CN110759928A - Preparation of Camptothecin Drug Nanocrystals by Reversible Decomposition Method - Google Patents

Abstract

The invention provides a method for preparing nanocrystals of camptothecin drugs by using a reversible decomposition method. The nanocrystals provided by the invention are needle-shaped or rod-shaped and can be free of any excipients, thereby retaining the antitumor activity of the drug, enhancing the tumor penetration effect of the drug, and reducing the toxic and side effects. The preparation process is simple, suitable for industrial production, and has broad application prospects.

Description

Preparation of Camptothecin Drug Nanocrystals by Reversible Decomposition Method

technical field

The invention specifically relates to nanocrystals of camptothecin drugs, a preparation method and application thereof, and belongs to the technical field of medicine.

Background technique

Anti-tumor therapy mainly relies on small-molecule anti-tumor drugs. Usually, anti-tumor drugs have many clinical application defects and limitations, such as: low drug solubility, difficult to dissolve in clinically available solvents; short plasma half-life of drugs, which must be administered frequently To maintain effective blood drug concentration; anti-tumor drugs have side effects on the body, and frequent administration of toxic and side effects aggravates. Some highly active antitumor drugs, such as camptothecin, paclitaxel, etc., cannot be directly administered intravenously due to the low solubility of the drug in water, and the solubility of the drug must be improved by means of chemical modification, solubilizer or new formulation technology.

Camptothecins are quinoline alkaloids with cytotoxicity, which are specific inhibitors of topoisomerase I and have certain inhibitory effects on various tumor cells. At present, the FDA-approved camptothecin drugs are irinotecan (CPT-11) and topotecan, which are obtained by chemical modification of 7-ethyl-10-hydroxycamptothecin (SN38). prodrugs. The in vitro antitumor activity of CPT-11 is one percent or even one thousandth of that of SN38. In vivo, it needs to be metabolized by carboxylesterase II and liver microsomes to the parent drug SN38 to exert its efficacy. However, human carboxylesterase activity is not as strong as that of murine carboxylesterase, so the human pharmacological activity of CPT-11 is not as obvious as animal experiments. Studies have shown that the proportion of CPT-11 converted to SN38 within 24h is only about 2-8%. Moreover, under physiological conditions, camptothecin drugs have a dynamic process of "E-ring" ring opening/closing, and the ring-opened carboxylic acid form of camptothecin drugs loses their pharmacological activity and can be quickly eliminated in vitro. Therefore, there is an urgent need to find a camptothecin drug preparation with strong antitumor activity and suitable for clinical application.

CN106963731 A provides a precursor compound 7-ethyl-10 tert-butoxycarbonyl camptothecin obtained by derivatizing and modifying SN38 to form pH-sensitive micelles. The prodrug micelle group had obvious antitumor activity relative to the physiological group. CN105777770 A provides a SN38 prodrug modified by a saturated long-chain fatty acid prepared by chemical modification of SN38, and a long-circulating liposome for preparing the prodrug. In vitro experiments showed that the prototype ring-opening speed of the prodrug long-circulating liposomes was greatly reduced, effectively increasing the concentration of the active drug. However, these preparation methods have potential safety hazards such as low drug loading, introduction of new excipients, organic solvent residues (such as chloroform, dichloromethane) and formulation stability.

In recent years, nanocrystalline drugs (NCs) have attracted much research attention due to their drug loading as high as 100%, which can significantly increase the efficiency of drug entry into cells, maximize drug efficacy, and reduce toxic and side effects (Muller RH, Gohla S, Keck CM. State of the art of nanocrystals-Special features, production, nanotoxicology aspects and intracellular delivery. European Journal of Pharmaceutics and Biopharmaceutics. 2011;78:1-9.). As a preparation intermediate, NCs can be further prepared into preparations suitable for oral, injection, spray, transdermal and other administration routes. At present, 6 nanocrystal intermediate drug varieties have been approved by the FDA for marketing. Therefore, by developing a safe, low-toxic, and high-drug loading camptothecin-based drug nanocrystal, it is expected to prepare a camptothecin-based drug preparation with high antitumor activity and suitable for clinical application.

The main preparation theories of nanocrystals are: "top-to-bottom", "bottom-to-top" and comprehensive use of two preparation methods. The crystals formed under supersaturated conditions are usually needle-like or dendrite-like, which are highly targeted to the mononuclear macrophage system (WangYC, Zheng Y, Zhang L, Wang QW, Zhang DR. Stability of nanosuspensions in drug delivery. Journal of Controlled Release.2013;172:1126-41.). CN106466296 A and literature (Yang Xiaofeng, Guo Ruiqi, Su Wenjing, etc.. Screening of hydroxycamptothecin nanocrystal formulations and preliminary industrialization. Pharmacy Herald, December 2016, Vol. 35, Issue 12. End.) Preparation of spherical nanocrystals of camptothecin-based drugs containing stabilizer by "two acid precipitation". The provided hydroxycamptothecin nanocrystals are spherical, and when the stabilizer is not included, the stability is poor at 4°C. Literature studies have pointed out that acidic substances can accelerate the Ostwald ripening process of nanocrystals and affect the stability of nanocrystals (M.P.Hendricks, B.M.Cossairt, J.S.Owen. The importance of nanocrystalprecursor conversion kinetics: mechanism of the reaction between cadmiumcarboxylate and cadmium bis(diphenyldithiophosphinate) .Acs Nano. 2012;6(11):10054-10062.). Nanocrystals prepared according to conventional theory usually have crystalline or amorphous structure characteristics. Studies have reported that nanorods with sharp edges or irregular shapes have significantly better passive transport efficiency and tumor penetration than spherical nanoformulations with regular shapes. Although the preparation theory of nanocrystals is relatively mature, few nanocrystals have a particle size below 220 nm. In addition, due to the influence of Ostwald ripening, nanocrystals generally have the defect of rapid particle size growth, and stabilizers need to be added to maintain the relative stability of particle size. Numerous factors limit the clinical application of nanocrystal formulations (Al-Kassas R, Bansal M, Shaw J. Nanosizing techniques for improving bioavailability of drugs. Journal of controlled release: Official Journal of the Controlled Release Society. 2017;260:202-12.) .

After a lot of experiments, the inventor of the present invention accidentally found that the "E" ring of camptothecin drugs has the structural characteristics of "closed ring/open ring" dynamic conversion under physiological conditions. The nanocrystals of camptothecin drugs with a particle size of about 150 nm and good stability, in the form of needles or rods, can promote the penetration of drugs in the tumor site, reduce the toxic and side effects, and have good clinical application prospects.

SUMMARY OF THE INVENTION

One of the objectives of the present invention is to provide a nanocrystalline preparation of acicular or rod-shaped camptothecin drugs with a particle size of 100-250 nm, and the nanocrystalline preparation is composed of only camptothecin drugs.

One of the objectives of the present invention is to provide a method for preparing nanocrystals of camptothecin drugs.

One of the objectives of the present invention is to provide a nanocrystal of camptothecin drugs, which is mainly prepared by a reversible decomposition method.

Wherein, the reversible decomposition method refers to: under a certain pH condition, the "E ring" of camptothecin drugs has a dynamic conversion of prototype/carboxylic acid type (taking the dynamic conversion of SN38 as an example, as shown below, under physiological conditions The dynamic balance of "ring opening/closing"), based on this characteristic, the prototype/carboxylate camptothecin mixture can be prepared. The ring-opened carboxylic acid-type camptothecin drug has a hydrophobic condensed ring structure and a hydrophilic "E-ring" ring-opening structure, and has characteristics similar to surfactants. When the mixture is dispersed in water, there is a strong intermolecular force between the hydrophobic end of carboxylate camptothecin and the prototype drug, forming a stable hydrophobic inner core; the carboxylic acid structure of the E ring-opening has strong hydrophilic ability , the hydrophilic end of the outer ring can be formed to solubilize the prototype drug, and a highly dispersed, self-assembled hybrid drug nanocrystal can be obtained. Finally, the volatile alkaline reagent is removed by the preparation method, the carboxylic acid-type camptothecin drug with no pharmacological activity is reversed to the prototype drug, and the solubilized mixed-type drug nanocrystals are reversed to the prototype camptothecin drug nanocrystals, which are retained. Pharmacological activity of drugs.

The nanocrystals of camptothecin drugs provided by the invention are characterized in that the nanocrystals only consist of 100% of the drugs. The shape of the nanocrystals is needle-like or rod-like. The particle size of the nanocrystals is 100-250 nm. The drugs constituting the nanocrystals mainly exist in the form of prototype drugs, and the proportion of prototype drugs in camptothecin drugs is greater than 95%.

The camptothecin-based drug nanocrystal provided by the invention is characterized in that the nanocrystal can be composed of a camptothecin-based drug and a freeze-drying protective agent. The particle size of the nanocrystals is 100-250 nm.

Wherein, the camptothecin drugs are selected from irinotecan (CPT-11), 7-ethyl-10-hydroxycamptothecin (SN38), 10-hydroxycamptothecin (HCPT), belonotecan One or more of (CDK-602) and topotecan (TPT) are mixed, and the camptothecin can also be selected from the acid salt form of the above-mentioned raw materials, preferably hydrochloride. Wherein, the structural formula of the camptothecins is as follows:

Wherein, the freeze-drying protective agent is selected from one or more mixtures of trehalose, sucrose, mannitol, lactose, maltose, glucose or human serum albumin, preferably trehalose or human serum albumin; freeze-drying protection The mass ratio of the agent to the camptothecin drugs is (0:1)～(100:1), and the preferred mass ratio of the lyophilized protective agent is (15:2)～(50:1).

One of the objectives of the present invention provides a method for preparing nanocrystals of camptothecins by reversible decomposition method, comprising the following steps:

(1) the camptothecin drug is dispersed in the reaction system, stirred at room temperature in the dark until the solution is clear, and the reaction is stopped; the reaction system includes a reaction reagent and a reaction solvent;

(2) Removing part of the reaction reagents and reaction solvent in the reaction (1) to obtain a solid powder or a suspension solution. Among them, the mass ratio of the carboxylic acid type drug to the prototype drug is 1:5 to 10:1;

(3) disperse the solid powder obtained in step (2) in water, and disperse uniformly to obtain solution I;

(4) Filtration of solution I, freeze-drying, to obtain camptothecin-based drug nanocrystal freeze-dried powder;

(5) Before use, add a solvent for injection to reconstitute the nanocrystal freeze-dried powder of camptothecin drugs obtained in step (4), disperse evenly, and then use.

In the present invention, the reaction reagent described in step (1) is a low-boiling, basic compound, including but not limited to triethylamine, diethylamine, ethylamine, trimethylamine, dimethylamine, monomethylamine or ammonia, preferably It is one of monomethylamine, ammonia water or its mixture; the volume/mass ratio of the reaction reagent and camptothecins is (2:1)～(20:1) (mL/g), preferably, the volume/mass ratio is The mass ratio is (5:1) to (10:1) (mL/g).

The characteristics of the reaction solvent are: a solvent with strong polarity and mutual solubility with the alkaline reaction reagent, such as methanol, ethanol, tetrahydrofuran or water, preferably one of methanol, ethanol and water.

The method of removing the reaction solvent in step (2) is freeze-drying, vacuum drying, and rotary evaporation under reduced pressure, preferably a rotary evaporation method under reduced pressure. The rotary evaporation temperature is 30-60°C, preferably 37-50°C.

The dispersion method in step (3) includes but is not limited to one or more of rapid stirring, water bath ultrasound, probe ultrasound or high pressure homogenization, preferably probe ultrasound and/or high pressure homogenization.

The filtering method in step (4) is selected from sterile filtration with a 0.22 μm microporous membrane.

In step (5), the solvent for injection includes, but is not limited to, one or more of 5% glucose solution, 0.9% sodium chloride solution or water for injection, preferably one or more of 5% glucose solution and water for injection.

A freeze-drying protective agent may also be added in the step (3), specifically, the solid powder obtained in the step (2) is dispersed in an aqueous solution containing the freeze-drying protective agent. The freeze-drying protective agent is selected from one or more of trehalose, sucrose, mannitol, lactose, maltose, glucose or human serum albumin. The prepared nanocrystal solution containing lyoprotectant can be stored at -20°C. The mass ratio of the freeze-drying protective agent to the camptothecin drugs is (0:1)～(100:1). When adding a lyoprotectant, the preferred mass ratio is (15:2) to (50:1).

Invention Benefit

(1) The needle-shaped or rod-shaped camptothecin drug nanocrystals provided by the present invention not only retains the anti-tumor activity of the drug, reduces the toxic and side effects, and promotes the penetration of the drug at the tumor site, and the preparation process is simple, and the preparation equipment requires It is suitable for industrial production and has broad application prospects.

(2) The nanocrystal preparation of camptothecin drugs provided by the present invention may not add any auxiliary materials, has high drug loading and good safety, and effectively avoids the safety risks caused by the auxiliary materials.

(3) The method for preparing camptothecin-based drug nanocrystals by the reversible decomposition method provided by the present invention has the advantages of simple operation and low requirements for equipment and equipment. Based on the reversible decomposition properties of the prototype camptothecin-based drug, a carboxylic acid type with increased polarity is obtained. The compound, using its amphiphilicity, can solubilize the poorly soluble prototype drug, and then reversibly decompose into the prototype drug, avoiding the use of surfactants and other excipients, avoiding the introduction of precursor compounds or derivatives, and avoiding the use of acidic reagents. The introduction is beneficial to improve the stability of the nanocrystal preparation, to improve the safety of the preparation, to retain the antitumor activity of the drug, and to reduce the toxic and side effects.

Description of drawings

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, wherein:

Figure 1: Thermogram of the SN38 prototype/carboxylate mixture. (A) Differential scanning calorimetry analysis of SN38 drug substance. (B) Differential scanning calorimetry analysis of the SN38 prototype/carboxylic acid type mixture. (C) Thermogravimetric analysis of SN38 drug substance. (D) Thermogravimetric analysis of the SN38 prototype/carboxylate mixture.

Figure 2: Diffraction analysis pattern of SN38 nanocrystalline powder.

Figure 3: Schematic diagram of HPLC before and after lyophilization of the SN38 prototype/carboxylate mixture. (A) Schematic diagram of HPLC of the SN38 prototype/carboxylic acid type mixture. (B) Schematic diagram of HPLC of SN38 nanocrystal solution.

Figure 4: Formulation characterization of nanocrystals. (A) Particle size distribution of SN38 nanocrystal solutions. (B) Zeta potential distribution of SN38 nanocrystal solution. (C) Particle size distribution of SN38/albumin nanocrystal solutions. (D) Zeta potential distribution of SN38/albumin nanocrystal solutions. (E) TEM image of SN38 nanocrystal solution. (F) TEM image of SN38/albumin nanocrystal solution. (G) TEM image of HCPT nanocrystal solution. (H) Scanning electron microscope analysis of freeze-dried samples of SN38 nanocrystals. (I) Scanning electron microscope analysis image of SN38 nanocrystal solution.

Figure 5: Drug concentration-time curves of SN38 nanocrystal formulation and irinotecan hydrochloride in healthy SD rats (n=5).

Figure 6: Organ distribution map of SN38 nanocrystal preparation and irinotecan hydrochloride at different time points in tumor-bearing mice (n=5). (A-D) is the statistic chart of drug concentration of SN38 in each main organ at 1h, 4h, 8h and 24h. (E) Statistical results of SN38 concentration at each time point in the tumor.

Figure 7: Pharmacodynamic experiments of SN38 nanocrystal formulation and irinotecan hydrochloride in tumor-bearing mice (n=5). (A) Tumor volume-time curve. (B) Body weight-time curves of tumor-bearing mice. (C) Tumor display diagram of each administration group. (D) Tumor weight of each administration group. (E) Tumor inhibition rate of each administration group.

Detailed ways

The following examples are further illustrations of the present invention, but in no way limit the scope of the present invention. The present invention is further described in detail below with reference to the examples, but those skilled in the art should understand that the present invention is not limited to these examples and the used preparation methods. Moreover, those skilled in the art can make equivalent substitutions, combinations, improvements or modifications to the present invention based on the description of the present invention, but these will all be included in the scope of the present invention.

Example 1

(1) Preparation of SN38 prototype/carboxylic acid type mixture

Take 1 mL of ammonia water, dissolve it in 5 mL of water, weigh SN38 (0.2 g) and disperse it in the aqueous ammonia solution, react at room temperature in the dark, until the solution is clear, stop the reaction, and remove the reaction solvent by rotary evaporation under reduced pressure at 60 °C to obtain SN38 prototype/carboxylic acid type mixture (light yellow flake solid), in which the mass percentage of the prototype drug is 82.1%.

(2) Preparation of SN38 nanocrystal formulations

The mixture (20 mg) obtained in step (1) was dispersed in 20 mL of purified water, and the probe was sonicated for 10 min (100 W) to obtain a blue-green opalescent solution. Subsequently, the preparation solution was filtered through a 0.22 μm filter membrane, and freeze-dried to obtain SN38 nanocrystal freeze-dried powder. Take 20 mL of 5% glucose solution for injection to reconstitute the lyophilized powder, shake and disperse to obtain SN38 nanocrystal solution.

Example 2

(1) Preparation of Camptothecin Drug Prototype/Carboxylic Acid Mixture

Take 2 mL of ammonia water, dissolve it in 5 mL of water, weigh camptothecin drugs (0.2 g, including CPT-11, HCPT, CDK-602 or TPT) and disperse them in the aqueous ammonia solution, react at room temperature in the dark, until the solution is clear, stop the reaction , After the reaction solvent was removed by rotary evaporation under reduced pressure at 60°C, the solid powder or suspension of the camptothecin drug prototype/carboxylic acid type mixture (light yellow flake solid or light yellow solution) was obtained.

(2) Preparation of camptothecin drug nanocrystal preparation

Disperse the mixture (20 mg) obtained in step (1) in 20 mL of purified water (or dilute the suspension to 250 mL and measure 25 mL), and ultrasonicate the probe for 10 min (100 W) to obtain a blue-green opalescent solution. Subsequently, the preparation solution was filtered through a 0.22 μm filter membrane, and freeze-dried to obtain a freeze-dried powder of nanocrystals. Take 5 mL of 5% glucose solution for injection to reconstitute the freeze-dried powder, and ultrasonically disperse to obtain the corresponding drug nanocrystal solution.

Table 1. Particle size and Zeta distribution of camptothecin nanocrystal solution prepared in Example 2

Example 3

(1) Preparation of SN38 prototype/carboxylic acid type mixture

Take 4 mL of triethylamine, dissolve it in 4 mL of water, weigh SN38 (0.2 g) and disperse it in an aqueous triethylamine solution, stir at room temperature in the dark, until the solution is clear, stop the reaction, and remove most of the alkaline reagents by vacuum drying to obtain SN38 Prototype/carboxylic acid type mixture suspension (light yellow liquid), wherein the mass percentage of prototype drug is 18.3%.

(2) Preparation of SN38 nanocrystal formulations

The mixture suspension obtained in step (1) was adjusted to 50 mL, and (25 mL of aqueous solution (with human serum albumin 50 mg) was added), and stirred rapidly to obtain a blue-green opalescent preparation solution. Subsequently, the preparation solution was freeze-dried to obtain SN38/albumin nanocrystal freeze-dried powder. Take 30mL of 0.9% physiological saline to redissolve the freeze-dried powder, shake and disperse to obtain a SN38 nanocrystal solution with a particle size of 135.8nm and a PDI of 0.102. The above steps can be obtained as required. The nanocrystal solution was frozen at -20°C.

Example 4

(1) Preparation of SN38 prototype/carboxylic acid type mixture

Same as Example 1.

(2) Preparation of SN38 nanocrystal formulations

The SN38 mixture (10 mg) obtained in the weighing step (1) was dispersed in 200 mL of aqueous solution (containing 500 mg of trehalose), and homogenized under high pressure (pressure 1000 bar, homogenized 5 times) to obtain a blue-green opalescent preparation solution. Subsequently, the preparation solution was filtered through a 0.22 μm filter membrane, and freeze-dried to obtain SN38/trehalose nanocrystal freeze-dried powder. Take 10 mL of water for injection to reconstitute the lyophilized powder, and ultrasonically disperse to obtain a SN38 nanocrystal solution with a particle size of 165.1 nm and a PDI of 0.183. The nanocrystal solution prepared in the above steps can be frozen at -20°C as required.

Example 5

(1) Preparation of SN38 prototype/carboxylic acid type mixture

Take 4 mL of triethylamine, dissolve it in 5 mL of methanol, weigh SN38 (0.2 g) and disperse it in triethylamine/methanol solution, stir the reaction at room temperature away from light until the solution is clear, stop the reaction, and freeze-dry to obtain SN38 prototype/carboxylic acid Type mixture (light yellow flaky solid), in which the prototype SN38 accounts for 52.6% of the theoretical mass percentage of the mixture

The above-mentioned alkaline reaction reagent can be selected from diethylamine, ethylamine, trimethylamine, dimethylamine or methylamine, and the reaction solvent can be selected from one or more of water, tetrahydrofuran, methanol or ethanol. The reaction time is adjusted according to the actual situation, and the reaction can be stopped when the HPLC monitoring shows that the mass percentage of the prototype drug is less than 10%. The prototype/carboxylic acid type mixture solid or suspension is obtained by rotary evaporation, and after the nanocrystals are prepared by metathesis method, the mass fraction of the carboxylic acid type drug is less than 5% (as shown in Table 2).

Table 2. Percentage of SN38 nanocrystal AC-SN38 prepared by different reaction conditions

Note: R-AC is the mass percentage of AC-SN38 in the reaction system when the reaction stops; M-AC is the mass percentage of AC-SN38 in the mixture or suspension after rotary evaporation; NCs-AC is the lyophilized nanocrystalline powder or The mass percentage of AC-SN38 in the reconstituted nanocrystal solution.

(2) Preparation of SN38 nanocrystal formulations

The mixture (10 mg) prepared by weighing (1) was dispersed in 10 mL of aqueous solution (containing 1 g of sucrose), and stirred rapidly for 20 min to obtain a blue-green opalescent preparation solution. Subsequently, the preparation solution was filtered through a 0.22 μm filter membrane, and freeze-dried to obtain SN38/sucrose nanocrystal freeze-dried powder. Take 10 mL of 5% glucose solution to redissolve the lyophilized powder, and ultrasonically disperse to obtain the SN38 nanocrystal solution.

The above-mentioned freeze-drying protection agent can be selected from mannitol, lactose, maltose, glucose or without the addition of the freeze-drying protection agent, and other operations are the same as above. After reconstitution, nanocrystals can be prepared with a particle size of 150-250 nm (as shown in Table 3), and the addition of a freeze-drying protective agent can be used to freeze the nanocrystal solution at -20°C.

Table 3. Average particle size of SN38 nanocrystals prepared by different types of lyoprotectants

Control example

About 17 mg of irinotecan hydrochloride (CPT-11) was weighed, dissolved in 10 mL, pH 6.0 5% glucose solution, and prepared into an equimolar solution with SN38 (1 mg/mL).

Test Example 1

Take the SN38 prototype/carboxylic acid type mixture (M-SN38) prepared in Example 1 for ¹ H-NMR detection and analysis, (DMSO-d ₆ , 400MHz): 10.29 (brs, 1H), 8.02 (d, J=6.0Hz) ,1H),7.41-7.39(m,2H),7.24(s,1H), 6.50(s,1H),5.42(s,2H),5.72(s,2H),3.09-3.07(m,2H), 1.88-1.85(m, 2H), 1.28(t, J=7.2Hz, 3H), 0.88(t, J=7.2Hz, 3H). The comparison results show that the parent structure SN38 of the SN38 prototype/carboxylic acid type mixture has no other structural changes.

Test Example 2

After vacuum drying the SN38 prototype/carboxylic acid type mixture (M-SN38) and SN38 bulk drug prepared in Example 1, weigh 10 mg of the powder and place it in an aluminum pan. /min, nitrogen atmosphere, differential scanning calorimetry analysis and thermogravimetric analysis. As shown in Figure 1, SN38 API (A, C) loses about 4.7% in weight at 115-145°C, loses a molecule of crystal water (theoretical weight loss is 4.39%), is in a molten state at 206-209°C, and decarboxylates and degrades under high heat conditions . Figure 1(B, D) shows that the SN38 prototype/carboxylic acid type mixture exotherms at 60-85 °C, and the weight loss is about 4.2%. The precondition for the existence of the type; the weight loss is about 0.72% at 101-127 °C, which is the loss of crystal water; 237-290 °C is in a molten state with endothermic, followed by decarboxylation and degradation to release heat.

Test Example 3

Take a small amount of SN38 nanocrystal freeze-dried powder and SN38 bulk drug obtained in Example 1, respectively coat them on the substrate, press them, and place them on an X-ray diffractometer for small-angle X-ray diffraction experiments, which are recorded as NCs-0% as shown in the figure. 2 shown. The Bragg parameters of the prepared NCs-0% are completely consistent with the Bragg parameters of the SN38 API reference substance. Combined with the analysis of the high performance liquid chromatogram in Figure 3, it can be speculated that when the SN38 prototype/carboxylic acid type mixture coexists, the carboxylic acid type SN38 with better hydrophilicity has a reversible hydrophilic end and SN38 self-fused ring hydrophobic end, which can be It acts as a surfactant to obtain highly dispersed mixed nanocrystals. After freeze-drying to remove volatile basic substances, the carboxylic acid type SN38 can be reversed to the prototype SN38 to obtain SN38 nanocrystals with a particle size of about 150 nm.

Test Example 4

Use dynamic light scattering to measure the particle size and distribution of the nanosuspension, take the SN38 nanocrystal freeze-dried solid powder and solution prepared in Example 1 and Example 3, the SN38/albumin nanocrystal solution and the SN38 nanocrystal solution prepared in Example 2. The HCPT nanocrystal solution was formulated for characterization, and the SN38 and HCPT nanocrystal solutions prepared in Example 1 and Example 2 were placed at 4 °C, and the particle size and PDI changes in the nanocrystal solution were monitored within 15 days, as shown in Table 4. shown. It was measured that the average particle size of the particles prepared in Example 1 in the aqueous solution was 150.9 nm, the PDI was 0.086, and the Zeta potential was -16.5 mV, as shown in Figure 4 (A, B), and the transmission electron microscope was shown in Figure 4 (E). The SEM images of nanocrystalline solid powder and nanocrystalline solution are shown in Fig. 4(H,I). The SN38 albumin nanocrystal particles prepared in Example 3 have an average particle size of 169.4 nm in an aqueous solution, a PDI of 0.193, and a Zeta potential of -19.4 mV, as shown in Figure 4 (C, D), and the transmission electron microscope is shown in Figure 4 ( F) shown. The HCPT nanocrystal solution in Example 2 was characterized by transmission electron microscopy, as shown in FIG. 4(G). Taking advantage of the reversible ring-opening/closing characteristics of camptothecins and introducing volatile alkaline reagents, the prepared nanocrystalline powders of camptothecins have a loose and porous structure. Needle-shaped or rod-shaped nanocrystals with better stability are formed.

Table 4. Particle size stability of NCs stored in the dark at 4°C (n=3)

Test Example 5

The SN38 nanocrystal solution prepared in Example 1 and the control example were used to investigate the drug plasma clearance experiment. Take healthy SD rats and inject an equimolar amount of SN38 (6.5mg/kg) into the tail vein, respectively at 10min, 20min, 30min, 1h, 2h, 4h, 8h, 12h, 24h, 36h. liter, add 2 times the volume of organic solvent (acetonitrile: methanol = 1:1, 0.5% acetic acid, v/v), mix well, let stand in the dark for 1 h, centrifuge at 13500 rpm for 10 min, take the supernatant for LC-MS/MS Quantitative analysis, the experimental results are shown in Figure 5.

It can be seen from FIG. 5 that irinotecan can be quickly cleared in vivo, and the nanocrystals prepared by the present invention can improve the retention time of SN38 in blood. As can be seen from Table 5, the blood elimination half-life of the nanocrystal preparation drug of the present invention is about 2 times that of irinotecan hydrochloride, and the area under the drug concentration-time curve (AUC) is more than 5 times that of irinotecan hydrochloride.

Table 5. Pharmacokinetic parameters of SN38 nanocrystal formulations and irinotecan hydrochloride (n=5)

AUC _0→t : area under the drug concentration-time curve

t _1/2 : blood concentration half-life

Test Example 6

The SN38 nanocrystal preparation prepared in Example 1 and the control example were used to investigate the distribution of the drug in the main organs in the body. Select Balb/c female mice in good physical condition and inoculate 4T1 tumor cells (about ₅ *105 tumor cells per mouse ⁾ on the left side of the mouse near the axillary mammary fat pad. When the tumor volume grows to After 200-300mm ³ , the mice were administered by tail vein injection (the equivalent dose of SN38 was 10mg/kg), and the mice were sacrificed 1h, 4h, 8h, and 24h after administration, and the blood, heart, liver, spleen, lung, and kidney were collected. and tumor were used for subsequent LC/MS/MS quantitative detection and analysis, and the distribution experiment was investigated. The experimental results are shown in Figure 6.

It can be seen from Figure 6 (A ~ D) that irinotecan hydrochloride in the control example is rapidly distributed in the organs and cleared rapidly, and the SN38 nanocrystal preparation prepared by the present invention is mainly distributed in the liver, spleen and lung, and the retention time in the organs is longer. It can be seen from Figure 6(E) that the SN38 nanocrystal preparation prepared by the present invention has a longer accumulation time in tumors, and the accumulation concentration has obvious advantages over irinotecan hydrochloride.

Test Example 7

The nanocrystal preparations of CPT-11, HCPT, CDK-602 and TPT prepared in Example 2 and the control examples were used to investigate the antitumor efficacy of tumor-bearing mice in vivo. Select Balb/c female mice in good physical condition and inoculate 4T1 tumor cells (about ₅ *105 tumor cells per mouse ⁾ on the left side of the mouse near the axillary mammary fat pad. When the tumor volume grows to After 50-100 mm ³ , the mice were injected into the tail vein (the equivalent dose converted to SN38 was 5 mg/kg), and the mice were administered every two days for a total of 3 times. The mice were sacrificed on the 20th day after tumor inoculation, and the tumors were separated. , record the tumor weight, and calculate the tumor inhibition rate. The calculation formula is: TGI%=(m _S -m)/m _s *100% (TGI is the tumor inhibition rate, m _s is the tumor weight in the physiological group, m is the tumor in the administration group Weight), tumor inhibition rate statistics are shown in the table below.

Table 6. Statistical results of tumor inhibition rate of several camptothecin drug nanocrystals in vivo (n=5)

Test Example 8

The SN38 nanocrystal preparation prepared in Example 1 and the control example were used to investigate the anti-tumor efficacy in tumor-bearing mice. Select Balb/c female mice in good physical condition and inoculate 4T1 tumor cells (about ₅ *105 tumor cells per mouse ⁾ on the left side of the mouse near the axillary mammary fat pad. When the tumor volume grows to After 50-100 mm ³ , the tail vein injection was started (the equivalent dose of SN38 was 5 mg/kg), administered every two days for a total of 5 times, and the size of the tumor and the body weight of the mice were recorded every other day. The formula for calculating the tumor volume of mice is: V=(L*D ² )/2 (V is the tumor volume, L is the long diameter of the tumor, and D is the short diameter of the tumor), and draw the tumor volume-time curve and body weight-time curve, such as Figure 7 (A,B). The mice were sacrificed on the 29th day after tumor _inoculation , the tumor was isolated, the tumor weight was recorded, and the tumor _{inhibition rate} was calculated. The tumor weight of the physiological group, m is the tumor weight of the administration group), as shown in Figure 7 (E, C, D).

As can be seen from Figure 7, under the set administration concentration, the tumor size of irinotecan hydrochloride has no significant difference with the normal saline group, and there is no obvious tumor suppressing effect, and the tumor growth rate is faster, and the SN38 nanocrystal preparation provided by the present invention is. The tumor growth rate of the group was slow during the administration period, and the tumor growth rate was slower after the administration was stopped. The tumor inhibitory effect was significantly different from that of the normal saline and irinotecan hydrochloride groups. It shows that the SN38 nanocrystal provided by the present invention has excellent antitumor activity.

Claims (10)

1. A nanocrystal formulation consisting solely of camptothecin drugs, which is needle-like or rod-like in shape.

2. The nanocrystal formulation of claim 1 having a particle size of 100 to 250 nm.

3. The nanocrystal formulation of claim 1, wherein the camptothecin drug is selected from one or more of irinotecan, 7-ethyl-10 hydroxycamptothecin, 10-hydroxycamptothecin, belotecan, topotecan, or salt forms thereof.

4. The nanocrystal preparation according to claim 1, wherein the nanocrystal further comprises a lyoprotectant, the lyoprotectant is selected from one or more of trehalose, sucrose, mannitol, lactose, maltose, glucose or human serum albumin, preferably trehalose or human serum albumin, and the mass ratio of the lyoprotectant to the drug is (0:1) - (100:1), preferably (15:2) - (50: 1).

5. A method for preparing the nanocrystal formulation according to any one of claims 1 to 4, wherein the nanocrystal formulation utilizes the property of reversible ring opening/closing of camptothecin drugs, and a basic agent is added to obtain a part of carboxylic acid type compounds with increased polarity, and the remaining part of the original drug is solubilized by the part of carboxylic acid type compounds with increased polarity to obtain highly dispersed, self-assembled mixed drug nanocrystals, and the volatile basic agent is removed by means of formulation means to convert the carboxylic acid type camptothecin drugs without pharmacological activity to the original drug, and the solubilized mixed drug nanocrystals are converted to the original camptothecin drug nanocrystals and dispersed in the solvent for injection.

6. A method of preparing a nanocrystal formulation of any one of claims 1-4, comprising the steps of:

(1) dispersing the camptothecin medicament in a reaction system consisting of a reaction reagent and a reaction solvent, and carrying out a light-resistant reaction at room temperature;

(2) removing part of reaction reagents and solvents in the reaction (1) to obtain solid powder or suspension, wherein the mass ratio of the carboxylic acid type drug to the prototype drug in the solid powder or suspension is 1: 5-10: 1;

(3) fully dispersing the mixture powder or suspension obtained in the step (2) in water;

(4) freeze drying to obtain nanometer camptothecin medicine crystal preparation.

7. The preparation method according to claim 6, wherein an injection solvent is added to reconstitute the camptothecin analogue drug nanocrystal preparation obtained in step (4) of claim 6, and the dispersion is uniform, so that the camptothecin analogue drug nanocrystal preparation can be used, wherein the injection solvent is one or more selected from a 5% glucose solution, a 0.9% sodium chloride solution or water for injection, and preferably is a 5% glucose solution or water for injection.

8. The preparation method according to claim 6, wherein the reaction reagent is one or more selected from triethylamine, diethylamine, ethylamine, trimethylamine, dimethylamine, monomethylamine and ammonia water, preferably monomethylamine or ammonia water; the reaction solvent is selected from one or more of methanol, ethanol, tetrahydrofuran or water, preferably one or more of methanol, ethanol and water; the volume/mass ratio of the reaction reagent to the sciophiline medicine is (2:1) - (20: 1) (mL/g), and preferably the volume/mass ratio is (5:1) - (10: 1).

9. The preparation method according to claim 6, wherein the partial reaction reagent and solvent in the reaction (1) are removed in the step (2) by freeze drying, vacuum drying or reduced pressure rotary evaporation, preferably reduced pressure rotary evaporation, wherein the rotary evaporation temperature is 30-60 ℃, preferably 37-50 ℃; the mass ratio of the volume of the aqueous solution to the prototype/carboxylic acid type mixture in the step (4) is (10:1) - (1: 2) (mL/mg), and the preferred volume/mass ratio is (5:1) - (1: 1); the dispersion mode is selected from one or more of rapid stirring, water bath ultrasound, probe ultrasound or high pressure homogenization, and is preferably probe ultrasound or high pressure homogenization.

10. Use of the nanocrystal formulation of any one of claims 1 to 9 for the manufacture of a medicament for the treatment of colorectal cancer, metastatic colorectal cancer, bladder cancer, gastric cancer, esophageal cancer, tonsil cancer, nasopharyngeal cancer, non-small cell lung cancer, pancreatic cancer, breast cancer, chronic myelogenous leukemia, lymphoma, skin cancer.

Images