制剂，其特征在于，所述的磷脂 选自氢化大豆磷脂、氢化蛋黄磷脂、二棕榈酰磷脂酰胆碱、磷脂酰胆碱、蛋黄磷脂、大豆磷脂、磷脂酰丝氨酸、二肉豆蔻酰磷脂酰胆碱、二硬脂酰磷脂酰胆碱、磷脂酰乙醇胺、鞘磷脂中的一种或一种以上。
  12. 根据权利要求8-10任一所述的考布他汀A4衍生物制剂，其特征在于，所述的pH调节剂选自枸橼酸、盐酸、磷酸、磷酸氢二钠、磷酸二氢钠、磷酸氢二钾、磷酸二氢钾、枸橼酸二钠、枸橼酸三钠、氢氧化钠中的一种或一种以上；调节pH值至3-7。
  13. 根据权利要求8-10任一所述的考布他汀A4衍生物制剂，其特征在于，所述的冻干保护剂选自海藻糖、蔗糖、乳糖、甘露醇、右旋糖酐40、木糖醇、山梨醇中的一种或一种以上。
  14. 一种如权利要求8-13任一所述的考布他汀A4硬脂酸酯纳米制剂的制备方法，其特征在于，包括如下步骤：
    称取配方量的考布他汀A4硬脂酸酯、磷脂、DSPE-PEG2000、胆固醇，加适量有机溶媒使溶解，得有机相；称取配方量的冻干保护剂，置于适量注射用水中，搅拌溶解，加热至40-80℃，得水相；将有机相在搅拌条件下注入水相中，混匀，注射用水定容；用pH调节剂调节pH值；过滤、除菌；分装、封口，即得考布他汀A4硬脂酸酯纳米制剂注射液；或者冻干制备成考布他汀A4硬脂酸酯纳米制剂冻干粉针。
  15. 根据权利要求14所述的考布他汀A4硬脂酸酯纳米制剂的制备方法，其特征在于，所述的有机溶媒选自无水乙醇、叔丁醇、丙二醇中的一种或一种以上，用量为总体积的1-10％。
  16. 根据权利要求14所述的考布他汀A4硬脂酸酯纳米制剂的制备方法，其特征在于，所述的水相温度是50-70℃。
  17. 根据权利要求1-13任一所述的考布他汀A4衍生物制剂在制备抗肿瘤药物中的应用。
  18. 一种抗肿瘤药物制剂，是以考布他汀A4硬脂酸酯作为唯一活性成份。
PCT/CN2016/101907 2015-10-14 2016-10-12 一种考布他汀a4衍生物及其制剂 WO2017063559A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201510661988.0A CN106580945B (zh) 2015-10-14 2015-10-14 一种考布他汀a4衍生物及其制剂
CN201510661988.0 2015-10-14

Publications (1)

Publication Number Publication Date
WO2017063559A1 true WO2017063559A1 (zh) 2017-04-20

Family

ID=58517098

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2016/101907 WO2017063559A1 (zh) 2015-10-14 2016-10-12 一种考布他汀a4衍生物及其制剂

Country Status (2)

Country Link
CN (1) CN106580945B (zh)
WO (1) WO2017063559A1 (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106242972B (zh) * 2016-07-29 2019-02-26 浙江大学 康普瑞汀药物前体、药物制剂和制备方法
CN109453123B (zh) * 2018-11-19 2021-05-11 中国药科大学 一种康普瑞汀类衍生物冻干粉针及其制备方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007059118A1 (en) * 2005-11-14 2007-05-24 Abraxis Bioscience, Inc. Combretastatin derivatives and related therapeutic methods
CN101327190A (zh) * 2008-07-29 2008-12-24 北京大学 一种供注射用的抗肿瘤长循环靶向脂质体

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101439182B (zh) * 2008-12-18 2012-02-01 北京大学 一种生长抑素受体介导的肿瘤靶向药物组合物

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007059118A1 (en) * 2005-11-14 2007-05-24 Abraxis Bioscience, Inc. Combretastatin derivatives and related therapeutic methods
CN101327190A (zh) * 2008-07-29 2008-12-24 北京大学 一种供注射用的抗肿瘤长循环靶向脂质体

Also Published As

Publication number Publication date
CN106580945A (zh) 2017-04-26
CN106580945B (zh) 2019-11-08

Similar Documents

Publication Publication Date Title
CN103221040B (zh) 双硫仑制剂及用途
CN103083239B (zh) 一种蟾毒灵脂质体及其制备方法和应用
MX2010011165A (es) Composiciones de derivados de taxano hidrofobos y sus usos.
PT706373E (pt) Taxol encapsulado num liposoma e um metodo
EP1539102A2 (en) Pharmaceutically active lipid based formulation of sn38
CN106946975B (zh) 一种雷公藤甲素衍生物及其制备方法与制剂
KR20150085122A (ko) 비뇨 생식기암 및 이의 전이 치료용 비소산, 이것의 나트륨염 및 이것의 유도체를 함유하는 약학 조성물
CA2779166C (en) Submicro emulsion of paclitaxel using steroid complex as intermediate carrier
US12029724B2 (en) Method for inhibiting tumor growth
WO2015172712A1 (zh) 维生素c与抗肿瘤药物协同作用的注射用药物组合物
NZ586859A (en) Drug delivery system for administration of poorly water soluble pharmaceutically active substances
Zhao et al. Reactive oxygen species‐responsive mitochondria‐targeted liposomal quercetin attenuates retinal ischemia–reperfusion injury via regulating SIRT1/FOXO3A and p38 MAPK signaling pathways
WO2017097196A1 (zh) 脂质体的制备方法
EP4382094A1 (en) Atherosclerosis-targeted liposome nanocarrier delivery system and preparation method therefor
WO2017063559A1 (zh) 一种考布他汀a4衍生物及其制剂
WO2020103175A1 (zh) 一种康普瑞汀类衍生物冻干粉针及其制备方法
CN1931157A (zh) 多烯紫杉醇脂质体及其制备方法
US20230226031A1 (en) Formulated and/or Co-Formulated Liposome Compositions Containing Immunogenic Cell Death (ICD) Inducing Prodrugs Useful In The Treatment of Cancer and Methods Thereof
CN110548006A (zh) 一种科罗索酸脂质体及其制备方法和用途
CN107412172A (zh) 一种紫杉醇白蛋白纳米混悬剂冻干粉及其制备工艺
WO2019218857A1 (zh) 一种多西他赛棕榈酸酯脂质体及其制备方法
CN116115566A (zh) 一种七叶皂苷钠脂质体及其制备方法
JP6206925B2 (ja) ホウ素クラスター修飾ｐｅｇ脂質誘導体およびこれを用いた分子集合体
CN105944120B (zh) 一种载有十二硼烷马来酰亚胺丙酰哌嗪七甲川菁硫醚盐的叶酸脂质体及其制备方法和应用
EP2433619A1 (en) Novel antitumoral agent and its therapeutic uses

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16854936

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205N DATED 20.06.2018)

122 Ep: pct application non-entry in european phase

Ref document number: 16854936

Country of ref document: EP

Kind code of ref document: A1