CN111202849B

CN111202849B - Albumin-bound nitrosourea antitumor pharmaceutical preparation and preparation method thereof

Info

Publication number: CN111202849B
Application number: CN201811297805.1A
Authority: CN
Inventors: 龚涛; 宋旭; 郭晨琦; 杨琴; 周楚楚; 张志荣; 孙逊
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2018-11-02
Filing date: 2018-11-02
Publication date: 2022-06-03
Anticipated expiration: 2038-11-02
Also published as: CN111202849A

Abstract

The invention provides a nitrosourea drug albumin conjugate preparation and a preparation method thereof. The preparation prepared by the invention has tumor targeting property and good drug effect; and the biocompatibility is good, the preparation process is simple, the preparation method is suitable for industrial mass production, and the application prospect is wide.

Description

Albumin-bound nitrosourea antitumor pharmaceutical preparation and preparation method thereof

Technical Field

The invention relates to a high-stability albumin-bound nitrosourea antitumor pharmaceutical preparation applicable to industrial production and a preparation method thereof, belonging to the technical field of medicines.

Background

Nitrosoureas drugs are a class of commonly used anti-tumor alkylating agents in clinical practice, and are widely used for treating brain tumors, digestive tract cancers (gastric cancer, liver cancer, colon cancer and rectal cancer), lung cancer, malignant lymphoma, chronic leukemia and the like. The antineoplastic medicine has strong cytotoxicity, the action mechanism is mainly to alkylate DNA and inhibit the synthesis of DNA and RNA, thereby inhibiting the proliferation of tumor cells, and the antineoplastic medicine mainly comprises: nimustine (ACNU), dithiomustine (Ditiomustine), brivudine (Bofumustine), carmustine (BCNU), emestine (Elmustine), Ecomustine (Ecmustine), Fotemustine (Fotemustine), nemustine (Pentamustine, Neptamustine), Lomustine (Lomustine, CCNU), Semustine (Semustine), Ranimustine (Ranimustine), tautomustine (Tauromustine), and the like. However, like other cytotoxic antitumor drugs, nitrosoureas drugs lack selectivity for tumor tissues, and cause toxic and side effects such as bone marrow suppression, nausea, vomiting, anorexia, fever, rash, alopecia, decrease in transaminase, increase in urea nitrogen, and the like, thus being greatly limited in clinical application.

The new dosage form of nitrosourea drugs disclosed at present mainly focuses on the combined administration of nitrosourea drugs and other drugs and the local sustained release application of nitrosourea drugs, for example, patent CN102219812A discloses a tumor-targeted deoxyglucose-nimustine anti-tumor compound drug and a preparation method thereof; patent CN101264328A discloses an anticancer sustained-release gel injection containing statin drugs; patent CN101176712A discloses a nimustine sustained release implant for treating solid tumor. Patent CN106137962A discloses a carmustine-loaded glioma targeted polymer micelle and a preparation method thereof, and patent CN1686134A discloses a nimustine brain sustained-release implant and a preparation method thereof, but the preparations still have the problems of complex material synthesis process, complex administration mode, low patient compliance and the like. Therefore, the development of a novel nitrosourea drug preparation which is safe and effective, easy for industrial production and high in patient compliance is still a problem to be solved urgently in pharmaceutics.

The albumin nanoparticles are solid spheres formed by using albumin as a carrier, encapsulating or adsorbing drugs and carrying out solidification separation. Albumin nanoparticles have become a relatively mature drug delivery system, and can transform vascular endothelial cell barrier at tumor into a natural biological pathway through a mechanism of transmembrane transport mediated by albumin receptors (gp 60, gp30, gp18 and cysteine-rich acidic Secreted Protein (SPARC)) on cell membranes, and transport drugs to tumor cells (xiu peak, et al, research progress of albumin nanoparticle drug delivery system, sheng yang university report 27 (2010) 968-78). The albumin material has the characteristics of safety, no toxicity, no immunogenicity, biodegradability, good biocompatibility and the like. However, the traditional preparation method of albumin nanoparticles usually involves chemical crosslinking or thermal denaturation, so that the albumin nanoparticles lose biological characteristics and do not have tumor tropism. The American bioscience GmbH discloses a disulfide bond formation-based albumin nanoparticle preparation technology (CN 1237901A), which takes drug nanoparticles as cores, and albumin is wrapped on the surfaces of the nanoparticles in a crosslinking way by disulfide bonds, so that taxol drugs are prepared into a stable albumin nanoparticle preparation suitable for clinical needs. Previous research in the laboratory (CN 102552154A) has demonstrated that lipophilic treatment of anti-tumor drugs is required, and surfactants other than albumin are required to be added to prepare albumin nanoparticle preparation with high encapsulation efficiency, uniform particle size, and stability. However, this method requires the addition of a large amount of auxiliary materials in addition to albumin, and the preparation method is cumbersome.

In conclusion, no new dosage form product of nitrosourea drugs for overcoming the defects is available on the market, so that a new preparation which has high efficiency, low toxicity, good stability, low production cost and simple preparation method and is suitable for large-scale industrial production and clinical use is urgently needed to be searched.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, and the nitrosourea pharmaceutical preparation which is high in efficiency, low in toxicity, good in drug stability, simple in preparation method and convenient to use clinically is prepared and used clinically.

In the research, the albumin, nitrosourea drugs and surfactants are unexpectedly found to form clear and transparent solutions and the preparation method is simple. Different from simple solubilization, the nitrosourea drug is combined with albumin, and the addition of the surfactant enables the nitrosourea drug to be combined with the albumin stably, so that the nitrosourea drug has good drug effect on brain glioma mice and reduces toxicity, which is an unexpected result in the prior art.

One purpose of the invention is to provide a nitrosourea antineoplastic drug albumin conjugate, which comprises 1 part of nitrosourea antineoplastic drug, 1-1000 parts of albumin and 1-100 parts of surfactant in parts by weight; preferably, 1 part of nitrosourea antineoplastic drug, 5-100 parts of albumin and 1-20 parts of surfactant.

The nitrosourea antineoplastic drugs comprise one or more of Nimustine (ACNU, Nimustine), dithiomustine (Ditiomustine), brivudine (Bofumustine), carmustine (BCNU, carmustine), emetine (Elmustine), efustine (Ecomustine), Fotemustine (Fotemustine), nemustine (Pentamustine, Neptamustine), lomustine (CCNU), Semustine (Semustine), Ranimustine (Ranimustine), and tautomustine (Tauromustine).

The albumin according to the invention is selected from human serum albumin and/or bovine serum albumin, preferably human serum albumin, added in an aqueous human serum albumin solution in a concentration range of about 0.1-25% (w/v), preferably in a range of 0.5-5% (w/v), more preferably 3% (w/v). The albumin is added to the aqueous phase to form a conjugate with the nitrosourea alkylating agent, thereby forming a clear and transparent solution.

The surfactant provided by the invention is selected from one or more of sodium dodecyl sulfate, Solutol HS15, Tween, Mizel, Brij, poloxamer and similar compounds; preferably Solutol HS15 and/or poloxamer; still more preferred is Solutol HS 15.

One of the purposes of the invention is to provide a nitrosourea antitumor drug albumin conjugate freeze-dried powder injection which comprises nitrosourea antitumor drugs, albumin, a surfactant, a freeze-drying protective agent and other pharmaceutically acceptable auxiliary materials, wherein the nitrosourea antitumor drug albumin conjugate freeze-dried powder injection comprises 1 part of nitrosourea antitumor drugs, 1-1000 parts of albumin, 1-100 parts of surfactant, 0-1000 parts of freeze-drying protective agent and a proper amount of other pharmaceutically acceptable auxiliary materials on the basis of parts by weight; preferably, 1 part of nitrosourea antineoplastic drugs, 5-100 parts of albumin, 1-20 parts of surfactants, 1-300 parts of freeze-drying protective agents and a proper amount of other pharmaceutically acceptable auxiliary materials.

The freeze-drying protective agent in the nitrosourea antitumor drug albumin conjugate freeze-dried powder injection is selected from the following components: one or more of glucose, sucrose, maltose, lactose, mannitol, trehalose, glycine and dextran. The preferred lyoprotectant is glucose. The dried powder can automatically disperse to form a solution when contacting an aqueous medium.

The other auxiliary materials are selected from conventional pharmaceutics such as an isotonic regulator, an antioxidant, a preservative, a pH regulator and the like.

The nitrosourea antineoplastic drug albumin conjugate preparation is administrated by injection, oral administration, in-vivo local administration, mucous membrane absorption administration and the like.

One of the purposes of the invention is to provide a nitrosourea antineoplastic drug albumin conjugate preparation, which is selected from injection preparations, oral preparations, sustained release preparations, controlled release preparations, mucosal absorption preparations and the like.

One of the purposes of the invention is to provide the application of the nitrosourea antineoplastic drug albumin conjugate preparation in preparing drugs for reducing the side effects of nitrosourea antineoplastic drugs, wherein the side effects comprise the lack of selectivity of the nitrosourea antineoplastic drugs on tumor tissues and the toxic and side effects of bone marrow suppression, nausea, vomiting, inappetence, fever, rash, alopecia, transaminase reduction, urea nitrogen increase and the like exist.

One of the purposes of the invention is to provide a preparation method of a nitrosourea antineoplastic drug albumin conjugate preparation, which is characterized by comprising the following steps:

(1) taking pharmaceutically acceptable salt of nitrosourea antineoplastic drugs, and neutralizing corresponding acid with alkaline substances; or dissolved in a fat-soluble solvent;

(2) dissolving a surfactant in a fat-soluble solution, and mixing with the solution obtained in the step (1);

(3) concentrating under reduced pressure to remove liposoluble solvent;

(4) dissolving albumin in appropriate amount of water;

(5) and (4) mixing the components (3) and (4), shaking until the solution is clear and transparent, filtering and sterilizing by a microporous filter membrane, and standing for a moment to remove bubbles in the preparation to obtain the nitrosourea antitumor drug albumin conjugate preparation.

Wherein, the alkaline substance in the step (1) is mainly used for neutralizing acid radicals in pharmaceutically acceptable salts of nitrosourea antitumor drugs to obtain free alkali in the form of drug molecules. The alkaline substance is selected from one or more of sodium hydroxide, sodium carbonate, sodium bicarbonate, potassium hydroxide, potassium carbonate, potassium bicarbonate, triethylamine and ammonia water; the alkaline substance in the step (1) is preferably sodium bicarbonate, and the adding amount of the alkaline substance can be determined according to the pharmaceutical properties.

In a specific embodiment, the microfiltration membrane is preferably a 0.22 μm microfiltration membrane.

The fat-soluble solvent of steps (1) and (2) of the present invention is selected from chloroform, dichloromethane, ethyl acetate, ethanol, methanol, tetrahydrofuran, dioxane, acetonitrile, acetone, dimethyl sulfoxide, dimethylformamide, methylpyrrolidone or similar solvents, and one or more mixtures thereof may be selected. Anhydrous ethanol is preferred. The temperature range of the reduced pressure concentration in the step (3) is 20-70 ℃, and preferably 50 ℃.

As a specific embodiment, the nitrosourea antineoplastic albumin conjugate preparation obtained by the above method can be prepared by adding a lyoprotectant in step (1), (4) or (5) and lyophilizing into powder by conventional methods in the art.

THE ADVANTAGES OF THE PRESENT INVENTION

The albumin material has the characteristics of safety, no toxicity, no immunogenicity, biodegradability, good biocompatibility and the like, so that the albumin material has the following advantages in the aspect of drug delivery:

(1) the aim of targeted delivery can be achieved, and the tumor distribution of the drug is improved;

(2) can reduce the toxic and side effects of nitrosourea antineoplastic drugs;

(3) the compound has good drug-loading capacity on nitrosourea drugs and salt forms thereof;

(4) the preparation method is simple and is suitable for large-scale industrial production;

(5) the treatment effect on tumor-bearing mice is good;

(6) can improve the stability of the medicine and is beneficial to storage;

(7) the medicine has high stability and good property.

Drawings

FIG. 1 is the appearance and electron micrograph of nimustine albumin conjugate preparation;

fig. 2 is a survival curve for C6 brain glioma-bearing mice.

Detailed Description

The following examples are further illustrative of the present invention and are in no way intended to limit the scope of the invention. The present invention is further illustrated in detail below with reference to examples, but it should be understood by those skilled in the art that the present invention is not limited to these examples and the preparation method used. Also, equivalent substitutions, combinations, improvements or modifications of the invention may be made by those skilled in the art based on the description of the invention, but these are included in the scope of the invention.

Example 1

Taking 300 mg of human serum albumin, adding 10mL of water for injection to dissolve to obtain a human serum albumin solution. Dissolving nimustine hydrochloride 15mg and Solutol HS 1530 mg in a proper amount of absolute ethyl alcohol, and removing the absolute ethyl alcohol by rotary evaporation at 50 ℃. Adding human serum albumin solution, shaking to obtain clear and transparent solution, filtering, and sterilizing. And (4) carrying out freeze drying under aseptic conditions to obtain the nimustine albumin conjugate preparation.

Example 2

Taking 300 mg of human serum albumin, adding 10mL of water for injection to dissolve to obtain a human serum albumin solution. Dissolving semustine 15mg and Solutol HS 1530 mg in chloroform, adding glucose 100 mg, and rotary evaporating at 70 deg.C to remove chloroform. Adding human serum albumin solution, shaking to obtain clear transparent solution, filtering, and sterilizing. And (4) carrying out freeze drying under aseptic conditions to obtain the semustine albumin conjugate preparation.

Example 3

Dissolving 500 mg of human serum albumin in 10mL of water for injection to obtain a human serum albumin solution. Dissolving nimustine hydrochloride 5mg in water 1mL, adding NaHCO 50mg/mL₃The solution was 20 μ L vortexed for one minute and centrifuged to obtain nimustine. Nimustine and 5mg tween 80 were dissolved in chloroform, 1.5 g lactose was added and chloroform was removed by rotary evaporation at 50 ℃. Adding human serum albumin solution, shaking to obtain clear and transparent solution, filtering with microporous membrane for sterilization, standing for a moment to remove air bubbles in the preparation, and freeze drying under aseptic condition to obtain nimustine albumin conjugate preparation.

Example 4

Taking 200 mg of human serum albumin, adding 10mL of water for injection to dissolve to obtain a human serum albumin solution. 5mg of carmustine and 155 mg of Solutol HS are dissolved in a proper amount of methanol, and methanol is removed by rotary evaporation at the temperature of 20 ℃. Adding human serum albumin solution, shaking to obtain clear and transparent solution, filtering, sterilizing, adding sucrose as freeze-drying protectant to obtain carmustine albumin binding solution containing 10% sucrose, and freeze drying under aseptic condition to obtain the carmustine albumin conjugate.

Example 5

Taking 50mg of human serum albumin, adding 10mL of water for injection to dissolve to obtain a human serum albumin solution. Dissolving nimustine hydrochloride 1 mg and Solutol HS 155 mg in appropriate amount of anhydrous ethanol, and removing the anhydrous ethanol by rotary evaporation at 45 ℃. Adding human serum albumin solution, shaking to obtain clear transparent solution, filtering with microporous membrane for sterilization, and standing for a moment to remove air bubbles in the preparation to obtain nimustine albumin conjugate preparation.

Example 6

Dissolving 500 mg of human serum albumin in 10mL of water for injection to obtain a human serum albumin solution. Dissolving fotemustine 5mg and sodium dodecyl sulfate 100 mg in appropriate amount of anhydrous ethanol, and removing the anhydrous ethanol by rotary evaporation at 50 ℃. Mixing the two, shaking to obtain clear and transparent solution, filtering with microporous membrane for sterilization, and standing for a moment to remove air bubbles in the preparation to obtain fotemustine albumin conjugate preparation.

Example 7

Taking 100 mg of bovine serum albumin, adding 10mL of water for injection to dissolve to obtain a bovine serum albumin solution. 5mg of lomustine and 6810 mg of F are dissolved in a proper amount of chloroform, and the chloroform is removed by rotary evaporation at 30 ℃. Adding human serum albumin solution, shaking to obtain clear transparent solution, filtering, and sterilizing. Adding lyophilized protectant 5% glucose and 5% mannose to obtain lomustine albumin conjugate solution containing 5% glucose and 5% mannose, which is clear and transparent, and lyophilizing under aseptic condition to obtain lomustine albumin conjugate preparation.

Example 8

Taking 500 mg of bovine serum albumin, adding 9 mL of water for injection to dissolve to obtain a bovine serum albumin solution. 2 mg of ranimustine and 20 mg of lauryl sodium sulfate are taken to be dissolved in a proper amount of methanol, and the methanol is removed by rotary evaporation at the temperature of 30 ℃. Mixing the two solutions, incubating for 5 min, filtering with microporous membrane for sterilization, and standing for a while to remove air bubbles in the solution to obtain clear and transparent ranimustine albumin conjugate.

Example 9

Taking 80 mg of bovine serum albumin, adding 10mL of water for injection to dissolve to obtain a bovine serum albumin solution. Dissolving nimustine hydrochloride 5mg in water 1mL, adding NaHCO 50mg/mL₃The solution is 40 mu L, vortexed for 1 min, and centrifuged to obtain nimustine. Nimustine and Solutol HS 1520 mg were dissolved in chloroform and the chloroform was removed by rotary evaporation at 50 ℃. Adding bovine serum albumin solution, shaking to obtain clear transparent solution, filtering, sterilizing, adding lyophilized protectant glucose to obtain nimustine albumin conjugate solution containing 10% glucose, and freeze drying under aseptic condition to obtain nimustine albumin conjugate preparation.

Example 10

Dissolving 5mg of human serum albumin in 10mL of water for injection to obtain a human serum albumin solution. Nimustine hydrochloride 1 mg and sodium dodecyl sulfate 2 mg are dissolved in 10mL water. Mixing the two solutions, incubating for 5 min, filtering with microporous membrane for sterilization, and standing for a while to remove air bubbles in the solution to obtain clear and transparent nimustine albumin conjugate.

Experimental example 1

Preparation example 3, test example 1 and test example 2 were carried out in accordance with the preparation method of example 3, the albumin solution in the formulation of example 3 was changed to an equal volume of water (test example 1), the formulation of example 3 was changed without adding solutol HS15 (test example 2), and the appearance of the product before lyophilization of the preparation formulation was observed. The results are shown in the following table:

TABLE 1 Albumin and nimustine interactions

The results show that example 3 was a yellow clear transparent solution before lyophilization; the drugs of test examples 1 and 2 were not dispersed and could not be prepared into solutions. As can be seen from the experimental results, clear and transparent solutions can not be formed by the preparation without the albumin and without the surfactant, and the uniform and transparent solution can be formed by the combined action of the drug, the surfactant and the albumin.

Experimental example 2

The centrifugal method is adopted to measure the binding rate of different nitrosourea antineoplastic drug albumin conjugates.

The products of the examples were centrifuged by ultrafiltration (6000 r/min, 10 min) to determine the drug concentrations C1, C2 of the filtrate and the products of the examples. Binding rate = C1/C2 × 100%.

TABLE 2 binding rates of different nitrosoureas antineoplastic drugs with albumin after preparation

The result shows that different nitrosourea antineoplastic drugs and albumin in the preparation can have high binding rate with the albumin to form stable solution.

Experimental example 3

We examined the stability of nimustine in the formulation of example 1. It can be seen that the compound preparation is clear and transparent solution after being redissolved by 10mL of injection water, the content of the main drug in the preparation has no obvious change, no new impurity appears, and the peak time and the content of the original impurity have no obvious change within two months after being placed at room temperature.

Experimental example 4

The cytotoxicity of the product of example 1 before lyophilization and the aqueous nimustine hydrochloride solution (taking the nimustine hydrochloride 15mg dissolved in 10mL water) at the same concentration (test example 3) was compared by MTT cytotoxicity test. Selecting 4T1 cells in good state and logarithmic growth phase, and dividing the cells into 1 × 10 cells⁴The concentration per well was seeded in 96-well plates at 37 ℃ in 5% CO₂And (5) standing and culturing in an incubator for 24 hours. The medium was discarded and the products of experimental example 3 and example 1 diluted in complete medium to a series of concentration gradients and the blank vehicle were added. Each group has 8 concentration gradients, each concentration is provided with 5 multiple holes, and then a group of positive control holes without medicine and negative control holes only with culture medium and without cells are arranged. After dosing, 5% CO at 37 ℃₂After CO-culturing the drug solution and cells in the incubator of (1) for 24 hours, the drug-containing medium was discarded, 100. mu.L of MTT solution (0.5 mg/mL) was added to each well, incubation was continued for 4 hours at 37 ℃ under 5% CO2, the 96-well plate was removed from the incubator, and the supernatant in the well was carefully aspirated. Add 150. mu.L of DMSO per well and shake for 15 min at 37 ℃ on a constant temperature shaker at 100 rpm to fully solubilize the crystalline formazan. The absorbance of each well at 490 nm was measured using an enzyme linked immunosorbent assay and the results recorded. The cell survival rate was calculated according to the following formula = (absorbance of experimental group-absorbance of negative control group)/(absorbance of positive control group-absorbance of negative control group) × 100%.

The results show that compared with the original drug group, the nimustine albumin conjugate preparation has obviously lower survival rate of tumor cells, and the preparation has better in-vitro anti-tumor effect compared with the original drug group.

Experimental example 5

We examined the antitumor effect in vivo.

Healthy male Kunming mice (28-30 g in weight) were anesthetized by intraperitoneal injection of 4% chloral hydrate (about 400 mg/kg). The hair of the mouse head is properly subtracted, after the hair is disinfected by 75% ethanol, the disinfected ophthalmic scissors are used for cutting the scalp by about 8 mm along the sagittal midline of the head, a cotton swab dipped with 10% hydrogen peroxide is used for quickly removing a tissue membrane to expose the skull, the cotton swab dipped with PBS is used for cleaning the hydrogen peroxide, the head of the mouse is fixed on a brain stereotaxic apparatus, the positions 1.8 mm to the right and 0.6 mm to the back of a bregma are positioned, a small hole is drilled at the positioning point by using a needle head of a 2 mL injector, and the drilling depth is controlled to avoid bleeding. A well-grown and log phase C6 cell suspension (5. mu.L) (1X 10) was aspirated by a microinjection needle⁶And (2) inserting the needle by 4 mm and withdrawing the needle by 1 mm in the direction vertical to the small hole, slowly injecting the cell suspension into the brain, and taking out the sample injection needle after the sample injection needle stays at the position of the small hole for 5 min. The wound of the mouse is smeared with a cotton swab dipped with the double antibody, and then the wound is sutured by using an operation line. Mice inoculated with C6 cells were randomly divided into 3 groups of 9 mice, and the following drugs were administered by tail vein injection on days 4, 7, and 10 after inoculation of C6 cells, respectively: the survival conditions of the mice in each group were observed and recorded in physiological saline, the product of test example 3 (15 mg/kg), and the product of example 1 (15 mg/kg), and survival curves were plotted. The results are shown in figure 2 and the following table:

TABLE 3 survival rates of different groups of tumor-bearing mice

On day 25 after inoculation, all mice in the saline group died, and all mice in the experimental example 3 group died on day 31, while the survival rate of the product group of example 1 was still over 75%. The survival rate of the mice in the product group of the example 1 is still 66.7% at day 78, which shows that the products of the example 1 and the example 1 can effectively prolong the survival period of the mice with the C6 glioma and the treatment effect is obviously improved compared with the product of the test example 3.

In conclusion, the nimustine albumin conjugate preparation obviously improves the drug effect of resisting brain glioma.

Experimental example 6

We examined toxicity.

Bone marrow suppression: healthy male Wistar rats which are raised for about one week and 200 +/-20 g are randomly divided into 5 groups of 8 rats, and the grouping condition is as follows: group A: a physiological saline solution group; group B: the original drug group (product of experimental example 3) (15 mg/kg); group C: formulation group (product of example 1) (15 mg/kg); group D: group of 3% albumin solutions (equivalent to administered amount of albumin in group C); group E: the original drug group and the 3% albumin mixed administration group (namely, the original drug group with the same dose and the 3% albumin solution are respectively injected into the bodies of rats).

Before administration, about 0.5 mL of whole blood is taken from the orbit of each group of rats and placed in an EDTA-2K tube to slightly shake so as to prevent blood coagulation, the level of blood leukocytes of each group of rats before administration is measured by a hematology analyzer, and the experimental result is recorded. Every two days after the administration, blood was taken for leukocyte measurement starting 24h, until the leukocyte level had decreased to a minimum and began to rise again. The results for the number of leukocytes in each group of rats are shown in the following table:

TABLE 4 leukocyte counts on days 7 and 9 of different groups administered

It can be seen that, at the same administration dose, there was no significant change in the white blood cells of the rats in both the normal saline group (group a) and the 3% albumin solution group (group D), while the white blood cell values of the remaining three groups all decreased to some extent after administration, reached the lowest value on the third day after administration, and then started to rise again, and by the seventh day after administration, the white blood cell count of the preparation group had risen to 7 (. 10) (-10)⁹L), in the normal range (5-25 x 10)⁹L) and the white blood cell count of the original drug group is 3.2 (. about.10)⁹L), the two are significantly different; by the ninth day after administration, the number of white blood cells of the preparation group was 18.5 (. about.10)⁹L), substantially returns to the pre-administration leukocyte level, and the drug substanceThe number of leukocytes in the group was 3.32 (. about.10)⁹/L), still below the normal range of values, with significant differences from the formulation group. The result shows that the nimustine hydrochloride raw drug has obvious bone marrow suppression effect, and the nimustine albumin conjugate preparation prepared by the nimustine hydrochloride raw drug can effectively reduce bone marrow suppression toxicity.

Experimental example 7

We have examined the distribution in vivo.

In vivo distribution experiments of nimustine albumin conjugate formulations: healthy male Kunming mice (28-30 g in weight) were anesthetized by intraperitoneal injection of 4% chloral hydrate (about 400 mg/kg). The hair of the mouse head is properly subtracted, after the hair is disinfected by 75% ethanol, the disinfected ophthalmic scissors are used for cutting the scalp by about 8 mm along the sagittal midline of the head, a cotton swab dipped with 10% hydrogen peroxide is used for quickly removing a tissue membrane to expose the skull, the cotton swab dipped with PBS is used for cleaning the hydrogen peroxide, the head of the mouse is fixed on a brain stereotaxic apparatus, the positions 1.8 mm to the right and 0.6 mm to the back of a bregma are positioned, a small hole is drilled at the positioning point by using a needle head of a 2 mL injector, and the drilling depth is controlled to avoid bleeding. A well-grown and log phase C6 cell suspension (5. mu.L) (1X 10) was aspirated by a microinjection needle⁶One), inserting the needle 4 mm and withdrawing the needle 1 mm along the direction vertical to the small hole, slowly injecting the cell suspension into the brain, and taking out the sample injection needle after the sample injection needle stays for 5 min at the position of the small hole. The wound of the mouse is smeared with a cotton swab dipped with double antibodies, and then the wound is sutured with an operation thread. Tumor-bearing mice successfully modeled were randomly divided into 6 groups of 6 mice each, of which 3 groups were administered with 15mg/kg of the product of test example 3 by tail vein injection, and the other 3 groups were administered with 15mg/kg of the product of example 1 by tail vein injection. Dissecting a group of tumor-bearing mice at 15 min, 1 h, and 4h after administration, collecting brain tissue, cleaning with normal saline, sucking water with filter paper, weighing, and freezing at-80 deg.C. The treatment was performed according to the biological sample treatment method and the drug concentration in brain was measured by LC-MS/MS. The results are shown in the following table:

the results show that the concentration of the drug in brain of each mouse in the preparation group is significantly higher than that in the original drug group at each time point compared with the free drug group, which indicates that the nimustine albumin conjugate preparation can penetrate through the blood brain barrier and reach the brain tumor for treatment.

In conclusion, the nitrosourea antitumor drug albumin conjugate preparation prepared by the invention has the advantages of small auxiliary material dosage, very simple preparation and good auxiliary material biocompatibility, and the preparation prepared by the invention has good drug effect on the mouse with the brain glioma, can obviously improve the survival rate of the mouse and reduce the toxicity. Therefore, the preparation prepared by the invention is expected to be used for industrial production and clinical application.

Claims

1. A nitrosourea drug albumin conjugate preparation is characterized in that non-particle conjugates dissolved in water are formed among nitrosourea drugs, albumin and surfactants through intermolecular force of non-covalent bonds, and the nitrosourea drug albumin conjugate preparation comprises, by weight, 1 part of nitrosourea drugs, 1-1000 parts of albumin and 1-100 parts of surfactants.

2. The nitrosourea-based albumin conjugate preparation according to claim 1, comprising 1 part of nitrosourea-based drug, 5 to 100 parts of albumin, and 1 to 20 parts of surfactant.

3. The nitrosourea-based pharmaceutical albumin conjugate formulation according to claim 1, wherein the nitrosourea-based drug is selected from one or more of Nimustine (ACNU, Nimustine), dithiomustine (Ditiomustine), brivudine (Bofumustine), carmustine (BCNU, carmustine), emestine (Elmustine), Ecomustine (Ecomustine), Fotemustine (Fotemustine), nemustine (Pentamustine, Neptamustine), lomustine (Lomustine, CCNU), Semustine (Semustine), ramustine (Ranimustine), tautomustine (Tauromustine).

4. A nitrosourea-based pharmaceutical albumin conjugate formulation as claimed in claim 1, wherein the albumin is selected from human serum albumin and/or bovine serum albumin; the albumin is added in a concentration range of 0.1-25% (w/v); the surfactant is selected from one or more of sodium dodecyl sulfate, Solutol HS15, tween, maize, beneze and poloxamer.

5. A nitrosourea-based pharmaceutical albumin conjugate formulation as claimed in claim 4, wherein the albumin is selected from human serum albumin.

6. A nitrosourea-based pharmaceutical albumin conjugate formulation as claimed in claim 4, wherein albumin is added at a concentration in the range 0.5-5% (w/v).

7. A nitrosourea-based pharmaceutical albumin conjugate formulation as claimed in claim 4, wherein the albumin is a 3% (w/v) aqueous solution of human serum albumin.

8. The nitrosourea-based pharmaceutical albumin conjugate formulation according to claim 4, wherein the surfactant is selected from Solutol HS15 or poloxamer.

9. The nitrosourea-based pharmaceutical albumin conjugate formulation of claim 4, wherein the surfactant is selected from Solutol HS 15.

10. The nitrosourea-based albumin conjugate preparation according to claim 1, which is a lyophilized powder injection.

11. The nitrosourea drug albumin conjugate formulation according to claim 10, wherein the formulation comprises a lyoprotectant selected from the group consisting of one or more of glucose, sucrose, maltose, lactose, mannose, trehalose, glycine, dextran, in an amount of 0-1000 parts by weight.

12. The nitrosourea-based pharmaceutical albumin conjugate formulation of claim 11, wherein the lyoprotectant is glucose.

13. The nitrosourea-based albumin conjugate preparation according to claim 11, wherein the lyoprotectant is present in an amount of 1 to 300 parts.

14. A nitrosourea-based pharmaceutical albumin conjugate formulation as claimed in claim 1, further comprising other excipients selected from one or more of an isotonicity adjusting agent, an antioxidant, a preservative, a pH adjusting agent to make a pharmaceutically acceptable formulation.

15. The nitrosourea-based pharmaceutical albumin conjugate formulation according to claim 1, wherein the formulation is selected from the group consisting of an injectable formulation, an oral formulation, a sustained release formulation, a controlled release formulation, a mucosal absorption formulation.

16. A process for the preparation of a nitrosourea drug albumin conjugate formulation as claimed in any one of claims 1 to 15, comprising the steps of:

(1) taking pharmaceutically acceptable salt of nitrosourea drugs, neutralizing corresponding acid with alkaline substances, and dissolving in a fat-soluble solvent; or directly dissolving nitrosourea drugs or pharmaceutically acceptable salts thereof in a fat-soluble solvent;

(3) concentrating under reduced pressure to remove liposoluble solvent;

(4) dissolving albumin in water;

(5) and (4) mixing the components (3) and (4), shaking until the solution is clear and transparent, filtering and sterilizing by using a microporous filter membrane, and standing for a moment to remove air bubbles in the preparation to obtain the nitrosourea drug albumin conjugate preparation.

17. The preparation method according to claim 16, wherein the fat-soluble solvent is selected from the group consisting of chloroform, dichloromethane, ethyl acetate, ethanol, methanol, tetrahydrofuran, dioxane, acetonitrile, acetone, dimethyl sulfoxide, dimethylformamide, and methyl pyrrolidone; in the step (3), the reduced pressure concentration temperature is 20-70 ℃; the microporous filter membrane is a 0.22 mu m microporous filter membrane.

18. The process according to claim 16, wherein the solvent is selected from the group consisting of absolute ethanol.

19. The method according to claim 16, wherein the concentration under reduced pressure in the step (3) is carried out at a temperature of 50 ℃.

20. Use of a formulation according to any one of claims 1 to 15 or a method of manufacture according to any one of claims 16 to 19 in the manufacture of a medicament for increasing the therapeutic effect of nitrosoureas which reduce toxicity, wherein toxicity includes bone marrow suppression, nausea, vomiting, anorexia, fever, rash, alopecia, decreased transaminases, increased urea nitrogen.