CN112245391A

CN112245391A - Antitumor lipid composition

Info

Publication number: CN112245391A
Application number: CN202011145564.6A
Authority: CN
Inventors: 夏桂民; 刘明亮; 王丹; 李岩
Original assignee: Institute of Medicinal Biotechnology of CAMS
Current assignee: Institute of Medicinal Biotechnology of CAMS
Priority date: 2020-10-23
Filing date: 2020-10-23
Publication date: 2021-01-22

Abstract

The present invention relates to an antitumor lipid composition. In particular, in one aspect, a lipid composition is provided comprising a compound of formula (I), a phospholipid, a pegylated phospholipid, cholesterol, and an excipient. The compound of formula (I) is shown as follows, wherein each substituent is described in the specification. Also provides a method for preparing the lipid composition, a method for improving the anti-tumor activity of an anti-tumor medicament by preparing the compound shown in the formula (I) into the lipid composition, and application of the lipid composition as the anti-tumor medicament. The method and lipid composition of the invention can effectively improve the antitumor activity of the medicament, and the method of the invention can prepareThe obtained lipid composition exhibits excellent pharmaceutical properties.

Description

Antitumor lipid composition

Technical Field

The invention belongs to the field of medical chemistry, relates to a method for improving antitumor activity of gemcitabine, and particularly relates to a group of gemcitabine carbamate compounds with antitumor activity, a preparation method thereof, and application thereof in antitumor aspect. In addition, the invention also provides a lipid composition of the gemcitabine carbamate compound and a preparation method of the lipid composition. The lipid composition of the present invention exhibits excellent technical effects

Background

Gemcitabine (gemcitabine), Chinese scholars name: 4-amino-1- (3, 3-difluoro-4-hydroxy-5-hydroxymethyltetrahydrofuran-2-yl) pyrimidin-2 (1H) -one, chemical name: 2' -deoxy-2 ',2' -difluorocytidine of the formula: c₉H₁₁F₂N₃O₄The novel fluoro nucleoside analogue has the chemical structure shown in the following formula (A):

gemcitabine has no pharmacological activity, is activated by deoxycytidine kinase after entering a human body, and is metabolized into corresponding monophosphate, diphosphate and triphosphate by cytidine deaminase to play a role. Gemcitabine is used in the treatment of various solid tumors (e.g., non-small cell lung cancer, pancreatic cancer, ovarian cancer, bladder cancer, breast cancer, etc.), and its hydrochloride is used clinically, but is currently limited to intravenous administration for the treatment of pancreatic cancer and non-small cell lung cancer. Patient compliance with this therapy is poor and there are several adverse effects, typical adverse effects include, the blood system: has bone marrow suppression effect, and can cause anemia, leukopenia and thrombocytopenia. ② gastrointestinal tract; patients of about 2/3 developed liver transaminase abnormalities, mostly mild, non-progressive lesions; nausea and vomiting reactions occur in about 1/3 patients, and 20% of patients require drug treatment. ③ Kidney: patients of about 1/2 develop mild proteinuria and hematuria, with some cases presenting renal failure of unknown cause. Fourthly, allergy: approximately 25% of patients develop a skin diagnosis, 10% of patients develop pruritus, and less than 1% of patients can develop bronchospasm. Fifthly, other: about 20% of patients have a performance similar to influenza; the incidence of edema/peripheral edema is about 30%; the incidence rates of alopecia, lethargy, diarrhea, oral toxicity and constipation were 13%, 10%, 8%, 7% and 6%, respectively. These adverse effects will seriously affect the benefit/risk ratio of clinical gemcitabine administration. In addition, it can be widely distributed in various tissues after intravenous injectionIt is rapidly metabolized by cytidine deaminase in the liver, kidney, blood and other tissues, and has a short plasma half-life (t)_1/2: 8-17min), multiple administrations are required (Gang Wang, et al.j.med.chem.2017, 60,2552; tang Li, et al, DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY, 2017, 43: 2016).

The prior art still expects new methods for treating tumors, which are expected to have some or some more excellent effects, for example, drugs having more excellent antitumor activity, such as gemcitabine derivatives, are expected to be applied to the clinic, including compositions of the gemcitabine derivatives adapted to the clinical application.

Disclosure of Invention

The object of the present invention is to provide a novel method which is expected to have some or some more excellent effects for treating tumors, for example, to provide a drug having more excellent antitumor activity for clinical use. The present inventors have surprisingly found that compounds having the structure of the present invention exhibit excellent antitumor activity, and also found that the preparation of the active compounds of the present invention into lipid compositions exhibits excellent technical effects.

To this end, the present invention provides in a first aspect the following compounds of formula (I) (which may also be referred to herein as gemcitabine carbamates, etc.),

wherein:

r represents C_10-22Saturated alkyl or unsaturated alkenyl, straight or branched, in which 1 or 2 CH's are present in the carbon chain₂Optionally replaced by O.

The compound according to the first aspect of the present invention, wherein said R is selected from: n-decyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, n-eicosyl, n-docosyl, n-hexadecyloxypropyl, n-octadecyl, n-9-ene-n-decyl, (11-ene) n-dodecyl, and (11-ene) n-dodecylethyl.

The compounds according to the first aspect of the invention may also exist in the form of solvates (e.g. hydrates), and thus such solvates (e.g. hydrates) are also included in the compounds of the invention.

A compound according to the first aspect of the invention, which is compound 1 to compound 12 selected from the group consisting of:

compound 1: n-decyl (1- (3, 3-difluoro-4-hydroxy-5-hydroxymethyltetrahydrofuran-2-yl) -2-oxo-1, 2-dihydropyrimidin-4-yl) carbamate,

compound 2: n-dodecyl (1- (3, 3-difluoro-4-hydroxy-5-hydroxymethyltetrahydrofuran-2-yl) -2-oxo-1, 2-dihydropyrimidin-4-yl) carbamate,

compound 3: n-tetradecyl (1- (3, 3-difluoro-4-hydroxy-5-hydroxymethyltetrahydrofuran-2-yl) -2-oxo-1, 2-dihydropyrimidin-4-yl) carbamate,

compound 4: n-hexadecyl (1- (3, 3-difluoro-4-hydroxy-5-hydroxymethyltetrahydrofuran-2-yl) -2-oxo-1, 2-dihydropyrimidin-4-yl) carbamate,

compound 5: n-octadecyl (1- (3, 3-difluoro-4-hydroxy-5-hydroxymethyltetrahydrofuran-2-yl) -2-oxo-1, 2-dihydropyrimidin-4-yl) carbamate,

compound 6: n-eicosyl (1- (3, 3-difluoro-4-hydroxy-5-hydroxymethyltetrahydrofuran-2-yl) -2-oxo-1, 2-dihydropyrimidin-4-yl) carbamate,

compound 7: n-docosyl (1- (3, 3-difluoro-4-hydroxy-5-hydroxymethyltetrahydrofuran-2-yl) -2-oxo-1, 2-dihydropyrimidin-4-yl) carbamate,

compound 8: n-hexadecyloxypropyl (1- (3, 3-difluoro-4-hydroxy-5-hydroxymethyltetrahydrofuran-2-yl) -2-oxo-1, 2-dihydropyrimidin-4-yl) carbamate,

compound 9: n-octadecyl oxyethyl (1- (3, 3-difluoro-4-hydroxy-5-hydroxymethyl-tetrahydrofuran-2-yl) -2-oxo-1, 2-dihydropyrimidin-4-yl) carbamate,

compound 10: (9-ene) n-decaalkyl (1- (3, 3-difluoro-4-hydroxy-5-hydroxymethyltetrahydrofuran-2-yl) -2-oxo-1, 2-dihydropyrimidin-4-yl) carbamate,

compound 11: (11-ene) n-dodecyl (1- (3, 3-difluoro-4-hydroxy-5-hydroxymethyltetrahydrofuran-2-yl) -2-oxo-1, 2-dihydropyrimidin-4-yl) carbamate,

compound 12: (11-ene) n-dodecylethyl (1- (3, 3-difluoro-4-hydroxy-5-hydroxymethyltetrahydrofuran-2-yl) -2-oxo-1, 2-dihydropyrimidin-4-yl) carbamate.

Further, the second aspect of the present invention provides a process for preparing the compound of formula (I), which is shown in the following reaction scheme 1.

Scheme 1:

in this scheme 1, R is as defined herein.

Specifically, the method of the present invention comprises the steps of:

1) dissolving a compound of formula (II) (obtained from a domestic commercial route) in a non-polar solvent (e.g., dichloromethane, chloroform, tetrahydrofuran, dioxane), and reacting with 1-2 equivalents of a compound of formula (III) (obtained from a domestic commercial route) in the presence of 1.5-3 equivalents of an organic base (e.g., triethylamine, N-lutidine, pyridine, 4-dimethylaminopyridine) at 0-40 deg.C for 3-10 hours with stirring to obtain a compound of formula (IV);

2) dissolving a compound of formula (IV) in a protic solvent (e.g., water, an alcohol or an alcohol-water mixed solvent, such as methanol; e.g., 10mmol of compound IV' is dissolved in 40 to 60mL, e.g., 50mL, of protic solvent), 0.1 to 2 equivalents of inorganic base (e.g., 0.1 to 0.5 equivalents; e.g., sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate), at 0 deg.C to room temperature for 0.5 to 3 hours with stirring to provide the compound of formula (I).

The process according to any one of the second aspect of the invention, wherein in step 2), ethyl acetate is also added with methanol and calcium chloride is also added with NaOH. For example, 10mL of ethyl acetate was also added with 50mL of methanol, and 0.25g of calcium chloride was also added with 0.1g of NaOH solid.

Further, in a third aspect, the present invention provides the use of a compound according to any one of the first aspect of the present invention or a compound prepared by the method according to any one of the second aspect of the present invention in the manufacture of a medicament for the treatment of a tumour.

The use according to the third aspect of the invention, wherein the tumour is a solid tumour.

The use according to the third aspect of the invention, wherein the tumour is a tumour selected from: non-small cell lung cancer, pancreatic cancer, ovarian cancer, bladder cancer, breast cancer, and liver cancer.

Further, the fourth aspect of the present invention provides a lipid composition comprising:

API (compound of formula (I) according to any one of the first aspect of the invention): 100 parts by weight of a water-soluble polymer,

phospholipid: 100 to 2000 parts by weight (for example, 200 to 1500 parts by weight),

pegylated phospholipids: 30 to 300 parts by weight (for example, 50 to 200 parts by weight),

cholesterol: 20 to 200 parts by weight (e.g., 50 to 150 parts by weight), and

and (3) an excipient.

The lipid composition according to the fourth aspect of the invention is a composition in a liquid state (e.g. is a lipid suspension), wherein the excipient is an aqueous vehicle. For example, it is selected from: water, 0.8-1% sodium chloride solution (e.g., 0.9% sodium chloride solution), 2-10% glucose solution (e.g., 5% glucose solution). For example, the amount of the aqueous solvent is such that the concentration of the compound of formula (I) in the liquid composition is 0.2-20 mg/ml, such as 0.25-15 mg/ml, such as 0.5-10 mg/ml, such as 0.5-5 mg/ml.

The lipid composition according to the fourth aspect of the invention, which is a composition in a solid state (e.g. is a freeze-dried composition), wherein the excipient is a freeze-dried excipient. For example, the lyophilized excipient is selected from: mannitol, sorbitol, lactose, glycine, dextran, sucrose, glucose, and the like. For example, the weight ratio of compound of formula (I) to lyophilized excipient is 1: 20-200, for example, in a weight ratio of 1: 30-150, for example, in a weight ratio of 1: 30 to 100.

The lipid composition according to the fourth aspect of the present invention, wherein the phospholipid is selected from the group consisting of: egg yolk lecithin, hydrogenated egg yolk lecithin, soy lecithin, hydrogenated soy lecithin, sphingomyelin, phosphatidylethanolamine, dimyristoylphosphatidylcholine (i.e., DMPC), dimyristoylphosphatidylglycerol (i.e., DMPG), dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoylphosphatidylcholine, dilauroylphosphatidylcholine, and combinations thereof.

The lipid composition according to the fourth aspect of the present invention, wherein the pegylated phospholipid (may be simply referred to as pegylated phospholipid) is a phospholipid modified with a molecular weight of 1000 to 10000 daltons, such as pegylated distearoylphosphatidylethanolamine, which may be expressed as distearoylphosphatidylethanolamine-polyethylene glycol (may be abbreviated as PEG-DSPE or DSPE-PEG). For example, the pegylated phospholipid is selected from: distearoylphosphatidylethanolamine-polyethylene glycol 1000 (abbreviated as PEG1000-DSPE, and the others may be similarly described), distearoylphosphatidylethanolamine-polyethylene glycol 2000, distearoylphosphatidylethanolamine-polyethylene glycol 3350, distearoylphosphatidylethanolamine-polyethylene glycol 4000, distearoylphosphatidylethanolamine-polyethylene glycol 5000, distearoylphosphatidylethanolamine-polyethylene glycol 6000, distearoylphosphatidylethanolamine-polyethylene glycol 8000, distearoylphosphatidylethanolamine-polyethylene glycol 10000.

The lipid composition according to the fourth aspect of the present invention is prepared by a process for preparing liposomes. The preparation of liposomes is well known in the art, such as, but not limited to: film dispersion method, extrusion preparation method, French pressure method, reverse phase evaporation method, chemical gradient method (for example, pH gradient method, ammonium sulfate gradient method).

The lipid composition according to the fourth aspect of the present invention is prepared by a thin film dispersion method (a classical liposome preparation method) comprising the steps of:

(21) dissolving phospholipid, pegylated phospholipid, cholesterol and active drug in an organic solvent (such as dichloromethane, chloroform, etc., in an amount of, for example, 2-4 times the amount of completely dissolved);

(22) evaporating the liquid obtained in the previous step on a rotary evaporator (for example, at 40-60 ℃, at a vacuum degree of 200-250 mbar, at a rotation speed of 250rpm) to remove the solvent, so that the residue forms a film on the inner wall of the container;

(23) preparation of lipid composition:

(23a) adding an aqueous solvent into a container, hydrating at 40-80 deg.C (e.g. 60-70 deg.C) for 1-5 hr (e.g. 1.5-2.5 hr), performing ultrasonic treatment for 15-60 min (e.g. 20-45 min), filtering for sterilization (e.g. using 220nm polyethersulfone microporous membrane), and collecting lipid composition in the form of liquid lipid suspension; or

(23b) Adding an excipient solution dissolved in water in advance into a container, hydrating at 40-80 deg.C (e.g. 60-70 deg.C) for 1-5 hr (e.g. 1.5-2.5 hr), performing ultrasonic treatment for 15-60 min (e.g. 20-45 min), filtering for sterilization (e.g. using 220nm polyethersulfone microporous membrane), packaging into glass bottles, and freeze-drying in a freeze-dryer to remove water to obtain solid lipid composition.

The lipid composition according to the fourth aspect of the present invention, wherein in the step (23b), the excipient concentration in the excipient solution previously dissolved with water is 3 to 20%, for example, 3 to 15%.

Further, the fifth aspect of the present invention provides a method for preparing the lipid composition according to any one of the fourth aspect of the present invention, which is carried out by a method selected from the group consisting of: film dispersion method, extrusion preparation method, French pressure method, reverse phase evaporation method, chemical gradient method (for example, pH gradient method, ammonium sulfate gradient method).

The method according to the fifth aspect of the present invention, wherein the thin film dispersion method comprises the steps of:

(21) dissolving phospholipid, pegylated phospholipid, cholesterol and active drug in an organic solvent (e.g., dichloromethane, chloroform, etc.);

(23) preparation of lipid composition:

The method according to the fifth aspect of the present invention, wherein in the step (23b), the excipient concentration in the excipient solution previously dissolved with water is 3 to 20%, for example, 3 to 15%.

Further, the sixth aspect of the present invention provides the use of the lipid composition according to any one of the fourth aspects of the present invention or the lipid composition prepared by the method according to any one of the fifth aspects of the present invention in the preparation of a medicament for treating tumors.

The use according to the sixth aspect of the invention, wherein the tumour is a solid tumour.

The use according to the sixth aspect of the invention, wherein the tumor is selected from: non-small cell lung cancer, pancreatic cancer, ovarian cancer, bladder cancer, breast cancer, and liver cancer.

The lipid composition according to any aspect of the present invention is a liquid or solid composition, which is diluted with water or dissolved to form a liquid medicine having a concentration of the compound of formula (I) of 0.2mg/ml or less, wherein the liquid medicine has an average particle size of less than 200nm (e.g., an average particle size of 20 to 200nm, e.g., an average particle size of 30 to 200nm, e.g., an average particle size of 40 to 200nm, e.g., an average particle size of 50 to 200nm, e.g., an average particle size of 30 to 180nm, e.g., an average particle size of 30 to 150nm), less than 5% of particles having a particle size of less than 10nm (e.g., less than 5% of particles having a particle size of less than 15 nm), and less than 5% of particles having a particle size of more than 500nm (e.g., less than 5%. This term may be referred to as particle size and particle size distribution.

The lipid composition according to any aspect of the present invention, which is a composition in a liquid or solid state, is diluted with water or dissolved to prepare a liquid medicine having a concentration of the compound of formula (I) of 0.2mg/ml or less, which liquid medicine is measured with a nano-particle sizer and calculates the particle diameters D10, D50, and D90 values of the nanoparticles (also commonly understood as a particle diameter where 10% of the particles are smaller than this value, a particle diameter where 50% of the particles are smaller than this value, or a median particle diameter, and a particle diameter where 90% of the particles are smaller than this value, respectively), the diameter Span value of the nanoparticles of the test article is calculated by the following formula: span ═ (Dv90-Dv10)/Dv 50; the composition has a Span of less than 5, particularly less than 3, more particularly less than 2.5, more particularly less than 2. Smaller Span means narrower particle size distribution of the particles and is more desirable in the art, and it is well known in the art that for injectable nanoparticle formulations, a Span of less than 3 is generally considered acceptable, a Span of less than 2.5 is generally considered satisfactory, and a Span of less than 2 is generally considered highly satisfactory. It has been surprisingly found that the compositions prepared by the process of the present invention have nanoparticles with an average particle size of less than 200nm, Span values of less than 2.5, and that some compositions exhibit substantially unchanged effect of average particle size and Span values after prolonged storage.

In any aspect of the present invention, the pharmaceutical composition prepared in liquid form or further prepared in the form of a freeze-dried powder injection may be prepared in a manner to control the preparation process to make the composition into a sterile preparation for use in a sterile manner. The process is easy to control, for example, the control mode is that each raw and auxiliary material is sterilized and then prepared into a sterile preparation by whole-process sterile operation; it may also be a post-controlled manner, i.e. the composition in liquid form as prepared is sterilized by filtration through, for example but not limited to, a microfiltration membrane. Thus, according to any aspect of the invention, the pharmaceutical composition prepared in liquid form or further formulated as a lyophilized powder for injection is a sterile formulation.

Any technical feature possessed by any one aspect of the invention or any embodiment of that aspect is equally applicable to any other embodiment or any embodiment of any other aspect, so long as they are not mutually inconsistent, although appropriate modifications to the respective features may be made as necessary when applicable to each other. Various aspects and features of the disclosure are described further below.

All documents cited herein are incorporated by reference in their entirety and to the extent such documents do not conform to the meaning of the present invention, the present invention shall control. Further, the various terms and phrases used herein have the ordinary meaning as is known to those skilled in the art, and even though such terms and phrases are intended to be described or explained in greater detail herein, reference is made to the term and phrase as being inconsistent with the known meaning and meaning as is accorded to such meaning throughout this disclosure.

In the present invention, references to% are weight/weight percentages, as not otherwise specified.

Gemcitabine, (+)2' -deoxy-2 '2' -difluorocytosine, is usually used clinically in the form of hydrochloride, and a common dosage form is gemcitabine hydrochloride freeze-dried powder injection. Can be clinically used for treating the following diseases: locally advanced or metastatic non-small cell lung cancer, locally advanced or metastatic pancreatic cancer, gemcitabine and paclitaxel in combination are suitable for the treatment of unresectable, locally recurrent or metastatic breast cancer that recurs after adjuvant/neoadjuvant chemotherapy.

Cellular metabolism and mechanism of action of gemcitabine: gemcitabine (dFdC) is a pyrimidine antimetabolite which is metabolized intracellularly to active nucleoside diphosphate (dFdCDP) and triphosphate (dFdCTP) by the action of nucleoside kinases. dFdCDP and dFdCTP inhibit DNA synthesis by two mechanisms of action, thereby achieving the cytotoxic effects of gemcitabine. First, dFdCDP inhibits the activity of ribonucleotide reductase, so that the production of deoxynucleoside triphosphate (dCTP), which is necessary for DNA synthesis, is inhibited. Second, dFdCTP competes with dCTP for incorporation into the DNA strand (self-enhancing effect). Likewise, small amounts of gemcitabine may also be incorporated into RNA molecules. Thus, a decrease in intracellular dCTP concentration is more favorable for incorporation of dFdCTP into the DNA strand. The DNA polymerase ε cannot remove the incorporated gemcitabine and repair the formed DNA strand. When gemcitabine is incorporated into a DNA strand, one nucleotide is added to the extended DNA strand. This added nucleotide can completely inhibit further synthesis of the DNA strand (cryptic strand termination). Gemcitabine induces apoptosis upon incorporation into a DNA strand.

Cytotoxic activity of gemcitabine on cultured cells: gemcitabine has significant cytotoxic activity against various cultured human and murine tumor cells. Its action is cell cycle specific, i.e., gemcitabine acts primarily on cells in the DNA synthesis phase (S-phase), under certain conditions, to prevent cell progression at the G1 phase/S phase junction. In vitro, the cytotoxic effect of gemcitabine depends on concentration and time.

Study of antitumor Activity of Gemcitabine in animal models: the antitumor activity of gemcitabine was found to be related to the mode of administration in studies in animal models of tumors. The daily administration results in high mortality and low antitumor activity in the animals. Gemcitabine has good antitumor activity against a variety of tumors in mice at non-lethal doses when administered once every 3-4 days.

Pharmacokinetic profile of gemcitabine: the pharmacokinetic profile of gemcitabine was evaluated in a total of 353 patients in 7 studies. Wherein 121 female patients and 232 male patients are between 29-79 years old. Of these patients, about 45% are non-small cell lung cancer patients and 35% are pancreatic cancer patients. The dosage range for obtaining the following pharmacokinetic parameters is 500-2,592mg/m²The infusion time is within 0.4-1.2 hours. The peak plasma concentration (obtained within 5 minutes after the end of infusion) was 3.2-45.5. mu.g/ml. According to 1000mg/m²The administration is carried out at a dose of 30min, the plasma concentration of the parent compound can be continuously higher than 5 mug/ml within 30min after the transfusion is finished, and the plasma concentration is also higher than 0.4 mug/ml within 1 hour after the transfusion is finished.

Distribution: the distribution volume of the central chamber is 12.4L/m for female²And 17.5L/m for men²(the inter-individual difference was 91.9%). The distribution volume of the peripheral compartment is 47.4L/m². The volume of the peripheral compartment is independent of gender. Plasma protein binding was negligible. Half-life: the half-life was 42-94 minutes, age and sex related. For the recommended dosing regimen, gemcitabine is completely cleared within 5-11 hours after infusion begins. Gemcitabine does not accumulate when administered once a week.

Gemcitabine is rapidly metabolized by cytidine deaminase in the liver, kidney, blood and other tissues. In cells, gemcitabine is metabolized intracellularly to produce gemcitabine monophosphate, diphosphate, and nucleoside triphosphate (dFdCMP, dFdCDP, and dFdCTP), where the dFdCDP and dFdCTP are active. Metabolites formed within these cells were not detected in either plasma or urine. The major metabolite, 2' -deoxy-2 ',2' -diflubenzuron (dFdU), is inactive and is detectable in both plasma and urine.

The systemic clearance rate of gemcitabine is 29.2L/hr/m²-92.2L/hr/m²Correlated with gender and age (individual differences 52.2%). Clearance is approximately 25% lower in women than in men. Although clearance is rapid, clearance decreases with age in both men and women. Gemcitabine is recommended to be administered at a dose of 1000mg/m²Intravenous drip for 30 minutes, without reducing the gemcitabine dose due to reduced clearance in men and women. Excretion via urine: less than 10% is excreted as bulk drug. Renal clearance: 2-7L/hr/m². Within one week after administration, 92% -98% of gemcitabine doses were detected, 99% of which were excreted mainly in the form of dFdU through urine and 1% through feces.

The present inventors have surprisingly found that a unique family of gemcitabine chemical structure modifications and the lipid compositions prepared therefrom have superior properties.

Detailed Description

The present invention will be further described by the following examples, however, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention. The present invention has been described generally and/or specifically with respect to materials used in testing and testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible.

In the following specific examples section, pharmaceutical compositions in liquid form or formulations in the form of lyophilized compositions are provided, as not otherwise mentioned, in amounts of the respective materials per 100mg or 100 parts by weight of the compound of formula (I) in the composition prepared; in the actual preparation, it is dosed in an amount to prepare a pharmaceutical composition comprising 10g of a compound of formula (I). When the pH value of the liquid medicine needs to be adjusted during the preparation of the composition, a 2M hydrochloric acid solution or a 2M sodium hydroxide solution is used. In the following examples, the content of organic solvent in the freeze-dried powder obtained by freeze-drying the composition was determined to be below the detection limit.

Example 1N-decyl (1- (3, 3-difluoro-4-hydroxy-5-hydroxymethyltetrahydrofuran-2-yl) -2-oxo-1, 2-dihydropyrimidin-4-yl) carbamate

An exemplary reaction scheme is as follows:

commercially available 2',2' -difluoro-2 '-deoxycytidine-3', 5 '-dibenzoate (compound of formula II, 9.4g, 0.02mol) was dissolved in 75mL of dichloromethane, triethylamine (3.0g, 0.03mol) was added, and then decyl chloroformate (III', 5.3g, 0.024mol) was added in portions, stirred at room temperature for 1.5 hours, and the TLC detection reaction was complete; then, the reaction mixture was washed with saturated brine and saturated sodium bicarbonate solution in this order, and the organic phase was concentrated and precipitated to obtain compound IV' which was used in the next reaction.

Dissolving the obtained 0.01mol of compound IV' in 50mL of methanol, adding a catalytic amount of NaOH solid (0.1g), stirring at room temperature for reaction for 0.5 hour, and detecting by TLC to finish the reaction; the reaction was then filtered through silica gel, the filtrate was concentrated and purified by column chromatography on silica gel (eluent CH2Cl 2: CH3OH ═ 20:1, v/v) to isolate and purify the title compound I' as a white solid, which in the present invention can be referred to as compound 1.

The yield of the two-step reaction from the compound of the formula II to the compound of the formula I in examples 1-12 is 50.6-53.7%, and the yield of the reaction from the compound of the formula IV to the compound of the formula I for removing Bz groups is 67.1-70.3%; for example, the two-step reaction of the compound of formula II of example 1 to the compound of formula I ' gave a yield of 52% and the reaction of the compound of formula IV ' to the compound of formula I ' gave a yield of 68.3% for the removal of the Bz group.

¹HNMR(500M,CD₃OD):δ8.25(d,J＝6.8Hz,1H),7.28(d,J＝6.8Hz,1H),6.20(brs,1H),4.31-4.27(m,1H),4.22-4.19(m,2H),3.98-3.96(m,1H),3.91(t,J＝7.5Hz,2H),1.72-1.70(m,2H),1.40-1.31(m,14H),0.88(t,J＝7.2Hz,3H).

MS-ESI(m/z):448.2(M+H)

[ HPLC-A method ]: method for determining impurity content in material

Taking a proper amount of a substance to be tested (such as a compound IV or a compound I obtained in the embodiment of the invention) in the preparation process, precisely weighing, adding a diluent (0.02% phosphoric acid-acetonitrile 70:30 solution) to dissolve and dilute the substance to be tested to prepare 1ml of solution containing 500 mu g of the substance to be tested as a test solution;

precisely measuring a proper amount of the solution, and adding a diluent to dilute the solution into a solution containing about 5 mu g of the solution per 1ml to serve as a control solution;

measuring by high performance liquid chromatography (0512 of the four-part general regulation of the Chinese pharmacopoeia 2015 edition), using octadecylsilane chemically bonded silica as a filler, 250mm × 4.6mm and 5 μm column specification, using 0.02% phosphoric acid-acetonitrile 70:30 solution as a mobile phase, with the flow rate of 1ml/min, the detection wavelength of 254nm and the column temperature of 30 ℃;

precisely measuring 20 mul of test solution, injecting into a liquid chromatograph, and recording the chromatogram until the retention time of the main component peak is 3 times;

and calculating the content of the specific impurities according to a comparison method of the main peak area of the comparison solution.

It has been found that during the hydrolysis of compound IV under basic conditions to obtain the debz group of compound I, a small amount of amide bond may be cleaved to yield gemcitabine (which is referred to as a specific impurity in the preparation of the present invention using the compound of formula I as a product), and the relative retention time of other substances, such as the compound of formula IV or the compound of formula I, is calculated based on the retention time of the specific impurity under the HPLC conditions as described above, and the relative retention time of the compound of formula IV or the compound of formula I is in the range of about 1.4 to about 1.7. The percentage of a particular impurity (relative to the compound of formula I) in the compound of formula I prepared in each example of the invention was determined using the [ HPLC-a method ]; alternatively, [ HPLC-A method ] may be used to determine the percent of a particular impurity (relative to the compound of formula IV) in compounds of formula IV prepared according to various embodiments of the invention. In addition, the chromatographic purity of the test sample can also be calculated by an area normalization method through the chromatogram of the test sample.

The percentage of specific impurities in the compounds of formula IV prepared in examples 1-12 of the present invention is less than 0.02%, for example, the percentage of specific impurities in the compound of formula IV' prepared in example 1 is less than 0.01%, which indicates that the specific impurities introduced by the raw materials or by-products before the compound of formula IV is prepared are quite small.

The percentage of specific impurities in the compounds of formula I obtained in examples 1 to 12 of the present invention was determined to be in the range of 0.62 to 0.96%, for example, the percentage of specific impurities in the compound of formula I' obtained in example 1 was determined to be 0.783%, indicating that specific impurities were significantly introduced during the preparation of the compound of formula I from the compound of formula IV.

In addition, the chromatographic purity of the compounds of formula I prepared in examples 1 to 12 of the present invention was determined to be within the range of 96.4 to 97.7%, for example, the chromatographic purity of the compound of formula I' obtained in example 1 was 97.13%, which shows relatively high purity, but does not meet the requirement that the purity of the pharmaceutical raw material is generally more than 98%.

Example 2N-dodecyl (1- (3, 3-difluoro-4-hydroxy-5-hydroxymethyltetrahydrofuran-2-yl) -2-oxo-1, 2-dihydropyrimidin-4-yl) carbamate

Proceeding according to the method of example 1, compound II is reacted with n-dodecyloxy chloroformate; the title compound is then prepared as a white solid by deprotection of the Bz protecting group, which in this invention may be referred to as compound 2.

¹HNMR(500M,CD₃OD):δ8.29(d,J＝6.5Hz,1H),7.25(brs,1H),6.22(brs,1H),4.33-4.21(m,3H),3.98-3.92(m,1H),3.84(t,J＝7.1Hz,2H),1.68-1.65(m,2H),1.40-1.31(m,16H),0.89(t,J＝7.2Hz,3H).

MS-ESI(m/z):476.2(M+H)

Example 3N-tetradecyl (1- (3, 3-difluoro-4-hydroxy-5-hydroxymethyltetrahydrofuran-2-yl) -2-oxo-1, 2-dihydropyrimidin-4-yl) carbamate

Proceeding according to the method of example 1, compound II is reacted with n-tetradecyloxy chloroformate; the title compound is then prepared as a white solid by deprotection of the Bz protecting group, which in this invention may be referred to as compound 3.

¹HNMR(500M,CD₃OD):δ8.30(d,J＝7.2Hz,1H),7.34(d,J＝6.8Hz,1H),6.25(brs,1H),4.32-4.26(m,1H),4.20-4.17(m,2H),3.98-3.96(m,2H),3.81(d,J＝12.5Hz,1H),1.69(q,J＝7.2Hz,2H),1.41-1.28(m,22H),0.89(t,J＝6.7Hz,3H).

MS-ESI(m/z):504.2(M+H)⁺.

Example 4N-hexadecyl (1- (3, 3-difluoro-4-hydroxy-5-hydroxymethyltetrahydrofuran-2-yl) -2-oxo-1, 2-dihydropyrimidin-4-yl) carbamate

Proceeding according to the method of example 1, compound II is reacted with n-hexadecyloxy chloroformate; the title compound is then prepared as a white solid by deprotection of the Bz protecting group, which in this invention may be referred to as compound 4.

¹HNMR(500M,CDCl₃):δ8.16(d,J＝7.2Hz,1H),7.36(d,J＝7.2Hz,1H),6.25(brs,1H),4.54-4.52(m,1H),4.22-4.20(m,2H),4.08-3.89(m,3H),1.77-1.54(m,3H),1.31-1.22(m,25H),0.89(t,J＝7.2Hz,3H).

MS-ESI(m/z):532.2(M+H)

Example 5N-octadecyl (1- (3, 3-difluoro-4-hydroxy-5-hydroxymethyltetrahydrofuran-2-yl) -2-oxo-1, 2-dihydropyrimidin-4-yl) carbamate

Proceeding according to the method of example 1, compound II is reacted with n-octadecyl chloroformate; the title compound is then prepared as a white solid by deprotection of the Bz protecting group, which in this invention may be referred to as compound 5.

¹HNMR(500M,CDCl₃):δ8.19(d,J＝7.0Hz,1H),7.35(d,J＝6.8Hz,1H),6.24(s,1H),4.55-4.50(m,1H),4.22-4.10(m,5H),1.72-1.64(m,2H),1.31-1.25(m,30H),0.88(t,J＝6.8Hz,3H).

MS-ESI(m/z):560.2(M+H)

Example 6N-eicosyl (1- (3, 3-difluoro-4-hydroxy-5-hydroxymethyltetrahydrofuran-2-yl) -2-oxo-1, 2-dihydropyrimidin-4-yl) carbamate

Proceeding according to the method of example 1, compound II is reacted with n-eicosoxy chloroformate; the title compound is then prepared as a white solid by deprotection of the Bz protecting group, which in this invention may be referred to as compound 6.

¹HNMR(500M,CDCl₃):δ8.17(d,J＝7.2Hz,1H),7.37(d,J＝7.2Hz,1H),6.26(s,1H),4.53-4.49(m,1H),4.22-4.19(m,3H),4.10-3.98(m,2H)1.73-1.65(m,2H),1.33-1.25(m,34H),0.89(t,J＝6.8Hz,3H).

MS-ESI(m/z):588.2(M+H)

Example 7N-docosyl (1- (3, 3-difluoro-4-hydroxy-5-hydroxymethyltetrahydrofuran-2-yl) -2-oxo-1, 2-dihydropyrimidin-4-yl) carbamate

Proceeding according to the method of example 1, compound II is reacted with n-docosanyloxychloroformate; the title compound is then prepared as a white solid by deprotection of the Bz protecting group, which in this invention may be referred to as compound 7.

¹HNMR(500M,CDCl₃):δ8.19(d,J＝7.0Hz,1H),7.38-7.36(m,1H),6.24(brs,1H),4.52-4.48(m,1H),4.20-3.99(m,5H),1.72-1.67(m,2H),1.31-1.24(m,38H),0.89(t,J＝7.0Hz,3H).

MS-ESI(m/z):616.2(M+H)

Example 8N-hexadecyloxypropyl (1- (3, 3-difluoro-4-hydroxy-5-hydroxymethyltetrahydrofuran-2-yl) -2-oxo-1, 2-dihydropyrimidin-4-yl) carbamate

Dissolving hexadecyloxy propanol in dichloromethane, adding 3 equivalents of triethylamine and 2 equivalents of diphosgene, reacting at room temperature for 1 hour, and directly spin-drying the solvent to obtain n-hexadecyloxy propyl chloroformate; then, the process of the present invention is carried out,

proceeding according to the method of example 1, compound II is reacted with n-hexadecyloxy chloroformate; the title compound is then prepared as a white solid by deprotection of the Bz protecting group, which in this invention may be referred to as compound 8.

¹HNMR(500M,CDCl₃):δ8.23(d,J＝7.2Hz,1H),7.37(d,J＝7.2Hz,1H),6.25(brs,1H),4.51-4.49(m,1H),4.20-4.17(m,3H),4.08-3.89(m,2H),3.76-3.71(m,4H),1.73-1.62(m,4H),1.31-1.24(m,26H),0.87(t,J＝7.0Hz,3H).

MS-ESI(m/z):590.2(M+H)

Example 9N-octadecyl oxyethyl (1- (3, 3-difluoro-4-hydroxy-5-hydroxymethyl-tetrahydrofuran-2-yl) -2-oxo-1, 2-dihydropyrimidin-4-yl) carbamate

Proceeding according to the method of example 8, compound II is reacted with n-octadecyl chloroformate; the title compound is then prepared as a white solid by deprotection of the Bz protecting group, which in this invention may be referred to as compound 9.

¹HNMR(500M,CDCl₃):δ8.21(d,J＝7.2Hz,1H),7.35(d,J＝7.0Hz,1H),6.25(brs,1H),4.53-4.50(m,1H),4.20-4.10(m,5H),3.99-3.89(m,6H),1.72-1.65(m,2H),1.33-1.25(m,30H),0.89(t,J＝7.2Hz,3H).

MS-ESI(m/z):604.2(M+H)

Example 10(9-ene) n-decaalkyl (1- (3, 3-difluoro-4-hydroxy-5-hydroxymethyltetrahydrofuran-2-yl) -2-oxo-1, 2-dihydropyrimidin-4-yl) carbamate

Proceeding according to the method of example 1, compound II is reacted with (9-ene) n-decaalkyl chloroformate; the title compound is then prepared as a white solid by deprotection of the Bz protecting group, which in this invention may be referred to as compound 10.

¹HNMR(500M,CDCl₃):δ8.22(d,J＝7.0Hz,1H),7.33-7.31(m,1H),6.25(brs,1H),5.22-5.17(m,1H),4.92-5.01(m,2H),4.56-4.51(m,1H),4.20-4.07(m,5H),1.92-1.66(m,4H),1.39-1.20(m,10H).

MS-ESI(m/z):446.2(M+H)

Example 11N-dodecyl (1- (3, 3-difluoro-4-hydroxy-5-hydroxymethyltetrahydrofuran-2-yl) -2-oxo-1, 2-dihydropyrimidin-4-yl) (11-ene) carbamate

Proceeding according to the method of example 1, compound II is reacted with (11-ene) n-dodecyl chloroformate; the title compound is then prepared as a white solid by deprotection of the Bz protecting group, which in this invention may be referred to as compound 11.

¹HNMR(500M,CDCl₃):δ8.24(d,J＝6.8Hz,1H),7.30(d,J＝6.8Hz,1H),6.25(brs,1H),5.26-5.23(m,1H),5.11-5.07(m,2H),4.55-4.52(m,1H),4.23-4.16(m,5H),2.11-2.02(m,2H),1.72-1.62(m,2H),1.38-1.23(m,14H).

MS-ESI(m/z):474.2(M+H)

Example 12N-dodecylethyl (1- (3, 3-difluoro-4-hydroxy-5-hydroxymethyltetrahydrofuran-2-yl) -2-oxo-1, 2-dihydropyrimidin-4-yl) carbamate (11-ene)

Proceeding according to the method of example 1, compound II is reacted with (11-ene) n-dodecylethylchloroformate; the title compound is then prepared as a white solid by deprotection of the Bz protecting group, which in this invention may be referred to as compound 12.

¹HNMR(500M,CDCl₃):δ8.26(d,J＝7.2Hz,1H),7.35(d,J＝7.2Hz,1H),6.31(brs,1H),5.28-5.25(m,1H),5.14-5.11(m,2H),4.54-4.50(m,1H),4.33-4.11(m,9H),2.09-2.02(m,2H),1.62-1.59(m,2H),1.35-1.27(m,14H).

MS-ESI(m/z):518.2(M+H)

EXAMPLE 21 preparation of Gemcitabine carbamates

Each of the title compounds of formula I was prepared starting from the compound of formula IV by the method of examples 1-12, respectively, and the remaining procedures were carried out as described in examples 1-12, except that in each Bz group removal reaction of this example, 10mL of ethyl acetate was added with 50mL of methanol, and 0.25g of calcium chloride was added with 0.1g of NaOH solid, to give each of the compounds of formula I.

The determination shows that the reaction yield of the compound of formula IV to the compound of formula I for removing Bz groups is 82.4-85.7%, for example, the reaction yield of the compound of example 21 for removing Bz groups is 84.8% in the method of example 1, which indicates that the reaction yield of the compound of example for removing Bz groups is significantly improved;

the percentage content of the specific impurities in the prepared compound of formula I is within the range of 0.106-0.141%, for example, the percentage content of the specific impurities in the compound of formula I' obtained by referring to the method of example 1 in this example 21 is 0.117%, which indicates that the method of this example can significantly reduce the content of the specific impurities in the product compound of formula I;

the chromatographic purity of the prepared compound of formula I is within the range of 98.3-99.6%, for example, the chromatographic purity of the compound of formula I' obtained in example 21 with reference to the method of example 1 is 99.27%, which shows significantly higher purity, and meets the purity requirement of pharmaceutical raw materials.

EXAMPLE 22 preparation of Gemcitabine carbamates

Each of the title compounds of formula I was prepared starting from the compound of formula IV by the methods of examples 1-12, respectively, in which 10mL of ethyl acetate was added along with 50mL of methanol in each Bz group removal reaction of this example, and the remaining operations were carried out as in examples 1-12, respectively, to give each of the compounds of formula I. The yield of the reaction of the compound of formula IV to the compound of formula I to remove Bz groups was determined to be 69.2-71.1%, for example 69.8% in the example 22 with reference to the method of example 1; the percentage content of the specific impurities in the prepared compound of formula I is within the range of 0.68-0.94%, for example, the percentage content of the specific impurities in the compound of formula I' obtained in example 22 with reference to the method of example 1 is 0.842%; the chromatographic purity of the compounds of formula I obtained is in the range of 96.7-98.1%, for example the chromatographic purity of the compound of formula I' obtained in example 22 with reference to the method of example 1 is 97.41%.

EXAMPLE 23 preparation of Gemcitabine carbamates

Each of the title compounds of formula I was prepared starting from the compound of formula IV by the method of examples 1-12, respectively, and in each of the Bz group removal reactions of this example, 0.25g of calcium chloride was added along with 0.1g of NaOH solid, and the remainder was carried out as in examples 1-12, respectively, to give the compound of formula I. The yield of the reaction of the compound of formula IV to the compound of formula I to remove Bz groups was found to be 67.6-69.8%, for example, the yield of the reaction of this example 23 to remove Bz groups in the method of example 1 was found to be 68.2%; the percentage content of the specific impurities in the prepared compound of formula I is within the range of 0.65-0.97%, for example, the percentage content of the specific impurities in the compound of formula I' obtained in example 23 with reference to the method of example 1 is 0.776%; the chromatographic purity of the compounds of formula I obtained is in the range of 96.4-97.8%, for example, the chromatographic purity of the compound of formula I' obtained in example 23 with reference to the method of example 1 is 97.13%. Surprisingly, it has been found that in the Bz group removing reaction for preparing gemcitabine carbamate, a proper amount of ethyl acetate is added with methanol, and calcium chloride is added with NaOH, so that the yield of the reaction can be improved, specific impurities can be reduced from being introduced into the final product, and the purity of the final product is high.

Example 31 antitumor Activity of drugs

1. Cells for assay

Human pancreatic cancer (AsPC-1) cells, human pancreatic ductal carcinoma (su.86.86).

2. Test article

Gemcitabine, 12 gemcitabine esters prepared in example 21.

3. Experimental methods

Culturing AsPC-1, SU.86.86, etc. tumor cells in vitro at 37 deg.C and 5% CO₂Cultured in a cell culture box under the condition to logarithmic phase. The cells were seeded in a 96-well plate at a density of 5000 cells/well and 100. mu.L/well, and then cultured in a cell incubator for 24 hours.

The toxicity of each test substance on the above tumor cells was determined by MTT method, which comprises setting gemcitabine ester compound and gemcitabine as positive control. Diluting the test object by using a cell culture solution in a multiple ratio to obtain a cell culture medium with gemcitabine concentration of 1-100 mu M. After 24h of cell plating, 200. mu.L of each drug-containing medium was used to replace the original medium in the corresponding wells, 4 duplicate wells were provided for each concentration, and a blank control was providedHoles and zero holes. After further incubation for 24, 48 and 72 hours, 20. mu.L of MTT solution with the concentration of 5mg/mL is added, culture is continued for 4 hours under the same condition, the culture solution is discarded, 150. mu.L of DMSO is added to each well to dissolve formazan, a plate shaking instrument is used for shaking for 10min, the absorbance value at the wavelength of 490nm is measured in an enzyme labeling instrument, and the cell growth inhibition percentage% and IC are calculated₅₀Value (. mu.M). IC of partial compound₅₀The (. mu.M) values are as follows:

IC for AsPC-1, gemcitabine₅₀(. mu.M) is 1198.0, IC of Compound 1₅₀(mu M) 86.2, IC of Compounds 2 to 12₅₀(mu M) is in the range of 75.1-132.7; IC for SU.86.86, gemcitabine₅₀(. mu.M) 124.1, IC of Compound 1₅₀(mu M) 5.8, IC of Compounds 2 to 12₅₀(mu M) is in the range of 4.3 to 7.5.

As can be seen from the above, the inhibition effect of the esters of the compounds 1-12 on AsPC-1 and SU.86.86 is obviously better than that of gemcitabine.

Example 32: pharmacodynamic test for inhibiting mouse transplantation tumor

1. Test materials

Medicine preparation: gemcitabine and five compounds of compound 2, compound 6, compound 9 and compound 11 obtained in example 21 were administered in a dose corresponding to 10mg gemcitabine/kg body weight/dose. Clean grade C57BL/6N mice. Tumor species: mouse transplantation tumor Lewis lung cancer, S180 sarcoma and H22 liver cancer.

2. Test method

(1) Mouse Lewis lung carcinoma:

male C57BL/6N mice weighing 18-22 g were divided randomly into 6 groups of 10 mice each, and each group was designated as a normal saline control group and five groups of the above five compounds.

Killing Lewis lung cancer by dislocation of neck, inoculating C57BL/6N tumor-bearing mice growing for 12d under the skin, taking fresh tumor tissue in a sterile manner, preparing cell homogenate by using a tissue grinder, and adjusting the content of living cells to 2-3 multiplied by 10 by using normal saline⁷and/mL, subcutaneously inoculated in the right axilla of C57BL/6N mice, 0.1mL each.

Dosage and method of administration: normal saline, and intragastric administration, 0.1mL/10g body weight/day daily, 1 time daily, 11 times total; the various reagents are injected intraperitoneally 1 time and 4 times a day.

The administration was started on day 2 of the inoculation, 24 hours after the last administration, the animals were sacrificed, the body weight and the tumor mass weight were weighed, and the tumor weight inhibition ratio was calculated according to the following formula:

the tumor weight inhibition ratio (%) was (1-tumor weight of test group/tumor weight of saline control group) × 100%.

The three tests were combined and statistically analyzed using SPSS10.0 software, and the experimental data for body weight and tumor weight were expressed as x + -s, and the differences between each administration group and the saline control group were compared by one-way analysis of variance.

(2) Mouse S180 sarcoma

Aseptically taking S180 sarcoma abdominal cavity, inoculating the ascites of mice growing for 8 days, and diluting the tumor cell content to 3-4 multiplied by 10 with normal saline⁷and/mL, inoculated subcutaneously in the right axilla of experimental mice, 0.1mL each. The administration period 11d, the grouping and administration condition and the statistical method are the same as those of the Lewis lung cancer test of mice.

(3) Mouse H22 liver cancer

Taking H22 liver cancer, inoculating abdominal cavity with mouse ascites of 9 days of growth, and diluting with normal saline until the content of tumor cells is 5-6 multiplied by 10⁷NIH mice were inoculated per mL. The administration period 11d, the grouping and administration condition and the statistical method are the same as those of the Lewis lung cancer test of mice.

3. Inhibition of mouse graft tumors

The tumor weight inhibition rate of gemcitabine on Lewis lung cancer is 65.3%, and the tumor weight inhibition rate of compounds 2, 6, 9 and 11 on Lewis lung cancer is 83-92% (for example, 88.6% of compound 2); the tumor weight inhibition rate of gemcitabine to S180 sarcoma is 54.7%, and the tumor weight inhibition rate of compounds 2, 6, 9 and 11 to S180 sarcoma is 74-79% (for example, 75.8% for compound 2); the tumor weight inhibition rate of gemcitabine on H22 liver cancer is 59.2%, and the tumor weight inhibition rate of compounds 2, 6, 9 and 11 on H22 liver cancer is 85-91% (for example, 90.6% of compound 2). Surprisingly, the compound prepared by the invention has obviously better tumor inhibition effect on various tumors than gemcitabine.

Example 41 preparation of lipid composition

The formula is as follows:

active drug: 100 parts by weight of a water-soluble polymer,

phospholipid (DMPC): 900 parts by weight of a water-soluble polymer,

pegylated phospholipid (DSPE-PEG)₂₀₀₀): 150 parts by weight of a non-woven fabric,

cholesterol: 90 parts by weight, and

excipient (aqueous solvent: 5% glucose solution to make liquid composition, the addition amount is to make the final concentration of active drug 1mg/ml, or lyophilized excipient: mannitol to make solid composition, the weight ratio of active drug and lyophilized excipient is 1: 40).

The preparation method adopts a film dispersion method and comprises the following steps:

(21) dissolving phospholipid, polyethylene glycol phospholipid, cholesterol and active drug in organic solvent (dichloromethane, the addition amount is 3 times of the complete dissolution degree);

(22) evaporating the liquid obtained in the previous step on a rotary evaporator (45 ℃, vacuum degree of 220mbar, rotation speed of 250rpm) to remove the solvent, so that the residue forms a film on the inner wall of the container;

(23) preparation of lipid composition:

(23a) adding aqueous solvent into the container, hydrating at 65 deg.C for 2 hr, performing ultrasonic treatment for 30min, filtering for sterilization (using 220nm polyethersulfone microporous filter membrane), and obtaining lipid composition in the form of liquid lipid suspension; or

(23b) Adding excipient solution (4% concentration) dissolved in water in advance, hydrating at 65 deg.C for 2 hr, performing ultrasonic treatment for 30min, filtering for sterilization (such as 220nm polyethersulfone microporous membrane), packaging in glass bottle, and freeze drying in freeze dryer to remove water to obtain solid lipid composition.

In this example 41, gemcitabine and 12 compounds obtained in example 21 of the present invention were used as active drugs, respectively, to prepare lipid compositions in the form of lipid suspensions in a liquid state (13 liquid compositions), and freeze-dried lipid compositions in a solid state (13 solid compositions).

Example 42 preparation of lipid composition

The formula is as follows:

active drug: 100 parts by weight of a water-soluble polymer,

phospholipid (DMPG): 200 parts by weight of a solvent, and a solvent,

PEGylated Phospholipids (PEG 1000-DSPE): 200 parts by weight of a solvent, and a solvent,

cholesterol: 50 parts by weight, and

excipient (aqueous solvent: 0.9% sodium chloride solution to make liquid composition, the addition amount is to make the final concentration of active drug 5mg/ml, or lyophilized excipient: glycine to make solid composition, the weight ratio of active drug and lyophilized excipient is 1: 30).

(21) dissolving phospholipid, polyethylene glycol phospholipid, cholesterol and active drug in organic solvent (chloroform, the amount of the chloroform is 2 times of the total dissolution);

(22) evaporating the liquid obtained in the previous step on a rotary evaporator (60 ℃, vacuum degree of 200mbar) to remove the solvent, so that the residue forms a film on the inner wall of the container;

(23) preparation of lipid composition:

(23a) adding aqueous solvent into container, hydrating at 60 deg.C for 1.5 hr, performing ultrasonic treatment for 20min, filtering for sterilization (using 220nm polyethersulfone microporous filter membrane), and obtaining lipid composition in the form of liquid lipid suspension; or

(23b) Adding excipient solution (15% concentration) dissolved in water in advance, hydrating at 70 deg.C for 2.5 hr, performing ultrasonic treatment for 45min, filtering for sterilization (such as 220nm polyethersulfone microporous membrane), packaging in glass bottle, and freeze drying in freeze dryer to remove water to obtain solid lipid composition.

In this example 42, gemcitabine and five compounds, compound 2, compound 6, compound 9 and compound 11, obtained in example 21, were used as active drugs to prepare lipid compositions in the form of lipid suspensions in a liquid state (5 liquid compositions) and freeze-dried lipid compositions in a solid state (5 solid compositions), respectively.

Example 43 preparation of lipid composition

The formula is as follows:

active drug: 100 parts by weight of a water-soluble polymer,

phospholipids (egg yolk lecithin): 1500 parts by weight of a reaction product of (B),

pegylated phospholipid (DSPE-PEG)₅₀₀₀): 50 parts by weight of a water-soluble polymer,

cholesterol: 150 parts by weight, and

excipient (aqueous solvent: water to make liquid composition, the addition amount is to make the final concentration of active drug be 0.5mg/ml, or lyophilized excipient: dextran, to make solid composition, the weight ratio of active drug and lyophilized excipient is 1: 100).

(21) dissolving phospholipid, polyethylene glycol phospholipid, cholesterol and active drug in organic solvent (dichloromethane with 4 times of the amount of completely dissolved);

(22) evaporating the liquid obtained in the previous step on a rotary evaporator (40 ℃, the vacuum degree is 250mbar) to remove the solvent, so that the residue forms a film on the inner wall of the container;

(23) preparation of lipid composition:

(23a) adding aqueous solvent into container, hydrating at 70 deg.C for 2.5 hr, performing ultrasonic treatment for 45min, filtering for sterilization (using 220nm polyethersulfone microporous filter membrane), and obtaining lipid composition in the form of liquid lipid suspension; or

(23b) Adding excipient solution (3% concentration) dissolved in water in advance, hydrating at 60 deg.C for 1.5 hr), performing ultrasonic treatment for 20min, filtering for sterilization (such as 220nm polyethersulfone microporous membrane), packaging in glass bottle, and freeze drying in freeze dryer to remove water to obtain solid lipid composition.

In this example 43, gemcitabine and five compounds, compound 2, compound 6, compound 9 and compound 11, obtained in example 21, were used as active drugs to prepare lipid compositions in the form of lipid suspensions in a liquid state (5 liquid compositions) and freeze-dried lipid compositions in a solid state (5 solid compositions), respectively.

Example 44 preparation of lipid composition

The formula is as follows:

active drug: 100 parts by weight of a water-soluble polymer,

phospholipid (soybean lecithin): 100 parts by weight of a water-soluble polymer,

pegylated phospholipid (DSPE-PEG)₄₀₀₀): 300 parts by weight of a solvent, and a solvent,

cholesterol: 200 parts by weight, and

excipient (aqueous solvent: 5% glucose solution to make liquid composition, the addition amount is to make the final concentration of active drug 10mg/ml, or lyophilized excipient: lactose to make solid composition, the weight ratio of active drug and lyophilized excipient is 1: 20).

(21) dissolving phospholipid, polyethylene glycol phospholipid, cholesterol and active drug in organic solvent (dichloromethane, the addition amount is 2 times of the complete dissolution degree);

(22) evaporating the liquid obtained in the previous step on a rotary evaporator (45 ℃, the vacuum degree is 230mbar) to remove the solvent, so that the residue forms a film on the inner wall of the container;

(23) preparation of lipid composition:

(23a) adding aqueous solvent into container, hydrating at 40 deg.C for 2 hr, performing ultrasonic treatment for 35min, filtering for sterilization (using 220nm polyethersulfone microporous filter membrane), and obtaining lipid composition in the form of liquid lipid suspension; or

(23b) Adding excipient solution (6% concentration) dissolved in water in advance, hydrating at 80 deg.C for 5 hr, performing ultrasonic treatment for 35min, filtering for sterilization (such as 220nm polyethersulfone microporous membrane), packaging in glass bottle, and freeze drying in freeze dryer to remove water to obtain solid lipid composition.

In this example 44, gemcitabine and five compounds, compound 2, compound 6, compound 9 and compound 11, obtained in example 21, were used as active drugs to prepare lipid compositions in the form of lipid suspensions in a liquid state (5 liquid compositions) and freeze-dried lipid compositions in a solid state (5 solid compositions), respectively.

Example 45 preparation of lipid composition

The formula is as follows:

active drug: 100 parts by weight of a water-soluble polymer,

phospholipid (dipalmitoylphosphatidylcholine): 2000 parts by weight of a non-woven fabric,

pegylated phospholipid (DSPE-PEG)₃₃₅₀): 30 parts by weight of a solvent, and a solvent,

cholesterol: 20 parts by weight, and

excipient (aqueous solvent: 0.9% sodium chloride solution to make liquid composition, the addition amount is to make the final concentration of active drug be 0.5mg/ml, or lyophilized excipient: mannitol to make solid composition, the weight ratio of active drug and lyophilized excipient is 1: 150).

(21) dissolving phospholipid, polyethylene glycol phospholipid, cholesterol and active drug in organic solvent (dichloromethane, the addition amount is 2.5 times of the total dissolution degree);

(22) evaporating the liquid obtained in the previous step on a rotary evaporator (50 ℃, vacuum degree 210mbar) to remove the solvent, so that the residue forms a film on the inner wall of the container;

(23) preparation of lipid composition:

(23a) adding aqueous solvent into container, hydrating at 80 deg.C for 2 hr, performing ultrasonic treatment for 40min, filtering for sterilization (using 220nm polyethersulfone microporous filter membrane), and obtaining lipid composition in the form of liquid lipid suspension; or

(23b) Adding excipient solution (with concentration of 3.5%) dissolved in water in advance, hydrating at 40 deg.C for 1 hr, performing ultrasonic treatment for 25min, filtering for sterilization (such as 220nm polyethersulfone microporous membrane), packaging in glass bottle, and freeze drying in freeze dryer to remove water to obtain solid lipid composition.

In this example 45, gemcitabine and five compounds, compound 2, compound 6, compound 9 and compound 11, obtained in example 21, were used as active drugs, respectively, to prepare lipid compositions in the form of lipid suspensions in a liquid state (5 liquid compositions) and freeze-dried lipid compositions in a solid state (5 solid compositions).

Example 51 characterization of lipid compositions

The lipid compositions in all liquid states and the lipid compositions in all solid states obtained in examples 41 to 45 were each diluted with water or dissolved and then diluted so that the concentration of the compound of formula (I) was 0.2mg/ml (the obtained liquid composition itself was not diluted if the concentration was lower than this concentration), the particle diameters of the fine particles in the drug solution were measured using a malvern Zetasizer Nano ZS Nano-particle size potentiometer, the average particle diameter was calculated, and the percentage of fine particles having a particle diameter of less than 10nm and the percentage of fine particles having a particle diameter of more than 300nm were counted.

As a result:

all the liquid lipid compositions obtained in examples 41 to 45 have less than 5% of particles having a particle size of less than 10nm and less than 5% of particles having a particle size of more than 300nm, for example, the liquid lipid composition obtained in example 41 for Compound 1 has 1.4% of particles having a particle size of less than 10nm and 0.7% of particles having a particle size of more than 300 nm;

all of the solid lipid compositions obtained in examples 41 to 45 had less than 5% of fine particles having a particle size of less than 10nm and less than 5% of fine particles having a particle size of more than 300nm, for example, example 41 showed 1.1% of the solid lipid composition having a particle size of less than 10nm and 0.8% of the solid lipid composition having a particle size of more than 300nm with respect to Compound 1;

the average particle size of all the liquid lipid compositions obtained in examples 41 to 45 was in the range of 74 to 122nm, for example, the average particle size of the particles in the liquid lipid composition obtained in example 41 for compound 1 was 86 nm.

The average particle size of the total solid lipid composition obtained in examples 41 to 45 is in the range of 91 to 131nm, for example, the average particle size of the particles in the liquid lipid composition obtained for compound 1 in example 41 is 107 nm; there was no significant difference in particle size between the various liquid compositions obtained in examples 41-45 and their corresponding solid compositions.

The above particle size measurements were made within 15 days of the preparation of the composition and these results do not reflect the stability in particle size properties of the composition, which corresponds to a 0 month result. When all the liquid lipid compositions obtained in examples 41 to 45 were left to stand at room temperature for 18 months, their particle sizes were measured, and as a result, the average particle size of each sample was increased by 27 to 35% as compared with that of the sample at 0 month, for example, the average particle size of the particles in the liquid lipid composition obtained in example 41 for compound 1 was increased by 31.7% after 18 months. In addition, when all the solid lipid compositions obtained in examples 41 to 45 were left to stand at room temperature for 18 months, and then their particle sizes were measured, the average particle size of each sample was increased by 31 to 36% as compared with the 0 month result thereof, for example, the average particle size of particles in the solid lipid composition obtained in example 41 for compound 1 after 18 months was increased by 33.8%.

Supplementary test (which may be referred to as example 46 in this application): with reference to the preparation methods of examples 41 to 45 herein, respectively, except that fructose was also added simultaneously (in an amount of 40% by weight of the active drug) at the time of adding the aqueous vehicle or excipient solution to the vessel in step (23a) or (23b), thereby obtaining lipid compositions in the form of lipid suspensions in the liquid state or lipid compositions in the solid state, and the average particle diameters, the percentages of particles smaller than 10nm, and the percentages of particles larger than 300nm of these lipid compositions in the form of lipid suspensions in the liquid state at 0 month and 18 months were determined as described above; as a result, the average particle size of all samples at 0 month and 18 month was less than 5% for particles having a particle size of less than 10nm, less than 5% for particles having a particle size of more than 300nm, and in the range of 86 to 133nm (for example, example 41 shows that the average particle size of particles in the liquid lipid composition obtained by adding fructose to compound 1 was 97 nm); the average particle size of all samples increased by-1.2% to 2.1% relative to their particle size at month 0 at 18 (showing no significant change, e.g., the average particle size of the 18 month particles of the liquid lipid composition of example 41 for compound 1 with fructose addition was 96nm, an increase of 1.23%). It has been surprisingly found that the results of this example 46 demonstrate that the addition of a small amount of fructose to a lipid composition helps to improve the particle size stability of the microparticles in the composition. According to the above results, although the lipid compositions of examples 41 to 45, to which fructose was not added, had an average particle size increased by 25% or more after 18 months of storage at room temperature, the average particle size after such an increase still satisfied the general requirements of pharmaceutical products, and therefore the lipid compositions of examples 41 to 45 and 46 were satisfactory from the clinical administration viewpoint, but it was still expected that better quality pharmaceutical products could be clinically applied. This surprising finding of the lipid composition of the invention was not at all foreseen by the prior art. Thus, according to any embodiment of any aspect of the invention, the lipid composition further comprises fructose; for example if the amount of sugar is 10 to 70%, for example 40%, by weight of the compound of formula (I); for example, the fructose is added together with the excipient. In addition, the solid and liquid lipid compositions of examples 41 to 45 and 46 were left at 2 to 8 ℃ for 24 months, and the average particle size of all samples was increased by-2.2 to 4.4% from the particle size at 0 month in 24 months, showing no significant change, for example, the average particle size of 24 month particles of the liquid lipid composition obtained by adding fructose to compound 1 in example 41 was increased by 0.73%, indicating that the particle size did not change significantly after the long-term storage at 2 to 8 ℃ which is a condition commonly used for liposome storage, regardless of the addition or non-addition of fructose.

Example 52 antitumor Activity of lipid composition

This example was carried out in conjunction with example 31.

1. Cells for assay

2. Test article

Gemcitabine, liquid (or solid) lipid compositions prepared in examples 41-45.

3. Experimental methods

The toxicity of each test substance on the above tumor cells was determined by MTT method, which comprises setting gemcitabine ester compound and gemcitabine as positive control. Diluting the test object by using a cell culture solution in a multiple ratio to obtain a cell culture medium with gemcitabine concentration of 1-100 mu M. After 24h of cell plating, the original medium in the corresponding wells was replaced with 200. mu.L of each drug-containing medium, 4 duplicate wells were set for each concentration, and blank control wells and zero-adjusted wells were set. After further incubation for 24, 48 and 72 hours, 20. mu.L of MTT solution with the concentration of 5mg/mL is added, culture is continued for 4 hours under the same condition, the culture solution is discarded, 150. mu.L of DMSO is added to each well to dissolve formazan, a plate shaking instrument is used for shaking for 10min, the absorbance value at the wavelength of 490nm is measured in an enzyme labeling instrument, and the cell growth inhibition percentage% and IC are calculated₅₀Value (. mu.M).

In the test of a tumor cell, the percentage obtained by dividing the IC50 (. mu.M) value of the liquid lipid composition obtained in examples 41 to 45 by the IC50 (. mu.M) value of the chemical substance and multiplying the result by 100% was defined as the relative inhibition percentage (%) of the liquid lipid composition, the smaller the relative inhibition percentage (%) indicates the smaller the half inhibitory concentration, indicating that the liposome has a stronger tumor cell inhibitory activity against the original compound, for example, the percentage of the IC50 value of the liquid lipid composition of Compound 1 prepared in example 41 against AsPC-1 divided by the IC50 value of Compound 1 against the tumor cells multiplied by 100%, that is the relative percent inhibition (%) of the compound 1 liquid lipid composition obtained in example 41, the relative inhibition percentage (%) was calculated for the solid lipid composition obtained by freeze-drying in the same manner. The results show that it is possible to display,

for AsPC-1 cells, the relative inhibition percentages of (i) the product of example 41, gemcitabine liquid and solid lipid composition (prepared with reference to example 41) were 91.2% and 89.7%, respectively; compounds 1-12 both produced in example 41 with a relative inhibition percentage in the range of 15.7-21.2% for both liquid and solid lipid compositions and no significant difference between solid and liquid forms of the same chemical, e.g., 18.4% and 19.7% for Compound 1 liquid and solid lipid compositions, respectively; (ii) the products of examples 42-45, gemcitabine liquid and solid lipid compositions both had relative percent inhibition in the range of 85.3-91.4%; the relative inhibition percentages for compound 2, 6 liquid and solid lipid compositions were all in the range of 13.7-18.2% and there was no significant difference between solid and liquid forms of the same chemical, e.g., compound 2 liquid and solid lipid compositions in example 42 had relative inhibition percentages of 16.2% and 15.6%, respectively; (ii) the product of example 46, involving typical compounds 2, 6, 9, 11, both had relative inhibition percentages in the range of 12.4-15.7% for both liquid and solid lipid compositions and there was no significant difference between solid and liquid forms of the same chemical, e.g., the relative inhibition percentages for compound 2 liquid and solid lipid compositions in example 41 of example 46 were 13.6% and 14.1%, respectively.

Relative inhibition percentages for su.86.86 cells, (a) the product of example 41, gemcitabine liquid and solid lipid composition (prepared according to example 41) were 88.7% and 90.2%, respectively; compounds 1-12 both produced in example 41 with relative inhibition percentages in the range of 4.4-7.2% for both liquid and solid lipid compositions and there was no significant difference between solid and liquid forms of the same chemical, e.g., compound 1 had relative inhibition percentages of 5.7% and 6.1% for the liquid and solid lipid compositions, respectively; (b) the products of examples 42-45, gemcitabine liquid and solid lipid compositions both had relative percent inhibition in the range of 88.2-91.6%; the relative inhibition percentages of compound 2, 6, liquid and solid lipid compositions were all in the range of 5.7-8.2% and there was no significant difference between solid and liquid forms of the same chemical, e.g., compound 2, liquid and solid lipid compositions in example 42 were 6.1% and 6.4%, respectively; (c) the product of example 46, relates to typical compounds 2, 6, 9, 11 with a percentage of relative inhibition in the liquid and solid lipid compositions ranging from 6.7 to 9.4% and no significant difference between the solid and liquid forms of the same chemical, for example 8.2% and 7.9% relative inhibition for compound 2 liquid and solid lipid compositions, respectively, of example 46, referred to in example 41. Shows that the addition of fructose in the lipid composition has no influence on the tumor inhibition effect.

The results show that the IC50 value of gemcitabine cannot be significantly reduced by adopting the lipid composition system, namely the effect of inhibiting tumor cells cannot be significantly improved, and the IC50 value of gemcitabine esters such as compounds 1-12 can be significantly reduced by adopting the lipid composition system, namely the effect of inhibiting tumor cells can be significantly improved. In other words, the lipid composition system of the present invention can significantly reduce the IC50 value of the gemcitabine ester of the present invention, i.e., can significantly improve the effect of inhibiting tumor cells, but it was surprisingly found that such lipid composition system could not effectively improve the effect of inhibiting tumor cells of gemcitabine, indicating that the lipid composition system of the present invention can significantly improve the antitumor activity of the gemcitabine ester compound of the present invention. The lipid composition system of the present invention can significantly improve the tumor cell-inhibiting effect of the ester compound, but cannot effectively improve the effect of gemcitabine itself, which cannot be predicted at all in the prior art.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims

1. A lipid composition comprising:

a compound of formula (I): 100 parts by weight of a water-soluble polymer,

phospholipid: 100 to 2000 parts by weight of a water-soluble polymer,

pegylated phospholipids: 30 to 300 parts by weight of a water-soluble polymer,

cholesterol: 20 to 200 parts by weight, and

an excipient;

the compound of formula (I) has the following structure:

wherein:

2. A lipid composition according to claim 1, wherein 1 CH in the carbon chain₂Optionally replaced by O.

3. The lipid composition according to claim 1, wherein: the R is selected from: n-decyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, n-eicosyl, n-docosyl, n-hexadecyloxypropyl, n-octadecyl, n-9-ene-n-decyl, (11-ene) n-dodecyl, and (11-ene) n-dodecylethyl.

4. The lipid composition according to claim 1, said compound of formula (I) being a compound 1 to a compound 12 selected from the group consisting of:

5. The lipid composition according to claim 1, which is a composition in a liquid state, wherein the excipient is an aqueous vehicle; for example, it is selected from: water, 0.8-1% sodium chloride solution (e.g., 0.9% sodium chloride solution), 2-10% glucose solution (e.g., 5% glucose solution); for example, the amount of the aqueous solvent is such that the concentration of the compound of formula (I) in the liquid composition is 0.2-20 mg/ml, such as 0.25-15 mg/ml, such as 0.5-10 mg/ml, such as 0.5-5 mg/ml; or

Which is a composition in a solid state (e.g., is a lyophilized composition), wherein the excipient is a lyophilized excipient; for example, the lyophilized excipient is selected from: mannitol, sorbitol, lactose, glycine, dextran, sucrose, glucose, and the like; for example, the weight ratio of compound of formula (I) to lyophilized excipient is 1: 20-200, for example, in a weight ratio of 1: 30-150, for example, in a weight ratio of 1: 30 to 100.

6. The lipid composition according to claim 1, wherein:

the phospholipid is selected from: egg yolk lecithin, hydrogenated egg yolk lecithin, soy lecithin, hydrogenated soy lecithin, sphingomyelin, phosphatidylethanolamine, dimyristoylphosphatidylcholine (i.e., DMPC), dimyristoylphosphatidylglycerol (i.e., DMPG), dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoylphosphatidylcholine, dilauroylphosphatidylcholine, and combinations thereof;

the pegylated phospholipid (may be abbreviated as pegylated phospholipid) is a phospholipid modified by a molecular weight of 1000 to 10000 dalton, such as pegylated distearoylphosphatidylethanolamine, which may be expressed as distearoylphosphatidylethanolamine-polyethylene glycol (may be abbreviated as PEG-DSPE or DSPE-PEG); for example, the pegylated phospholipid is selected from: distearoylphosphatidylethanolamine-polyethylene glycol 1000 (abbreviated as PEG1000-DSPE, and the others may be similarly described), distearoylphosphatidylethanolamine-polyethylene glycol 2000, distearoylphosphatidylethanolamine-polyethylene glycol 3350, distearoylphosphatidylethanolamine-polyethylene glycol 4000, distearoylphosphatidylethanolamine-polyethylene glycol 5000, distearoylphosphatidylethanolamine-polyethylene glycol 6000, distearoylphosphatidylethanolamine-polyethylene glycol 8000, distearoylphosphatidylethanolamine-polyethylene glycol 10000.

7. The lipid composition according to claim 1, which is prepared by a process selected from the group consisting of: a thin film dispersion method, an extrusion preparation method, a French pressure method, a reverse phase evaporation method, a chemical gradient method (for example, a pH gradient method, an ammonium sulfate gradient method); for example, it is prepared by a film dispersion method comprising the steps of:

(23) preparation of lipid composition:

(23b) Adding an excipient solution dissolved in water in a container, hydrating at 40-80 deg.C (e.g. 60-70 deg.C) for 1-5 hr (e.g. 1.5-2.5 hr), performing ultrasonic treatment for 15-60 min (e.g. 20-45 min), filtering for sterilization (e.g. using 220nm polyethersulfone microporous membrane), packaging in glass bottle, and freeze-drying in a freeze-dryer to remove water to obtain solid lipid composition; for example, in the step (23b), the excipient concentration in the excipient solution previously dissolved in water is 3 to 20%, for example, 3 to 15%.

8. A process for preparing a lipid composition according to any one of claims 1 to 7, which process is carried out by a process selected from the group consisting of: a thin film dispersion method, an extrusion preparation method, a French pressure method, a reverse phase evaporation method, a chemical gradient method (for example, a pH gradient method, an ammonium sulfate gradient method); for example, it is prepared by a film dispersion method comprising the steps of:

(23) preparation of lipid composition:

9. The method according to claim 8, wherein in step (23b), the excipient concentration in the excipient solution previously dissolved with water is 3-20%, such as 3-15%.

10. Use of a lipid composition according to any one of claims 1 to 7 or prepared by a process according to any one of claims 8 to 9 in the manufacture of a medicament for the treatment of a tumour; for example, the tumor is a solid tumor; for example, the tumor is selected from: non-small cell lung cancer, pancreatic cancer, ovarian cancer, bladder cancer, breast cancer, and liver cancer.