CABAZITAXEL WEAKLY- ALKALINE DERIVATIVE AND FORMULATION THEREOF

Abstract

A cabazitaxel weakly-alkaline derivative, preparation, and synthesis thereof, a liposome preparation containing the cabazitaxel weakly-alkaline derivative and application of the cabazitaxel weakly-alkaline derivative in a drug delivery system are provided. Cabazitaxel is connected with a weakly-alkaline intermediate through an ester bond, the ester bond can be broken under the action of esterase in vivo, and an active drug is released. A connecting group is C.sub.1-C.sub.4 alkyl, C.sub.3-C.sub.6 naphthenic base or phenyl; [N] is an N-methyl piperazinyl group, a piperidinyl group, a 4-(1-piperidinyl) piperidinyl group, a morpholinyl group, a pyrrolidine group or other tertiary amine structures. The cabazitaxel weakly-alkaline derivative can be prepared into the liposome preparation having high drug loading capacity, high encapsulation efficiency, and good stability. The in-vivo circulation time of the drug can be greatly prolonged, the accumulation amount of the drug at a tumor part is increased, and the anti-tumor effect and the tolerance dose are improved.

Claims

1. A cabazitaxel weakly-alkaline derivative or a pharmaceutically acceptable salt thereof, wherein the cabazitaxel weakly-alkaline derivative has a structural formula as follows: ##STR00006## wherein the linking group is C.sub.1-C.sub.4 alkyl, C.sub.3-C.sub.6 cycloalkyl or phenyl; and the [N] is N-methylpiperazinyl, piperidinyl, 4-(1-piperidinyl) piperidinyl, morpholinyl, tetrahydropyrrolyl, or other tertiary amine structure.

2. The cabazitaxel weakly-alkaline derivative or pharmaceutically acceptable salt thereof according to claim 1, wherein the cabazitaxel weakly-alkaline derivative has a structural formula as follows: ##STR00007##

3. The cabazitaxel weakly-alkaline derivative or pharmaceutically acceptable salt thereof according to claim 1, wherein the pharmaceutically acceptable salt is a salt formed by the cabazitaxel weakly-alkaline derivative and a pharmaceutically acceptable inorganic or organic acid.

4. A method for synthesizing a cabazitaxel weakly-alkaline derivative, the cabazitaxel weakly-alkaline derivative having a structural formula as follows: ##STR00008## the method comprising: performing esterification reaction between 4-(4-methylpiperazinylmethyl) benzoyl chloride and cabazitaxel under the catalysis of DMAP or triethylamine; and then performing separation and purification to obtain the cabazitaxel weakly-alkaline derivative, wherein the whole reaction process is performed under the protection of N.sub.2.

5. A liposome of a cabazitaxel weakly-alkaline derivative, wherein the cabazitaxel weakly-alkaline derivative is prepared into a liposome, the cabazitaxel weakly-alkaline derivative having a structural formula as follows: ##STR00009## the liposome comprising the cabazitaxel weakly-alkaline derivative, phospholipid, cholesterol, and PEGylated phospholipid; and the liposome is prepared by the steps of: (1) preparing a blank liposome having a gradient; (2) preparing an ethanol solution of the cabazitaxel weakly-alkaline derivative; and (3) incubating the ethanol solution of the cabazitaxel weakly-alkaline derivative and the blank liposome having a gradient.

6. The liposome of cabazitaxel weakly-alkaline derivative according to claim 5, wherein the liposome is prepared by the further steps of: (1) weighing a membrane material required for preparing the liposome, dissolving the membrane material into an organic solvent, and performing evaporation under reduced pressure to form a dry lipid membrane; (2) adding an internal aqueous phase solution to the dry lipid membrane acquired in step (1), performing hydration at a temperature higher than a phase transition temperature, and sequentially squeezing a resulting product through polycarbonate membranes with different pore sizes to form nano-sized small unilamelar liposome; (3) replacing an external aqueous phase of the small unilamelar liposome acquired in step (2) to acquire a blank liposome having a gradient between the internal aqueous phase and the external aqueous phase; and (4) adding an organic solution of the cabazitaxel weakly-alkaline derivative to the blank liposome having a gradient acquired in step (3), and performing incubation to acquire liposome preparation of cabazitaxel weakly-alkaline derivative, wherein the organic solvent may be removed by tangential ultrafiltration, dialysis, and the like.

7. The liposome of cabazitaxel weakly-alkaline derivative according to claim 6, wherein in step (2), the internal aqueous phase solution is a citric acid solution, an ammonium sulfate solution, a sulfobutyl ether-β-cyclodextrin triethylammonium salt solution, a sucrose octasulfate triethylammonium salt solution, or the like.

8. The liposome of cabazitaxel weakly-alkaline derivative according to claim 5, wherein a weight ratio of the cabazitaxel weakly-alkaline derivative to total lipids is 1:4-12, and the total lipids is a sum of phospholipid, cholesterol and PEGylated phospholipid.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The above-mentioned and other features and advantages of this invention, and the manner of attaining them, will become more apparent and the invention will be better understood by reference to the following description of embodiments of the invention taken in conjunction with the accompanying drawings, wherein:

[0014] FIG. 1 is a diagram showing structure formula of a cabazitaxel derivative (CN1) having a basic group part of 4-(4-methylpiperazinemethyl) phenyl, according to Example 1 of the present invention;

[0015] FIG. 2 is a diagram showing .sup.1H-NMR spectrum of the cabazitaxel derivative (CN1) having a basic group part of 4-(4-methylpiperazinemethyl) phenyl, according to Example 1 of the present invention;

[0016] FIG. 3 is a diagram showing structure formula of a cabazitaxel derivative (CN2) having a basic group part of 4-(1-piperidinyl) piperidinyl, according to Example 2 of the present invention;

[0017] FIG. 4 is a diagram showing .sup.1H-NMR spectrum of the cabazitaxel derivative (CN2) having a basic group part of 4-(1-piperidinyl) piperidinyl, according to Example 2 of the present invention;

[0018] FIG. 5 is a diagram showing structure formula of a cabazitaxel derivative (CN3) having a basic group part of 4-methylpiperazine-1-methyl, according to Example 3 of the present invention;

[0019] FIG. 6 is a diagram showing .sup.1H-NMR spectrum of the cabazitaxel derivative (CN3) having a basic group part of 4-methylpiperazine-1-methyl, according to Example 3 of the present invention;

[0020] FIG. 7 is a diagram showing a particle size, an encapsulation ratio and a storage time of a liposome (CN1-liposome) of the cabazitaxel derivative according to Example 5 of the present invention;

[0021] FIG. 8 is a diagram showing a change in tumor volume in an in-vivo anti-tumor experiment using the liposome of the cabazitaxel derivative according to Example 7 of the present invention; and

[0022] FIG. 9 is a diagram showing a change in body weight of a mouse in an in-vivo anti-tumor experiment using the liposome of the cabazitaxel derivative according to Example 7 of the present invention.

[0023] Corresponding reference characters indicate corresponding parts throughout the several views. The exemplifications set out herein illustrate embodiments of the invention and such exemplifications are not to be construed as limiting the scope of the invention in any manner.

DETAILED DESCRIPTION OF THE INVENTION

[0024] For a cabazitaxel weakly-alkaline derivative provided according to the present invention, cabazitaxel is connected to a weakly-alkaline intermediate through an ester bond, and the ester bond may be broken under the action of esterase in vivo so as to release an active drug. A structural formula of the cabazitaxel weakly-alkaline derivative is as follows:

##STR00004##

wherein [0025] the linking group is C.sub.1-C.sub.4 alkyl, C.sub.3-C.sub.6 cycloalkyl or phenyl; and [0026] the [N] is N-methylpiperazinyl, piperidinyl, 4-(1-piperidinyl) piperidinyl, morpholinyl, tetrahydropyrrolyl or other tertiary amine structure.

[0027] Further, according to the present invention, a cabazitaxel weakly-alkaline derivative having the following structure is provided:

##STR00005##

[0028] A synthesis method of the cabazitaxel weakly-alkaline derivative according to the present invention is as follows:

[0029] under the catalysis of DMAP, performing esterification reaction between 4-(4-methylpiperazinylmethyl) benzoyl chloride and cabazitaxel, and then performing separation and purification to obtain the cabazitaxel weakly-alkaline derivative, wherein the whole reaction process is performed under the protection of N.sub.2, and DMAP may be replaced with triethylamine.

[0030] Further, the present invention provides a liposome including a cabazitaxel weakly-alkaline derivative, wherein the liposome includes the cabazitaxel weakly-alkaline derivative, phospholipid, cholesterol, PEGylated phospholipid, and the like. The weight ratio of the derivative to total lipids is 1:4-12, and the total lipids is a sum of phospholipid, cholesterol, and PEGylated phospholipid. The phospholipid, cholesterol and PEGylated phospholipid are used in conventional amounts in the art.

[0031] The present invention further provides a preparation method of a liposome of the cabazitaxel weakly-alkaline derivative, comprising: [0032] (1) weighing a membrane material required for preparing the liposome, dissolving the membrane material into an organic solvent, and performing evaporation under reduced pressure to form a dry lipid membrane; [0033] (2) adding an internal aqueous phase solution to the dry lipid membrane acquired in step (1), performing hydration at a temperature higher than a phase transition temperature, and sequentially squeezing a resulting product through polycarbonate membranes with different pore sizes to form nano-sized small unilamelar liposome; [0034] (3) replacing an external aqueous phase of the small unilamelar liposome acquired in step (2) to acquire a blank liposome having a gradient between the internal aqueous phase and the external aqueous phase; and [0035] (4) adding an organic solution of the cabazitaxel weakly-alkaline derivative to the blank liposomes having a gradient acquired in step (3), and performing incubation to acquire a liposome preparation of the cabazitaxel weakly-alkaline derivative.

[0036] In some embodiments, in step (2), the internal aqueous phase solution may be a citric acid solution, an ammonium sulfate solution, a sulfobutyl ether-β-cyclodextrin triethylammonium salt solution, or a sucrose octasulfate triethylammonium salt solution.

[0037] In some embodiments, the internal aqueous phase solution is an ammonium sulfate solution.

[0038] In sonic embodiments, in step (3), the external aqueous phase solution may be a sucrose solution, HEPES buffer, phosphate buffer, or acetate buffer.

[0039] In some embodiments, the external aqueous phase solution is a sucrose solution.

[0040] In some embodiments, in step (4), a solvent of the organic solution may be methanol, ethanol, acetone, tetrahydrofuran, acetonitrile, or DMSO.

[0041] In some embodiments, a solvent of the organic solution is ethanol.

[0042] In the present invention, cabazitaxel is prepared into the weakly-alkaline derivative for preparing a liposome, which can not only avoid the side effects caused by Tween 80 in a cabazitaxel injection, but also increase the maximum tolerated dose of a drug. Thus, the anti-tumor effect of the drug is enhanced and great clinical application potential is achieved.

[0043] The liposome nano-drug delivery system provided according to the present invention has the advantages as follows: (1) the particle size is small and uniform (less than 100 nm), which is beneficial for enrichment of the drug to a tumor site through the EPR effect; (2) the drug loading capacity is high, which is beneficial for reduction of adverse reactions caused by excipients and biological materials; (3) complete drug encapsulation can be acquired, and good stability and ease industrialization are achieved; (4) the uptake by a reticuloendothelial system is effectively avoided, a long circulation effect is achieved in blood and the probability that the drug reaches the tumor site is increased; and (5) compared with commercially available preparations, the liposome nano-drug delivery system provided according to the present invention has an improved anti-tumor effect and reduced toxic and side effects.

Exemplary Embodiments

[0044] The following embodiments are intended to further illustrate the present invention without limiting the present invention in any way.

EXAMPLE 1: SYNTHESIS OF A CABAZITAXEL DERIVATIVE (CN1) HAVING A BASIC GROUP PART OF 4-(4-METHYLPIPERAZINEMETHYL) PHENYL

[0045] Cabazitaxel (200 mg, 0.24 mmol) and 4-(4-methylpiperazinylmethyl) benzoyl chloride (156 mg, 0.48 mmol) were weighed and dissolved in dichloromethane; 0.25 mL of triethylamine was added thereto; under an ice bath, a dichloromethane solution of DMAP (5.9 mg, 0.048 mmol) was slowly added dropwise to the thus obtained mixture; and stirring was performed at the room temperature overnight under N.sub.2 protection. After the reaction was completed, separation and purification were performed through column chromatography to acquire a cabazitaxel derivative in the form of a white powder (yield: 95.01%). The structure of the compound in Example 1 was determined through a nuclear magnetic resonance hydrogen spectrum. The results are shown in FIG. 2. Spectral analysis results are as follows:

.sup.1H NMR (Chloroform-d,400 MHz) δ8.11 (2H,d,J=7.4 Hz), 7.94 (2H,d,J=8.1 Hz), 7.61 (1H,t,J=7.4 Hz), 7.51 (1H,d,J=7.8 Hz), 7.48 (1H,d,J=7.8 Hz), 7.45-7.35(6H,m), 7.32-7.27(1H,m), 6.26 (1H,t,J=9.1 Hz), 5.64 (1H,d,J=7.0 Hz), 5.50 (1H,d,J=3.4 Hz), 5.43(1H,d,J=9.4 Hz), 5.30 (1H,s), 5.00(1H,d,J=8.8 Hz), 4.83 (1H,s), 4.31 (1H,d,J=8.4 Hz), 4.17(1H,d,J=8.3 Hz), 3.97-3.88(1H,m), 3.86 (1H,d,J=7.0 Hz), 3.56(2H,s), 3.43(3H,s), 3.31(3H,s), 2.82-2.62(2H,m), 2.54-2.38(6H,m), 2.31(3H,s), 2.25(1H,m), 2.04(3H,s), 1.82(1H,m), 1.79(1H,m), 1.71 (3H,s), 1.64 (2H,s), 1.36 (9H,s), 1.26(3H,s), 1.21(6H,s).

EXAMPLE 2: SYNTHESIS OF A CABAZITAXEL DERIVATIVE (CN2) HAVING A BASIC GROUP PART OF 4-(1-PIPERIDINYL) PIPERIDINYL

[0046] Cabazitaxel (200 mg, 0.24 mmol) and 4-piperidylpiperidinecarbonyl chloride (111 mg, 0.48 mmol) were weighed and dissolved in dichloromethane; 0.25 mL of triethylamine was added thereto; under an ice bath, a dichloromethane solution of DMAP (5.9 mg, 0.048 mmol) was slowly added dropwise to the thus obtained mixture; and stirring was performed at the room temperature overnight under N.sub.2 protection. After the reaction was completed, separation and purification were performed through column chromatography to acquire a cabazitaxel derivative in the form of a white powder (yield: 95%). The structure of the compound in Example 2 was determined through a nuclear magnetic resonance hydrogen spectrum. The results are shown in

[0047] FIG. 4. Spectral analysis results are as follows:

.sup.1H NMR (400 MHz, Chloroform-d) δ8.07-8.00 (m,2H), 7.54(t,J=7.4 Hz, 1H), 7.43 (t,J=7.6 Hz,2H), 7.33 (t,J=7.6 Hz,2H), 7.22 (m,3H), 6.32-5.95 (m, 1H), 5.56 (d,J=7.0 Hz,1H), 5.44-5.26 (m,2H), 5.23 (s,1H), 5.20 (d,J=3.8 Hz, 1H), 4.92 (d,J=9.4 Hz,1H), 4.74(s,1H), 4.23 (d,J=8.4 Hz,1H), 4.09 (d,J=8.4 Hz,2H), 3.83 (dd,J=10.7,6.4 Hz,1H), 3.76 (d,J=7.0 Hz,1H), 3.36(s,3H), 3.22(s, 3H), 2.77-2.49 (m,4H), 2.36 (s,3H), 2.27-2.05 (m,1H), 1.92(s,3H), 1.77-1.66 (m,1H), 1.64(s,3H), 1.55(s,1H), 1.52-1.39 (m,2H), 1.28(s,9H), 1.19(s,3H), 1.14 (s,3H), 1.12(s,3H).

EXAMPLE 3: SYNTHESIS OF A CABAZITAXEL DERIVATIVE (CN3) HAVING A BASIC GROUP PART OF 4-METHYLPIPERAZINE-1-METHYL

[0048] Cabazitaxel (200 mg, 0.24 mmol) and 4-methylpiperazine-1-carbonyl chloridel (78 mg, 0.48 mmol) were weighed and dissolved in dichloromethane; 0.25 mL of triethylamine was added thereto; under an ice bath, a dichloromethane solution of DMAP (5.9 mg, 0.048 mmol) was slowly added drop wise to the thus obtained mixture; and stifling was performed at the room temperature overnight under N.sub.2 protection. After the reaction was completed, separation and purification were performed through column chromatography to acquire a cabazitaxel derivative in the form of a white powder (yield: 93.8%). The structure of the compound in Example 3 was determined through a nuclear magnetic resonance hydrogen spectrum. The results are shown in FIG. 6. Spectral analysis results are as follows:

.sup.1H NMR (400 MHz,Chloroform-d) δ8.03 (d,J=7.5 Hz,2H), 7.54 (t,J=7.4 Hz, 1H), 7.43 (t,J=7.6 Hz,2H), 7.32 (t,J=7.6 Hz,2H), 7.26-7.20 (m,3H), 6.17(t,J=9.3 Hz,1H), 5.56 (d,J=7.0 Hz,1H), 5.36 (s,1H), 5.25(s,1H), 5.23(s,1H), 4.92(dd, J=9.5,2.0 Hz,1H), 4.74(s,1H), 4.23 (d,J=8.4 Hz,1H), 4.09 (d,J=8.4 Hz,1H), 3.83 (dd,J=10.7,6.3 Hz,1H), 3.76(d,J=7.0 Hz,1H), 3.39 (s,2H), 3.36 (s,3H), 3.22 (s,3H), 2.62 (ddd,J=14.1,9.8,6.3 Hz,1H), 2.36(m,1H), 2.35(s,3H), 2.22(s, 3H), 2.15 (t,J=7.6 Hz,2H), 2.00 (m,1H), 1.92 (s,3H), 1.78-1.66 (m,2H), 1.64(s, 3H), 1.52(s,1H), 1.28 (s,9H), 1.19(s,3H), 1.14(s,3H), 1.12(s,3H).

EXAMPLE 4: PREPARATION OF LIPOSOMES OF CABAZITAXEL DERIVATIVE

[0049] The preparation method of the liposomes of cabazitaxel derivate derivative according to the present example included the following steps: [0050] (1) preparation of blank liposomes: DSPC, cholesterol, DSPE-mPEG.sub.2000 (a mass ratio of 3:1:0.05) were weighted; chloroform was added to the above materials for dissolution; evaporation under reduced pressure was performed at 37° C. for removing an organic solvent to form a dry lipid membrane; 350 mM ammonium sulfate solution was added for hydration at 65° C. for 30 minutes; and whole particles were filtered through a polycarbonate membrane to form small unilamelar liposomes having both of an internal aqueous phases and an external aqueous phases being the ammonium sulfate solution; [0051] (2) preparation of gradient blank liposomes: the blank liposomes acquired in step (1) passed through an agarose CL-4B gel column previously equilibrated with 300 mM sucrose to acquire an ammonium ion gradient blank liposome having an internal aqueous phase of ammonium sulfate solution and an external aqueous phase of sucrose solution; [0052] (3) drug loading process: an ethanol solution of a cabazitaxel derivative was added to the ammonium ion gradient blank liposomes prepared in step (2); and incubation was performed for 20 min at 60° C. to finally acquire the liposomes of cabazitaxel derivative (CN1-liposomes, CN2-liposomes and CN3-liposomes).

EXAMPLE 5: COLLOIDAL STABILITY TEST OF SOMES OF CABAZITAXEL DERIVATIVE (CN1-LIPOSOMES)

[0053] The liposomes preparation prepared in Example 4 was subjected to sterile filtration and stored at 4° C. for 60 days. During this period, a change in particle size was measured through a dynamic light scattering method, and a change in encapsulation rate was measured through high-performance liquid chromatography. The results are shown in FIG. 7: the particle size and the encapsulation rate of the actively drug-loaded liposomes of cabazitaxel derivative were not significantly changed within 60 days, thus they exhibit good long-term storage stability.

EXAMPLE 6: PHARMACOKINETIC STUDY OF LIPOSOMES OF CABAZITAXEL DERIVATIVE (CN1-LIPOSOMES)

[0054] 10 healthy male rats with a weight of 200-250 g for each one, were randomly divided into 2 groups with 5 rats in each group, and these two groups of rats were injected with a commercially available preparation and the CN1-liposomes prepared in Example 4 at tail veins respectively, wherein the equivalent dosage of cabazitaxel was 5 mg/kg. Blood was taken from the orbits at a prescribed time and centrifuged to acquire plasma, and the concentration of a drug in the plasma was measured through high-performance liquid chromatography-mass spectrometry.

[0055] The results are shown in Table 1. A circulation time of the liposomes preparation in the body was significantly prolonged, and the area under the concentration-time curve (AUC) of the liposome preparation was significantly improved. The experimental results showed that the liposome preparation can significantly prolong the circulation time of the drug in the blood and increases the possibility that the drug accumulates to the tumor site through the EPR effect.

TABLE-US-00001 TABLE 1 pharmacokinetic parameters of liposomes of cabazitaxel derivative (CN1-liposomes) Commercially available preparation CN1-liposomes AUC.sub.(0-∞) 1342.167 ± 231.161  1277826.64 ± 433820.141 (ug/L*h) MRT.sub.(0-∞) (h) 2.845 ± 0.814 6.207 ± 0.946 t.sub.1/2 (h) 3.393 ± 1.562 4.497 ± 0.732 CLz (L/h/kg) 3.816 ± 0.667 0.005 ± 0.002 C.sub.max (ug/L) 1259.42 ± 227.789 270060.434 ± 142270.937

EXAMPLE 7: ANIMAL PHARMACODYNAMICS EXPERIMENT OF LIPOSOMES OF CABAZITAXEL DERIVATIVE

[0056] Mouse prostate cancer cells (RM-1, 5*10.sup.6 cells/100 μL PBS) were inoculated subcutaneously on the right ventral sides of male C57BL/6 mice. After the tumors grew to 60-80 mm.sup.3, the mice were randomly divided into 6 groups with 5 mice in each group: a blank control group, a CN1-solution group, a commercially available preparation group, a CN1-liposomes group, a CN2-liposomes group, and a CN3-liposomes group. The drugs were administered every three days for a total of 5 times, wherein the equivalent dosage of cabazitaxel was 6 mg/kg, and the high dosage was three times of the low dosage. After administration, the states of the mice were observed, the mice were weighed and the volumes of the tumors were measured. At the end of the last dosing cycle, the mice were killed, tumors and major organs were stripped for analysis and evaluation.

[0057] The results are shown in FIG. 8, which shows that the CN1-solution group has almost no antitumor activity, whereas the CN1-liposomes group exhibits better antitumor activity than the commercially available preparation group. As a result, although the alkalized CN2 and CN3 may be successfully prepared into liposomes preparations, the antitumor activity of the CN2-liposomes group and the CN3-liposomes group is weaker than that of the CN1-liposomes group and is also weaker than that of the commercially available preparation group.

[0058] As shown in FIG. 9, the body weight of the mice in the commercially available preparation group is decreased significantly, indicating that the preparation has certain toxicity to the mice, whereas no significant body weight change is observed in the liposome preparation group. In summary, the CN1-liposomes group also has better anti-tumor activity and exhibits the effects of “reducing toxicity and increasing efficacy”, and thus is a safe and effective anti-tumor drug delivery system and has good clinical application potential.

[0059] While this invention has been described with respect to at least one embodiment, the present invention can be further modified within the spirit and scope of this disclosure. This application is therefore intended to cover any variations, uses, or adaptations of the invention using its general principles. Further, this application is intended to cover such departures from the present disclosure as come within known or customary practice in the art to which this invention pertains and which fall within the limits of the appended claims.