CN104546719B - A kind of load Z GP Dox mixed micelle preparation and preparation method thereof - Google Patents

Abstract

The invention discloses a Z-GP-Dox-loaded mixed micelle preparation and a preparation method thereof. The Z-GP-Dox-loaded mixed micelle preparation is prepared from the following components: 0.10%-0.40% Z-GP-Dox, 0.62%-2.50% phospholipids, 0.06%-0.50% surface Active agent, the balance of injection solvent. The present invention only uses two kinds of amphiphilic molecules as the constituent materials of the mixed micelles, and under the principle of ensuring the use of less excipients, a Z-GP-Dox-loaded mixed micelles preparation of good quality is prepared, which is fully in line with the research and development technology of injections. The guiding principle of using excipients and prescription is simple, and provides a mixed micellar drug-loading system as the solution for intravenous delivery of Z-GP-Dox, in order to improve the apparent water solubility and drug-making possibility of Z-GP-Dox Lay the technical foundation.

Description

A kind of mixed micelle preparation loaded with Z-GP-Dox and preparation method thereof

technical field

The invention relates to a mixed micelle pharmaceutical preparation for injection, more specifically, to a Z-GP-Dox-loaded mixed micelle and a preparation method thereof.

Background technique

Fibroblast activation protein-α (fibroblast activation protein-α, FAPα) is a membrane protein specifically expressed on tumor-associated fibroblasts (carcinoma-associated fibroblasts, CAF) in the tumor stroma, and it has dipeptidase activity , can specifically recognize the glycylproline dipeptide (N-carbobenzoxoxy-glycyl-proline, Z-GP) whose N-terminus is blocked by benzyloxycarbonyl (Z), and exert the exopeptidase activity to hydrolyze Z-GP Peptide bond formed at the C-terminus of proline.

Doxorubicin is a broad-spectrum antineoplastic drug commonly used clinically, and is often used in the treatment of liver cancer, gastric cancer, breast cancer, lung cancer, ovarian cancer, etc. Z-GP-Dox (N-benzyloxycarbonyl-manprolyl doxorubicin) is a prodrug of doxorubicin, which can be recognized by FAPα in tumor tissue and enzymatically released the parent drug doxorubicin , in order to achieve the purpose of targeted therapy.

The molecular formula of Z-GP-Dox is C ₄₂ H ₄₅ N ₃ O ₁₅ , the molecular weight is 831.29, and its structural formula is shown in formula (I).

(I).

Previous studies have shown that Z-GP-Dox can effectively target and release the parent drug doxorubicin (Dox) under the hydrolysis of FAPα, thereby producing a significant cytotoxic effect; and further passed the 4T1 breast cancer tumor-bearing mice in vivo Pharmacodynamic studies have confirmed that Z-GP-Dox has good antitumor activity. At the same time, according to the acute toxicity study in mice, the median lethal dose of intravenous doxorubicin is about 13.4 mg/kg, and Z-GP-Dox is at the maximum dose of 25 mg/kg (equimolar to 16.3 mg/kg). Dox dosage) did not find the animal lethal situation, the result suggested that Z-GP-Dox has higher safety.

Z-GP-Dox has completed the main contents of pharmacodynamics, pharmacokinetics, general pharmacology and other druggability evaluations, showing its good druggability, but its poor water solubility is the biggest problem hindering its druggability. Z-GP-Dox is soluble in some organic solvents such as propylene glycol and methanol, and is almost insoluble in water for injection or normal saline. In the previous research, propylene glycol could only be used as a latent solvent for drug preparation and administration. Because excessive propylene glycol can produce side effects such as irritation and hemolysis, the dosage of administration is greatly limited.

Accordingly, we have explored a variety of dosage forms to improve the apparent water solubility of Z-GP-Dox for intravenous injection, such as the development of Z-GP-Dox nanosuspensions, solid lipid nanoparticles and other dosage forms, through polymer Oxyethylene castor oil solubilization, etc., but there are problems such as excessive particle size, toxic and side effects of auxiliary materials, or failure to achieve ideal solubility. The dosage form problem has become the biggest bottleneck in Z-GP-Dox research, and it has also affected its later preclinical research and product development.

Mixed micelles (mixed micelles) is a micellar system made by mixing two or more different surfactants (such as phospholipids, bile salts, etc.), which has thermodynamic stability and good nano-effects. An effective means for delivering poorly soluble drugs. As far as mixed micelles are concerned, phospholipids/bile salts are a typical combination. Currently, there are two products of such mixed micelles that are successfully marketed, namely Konakion ^® produced by F. Hoffmann-La Roche Ltd. MM (vitamin K ₁ ) and Cernevit ^® (multivitamin) from Baxter International Inc.

At present, there are no other relevant technical reports using a mixed micelle drug-loading system as a solution for intravenous delivery of Z-GP-Dox.

Contents of the invention

The technical problem to be solved in the present invention is to provide a Z-GP-Dox-loaded mixed micelle preparation for the lack of existing Z-GP-Dox application technology, and to realize the mixed micelle drug-loading system as Z-GP-Dox. Intravenous delivery dosage form solution to improve the apparent water solubility and druggability of Z-GP-Dox.

Another technical problem to be solved by the present invention is to provide a preparation method of the Z-GP-Dox-loaded mixed micelle preparation. Based on the preparation method of the present invention, a mixed micelle preparation with qualified particle size, uniform particle size distribution and stable physical properties can be prepared. bundle of products.

Another technical problem to be solved by the present invention is to solve the insoluble problem of the main drug Z-GP-Dox, and provide an intravenous injection preparation of Z-GP-Dox.

The purpose of the present invention is achieved through the following technical solutions:

A Z-GP-Dox-loaded mixed micelle preparation is provided, which is composed of amphiphilic phospholipid, surfactant, two kinds of micelle carrier materials, injection solvent and main drug Z-GP-Dox. Specifically, it is prepared from the following components: 0.10% to 0.40% of Z-GP-Dox, 0.62% to 2.50% of phospholipids, 0.06% to 0.50% of surfactants, the rest of the injection solvent, The percentages of each of the above components are calculated according to the mass-volume ratio (w/v), the mass unit is mg, and the volume unit is mL.

Wherein, the amphiphilic phospholipids include but not limited to egg yolk lecithin, soybean phosphatidylcholine, hydrogenated egg yolk lecithin, hydrogenated soybean phospholipid, cephalin, inositol phospholipid, serine phospholipid or polyene phosphatidylcholine. Soy phospholipids with a phosphatidylcholine content greater than 97% are preferred.

The surfactant includes, but is not limited to, one of bile salts, saturated or unsaturated fatty acid salts with a chain length of 8-18, such as sodium oleate, sodium cholate, or sodium taurocholate. Sodium oleate is preferred.

The injection solvent can be selected from water for injection, physiological saline for injection, 5% glucose aqueous solution for injection or phosphate buffer solution with pH 7.4-8.2.

The present invention also provides a preferred preparation method of the Z-GP-Dox-loaded mixed micelle preparation, which is prepared by solvent diffusion-evaporation method, using phospholipid and another surfactant as the micelle carrier material, and Z-GP-Dox is encapsulated in the hydrophobic core. Include the following steps:

S1. Fully dissolving Z-GP-Dox, phospholipid, and surfactant in an organic solvent to form solution A;

S2. Slowly add solution A obtained in S1 dropwise into the injection solvent stirred at high speed to form solution B;

S3. Evaporate under reduced pressure to remove the residual organic solvent in the solution B obtained in S2 and concentrate to an appropriate volume to obtain a mixed micelle preparation loaded with Z-GP-Dox.

Wherein, the phospholipid used in S1 includes but not limited to one of egg yolk lecithin, soybean phosphatidylcholine, hydrogenated egg yolk lecithin, hydrogenated soybean phospholipid, cephalin, inositol phospholipid, serine phospholipid or polyene phosphatidylcholine. Soy phospholipids with a phosphatidylcholine content greater than 97% are preferred.

The surfactants used in S1 include but are not limited to bile salts, one of saturated or unsaturated fatty acid salts with a chain length of 8 to 18, such as sodium oleate, sodium cholate or sodium taurocholate, etc., preferably sodium oleate.

The organic solvent described in S1 includes but is not limited to one of methanol, ethanol, ethyl acetate, acetone or chloroform, preferably ethanol.

The injection solvent described in S2 can be selected from water for injection, physiological saline for injection, 5% glucose aqueous solution for injection or phosphate buffer saline with pH 7.4 to 8.2.

The decompression evaporation described in S3 can be carried out using a rotary evaporator.

Based on the present invention, the problem of poor solubility of the main drug Z-GP-Dox can be solved, and an intravenous injection preparation of Z-GP-Dox is provided.

The present invention has following beneficial effect:

The invention provides a Z-GP-Dox-loaded mixed micelle preparation. One of the remarkable beneficial effects of the invention lies in that the composition of the prescription is simple. In the present invention, in addition to the main drug Z-GP-Dox, by determining the scientific components and proportions, only two amphiphilic molecules are used as the constituent materials of the mixed micelles. Under the principle of ensuring the use of less auxiliary materials, The Z-GP-Dox-loaded mixed micelle preparation of good quality was prepared, which fully complied with the guiding principles of injection research and development technology: under the condition of ensuring the quality of the injection, the injection prescription should use as few excipients as possible, and the prescription should be simple. The invention provides a mixed micelle drug-loading system as the intravenous delivery formulation solution of Z-GP-Dox, laying a technical foundation for improving the apparent water solubility and drug-making possibility of Z-GP-Dox.

The second beneficial effect of the present invention is: based on the prescription of the present invention, the present invention provides reasonable preparation steps and processes, the prepared mixed micelles have a smaller particle size of about 86.6 nm, a uniform particle size distribution, a PDI of 0.269, and a Zeta The potential is -48.4 mV, the appearance is a red clear suspension, with obvious Tyndall phenomenon, and the effect is stable.

Furthermore, the present invention uses fatty acid salt as a co-additive to prepare Z-GP-Dox-loaded mixed micelle preparations. Compared with bile salts commonly used in the prior art, fatty acid salt (Sodium Aliphatate) is less toxic, therefore, successfully Provide a technical solution for preparing injectable mixed micelles using fatty acid salts and phospholipids as co-additives, and provide technical support for the broad application prospects of Z-GP-Dox-loaded mixed micelles.

Description of drawings

Fig. 1 Particle size distribution diagram of Z-GP-Dox mixed micelles.

Fig. 2 Transmission electron microscope images of Z-GP-Dox mixed micelles.

detailed description

The present invention will be further described below in conjunction with specific examples. The following examples are only illustrative examples and do not constitute improper limitations to the present invention. The present invention can be implemented in various ways defined and covered by the content of the invention. Unless otherwise specified, the reagents, compounds and equipment used in the present invention are conventional reagents, compounds and equipment in the technical field.

Example 1

This embodiment provides a Z-GP-Dox-loaded mixed micelle preparation, which consists of amphiphilic phospholipids, surfactants, two micellar carrier materials, an injection solvent and the main drug Z-GP-Dox. The composition of the prescription is as follows:

The preparation steps are as follows:

S1. Fully dissolve Z-GP-Dox, soybean lecithin (phosphatidylcholine content greater than 98%), and sodium oleate in 90% (w/v) ethanol to form solution A;

S2. The solution A obtained in S1 is slowly added dropwise into the water for injection stirred at a high speed with a micro-injection pump to form a solution B;

S3. Use a rotary evaporator to evaporate under reduced pressure (37°C) to remove the residual ethanol in the solution B obtained in S2 and concentrate to about 75% of the original volume, so that the concentration of Z-GP-Dox is above 2.5 mg/mL, and the Z-GP-Dox concentration is obtained. - Mixed micellar formulation of GP-Dox.

Product Appearance: Red clear suspension with obvious Tyndall phenomenon.

Particle size: average particle size 76.91 nm, ξ potential: -27.5 mv.

Example 2

This embodiment provides a Z-GP-Dox-loaded mixed micelle preparation, which consists of amphiphilic phospholipids, surfactants, two micellar carrier materials, an injection solvent and the main drug Z-GP-Dox. The composition of the prescription is as follows:

The preparation steps are as follows:

S1. Fully dissolve Z-GP-Dox, egg yolk lecithin, and sodium oleate in 90% (w/v) ethanol to form solution A;

S2. Using a micro-injection pump, slowly drop the solution A obtained in S1 into the physiological saline for injection stirred at a high speed to form a solution B;

S3. Use a rotary evaporator to evaporate under reduced pressure (37°C) to remove the residual ethanol in the solution B obtained in S2 and concentrate to about 75% of the original volume, so that the concentration of Z-GP-Dox is above 2.5 mg/mL, and the Z-GP-Dox concentration is obtained. - Mixed micellar formulation of GP-Dox.

Product Appearance: Red transparent suspension with obvious Tyndall phenomenon.

Particle size: average particle size 78.82 nm, ξ potential: -29.3 mv.

Example 3

This embodiment provides a Z-GP-Dox-loaded mixed micelle preparation, which consists of amphiphilic phospholipids, surfactants, two micellar carrier materials, an injection solvent and the main drug Z-GP-Dox. The composition of the prescription is as follows:

The preparation steps are as follows:

S1. Fully dissolve Z-GP-Dox, hydrogenated soybean lecithin, and sodium cholate in 90% ethanol to form solution A;

S2. Slowly add the solution A obtained in S1 dropwise into the high-speed stirred 5% glucose aqueous solution for injection with a micro-injection pump to form solution B;

S3. Use a rotary evaporator to evaporate under reduced pressure (37°C) to remove the residual ethanol in the solution B obtained in S2 and concentrate to about 75% of the original volume, so that the concentration of Z-GP-Dox is above 2.5 mg/mL, and the Z-GP-Dox concentration is obtained. - Mixed micellar formulation of GP-Dox.

Product Appearance: Red transparent suspension with obvious Tyndall phenomenon.

Particle size: average particle size 72.76 nm, ξ potential: -26.8 mv.

Example 4

This embodiment provides a Z-GP-Dox-loaded mixed micelle preparation, which consists of amphiphilic phospholipids, surfactants, two micellar carrier materials, an injection solvent and the main drug Z-GP-Dox. The composition of the prescription is as follows:

The preparation steps are as follows:

S1. Fully dissolve Z-GP-Dox, distearoylphosphatidylcholine and sodium taurocholate in 90% ethanol to form solution A;

S2. Slowly add the solution A obtained in S1 dropwise into the phosphate buffer solution of pH 7.4 to 8.2 stirred at high speed with a micro-injection pump to form solution B;

S3. Use a rotary evaporator to evaporate under reduced pressure (37°C) to remove the 90% ethanol remaining in the solution B obtained in S2 and concentrate it to about 75% of the original volume, so that the concentration of Z-GP-Dox is above 2.5 mg/mL to obtain Z-GP-Dox-loaded mixed micelle formulation.

Appearance: Red transparent suspension with obvious Tyndall phenomenon.

Particle size: average particle size 92.11 nm, ξ potential: -31.4 mv.

Example 5

This embodiment provides a Z-GP-Dox-loaded mixed micelle preparation, which consists of amphiphilic phospholipids, surfactants, two micellar carrier materials, an injection solvent and the main drug Z-GP-Dox. The composition of the prescription is as follows:

The preparation steps are as follows:

S1. Fully dissolve Z-GP-Dox, cephalin, and sodium oleate in 90% ethanol to form solution A;

S2. Use a micro-injection pump to slowly drop the solution A obtained in S1 into the water for injection stirred at a high speed to form a solution B;

S3. Use a rotary evaporator to evaporate under reduced pressure (37°C) to remove the residual ethyl acetate in the solution B obtained in S2 and concentrate it to about 75% of the original volume, so that the Z-GP-Dox concentration is above 2.5 mg/mL to obtain Z-GP-Dox-loaded mixed micelle formulation.

Appearance: Red transparent suspension with obvious Tyndall phenomenon.

Particle size: average particle size 82.54 nm, ξ potential: -33.6 mv.

Embodiment 6 stability test

The long-term stability test results carried out by taking the Z-GP-Dox mixed micelle preparation prepared in Examples 1 to 5 as an example are shown in Table 1:

Table 1 Stability test results

Example 7

In this embodiment, based on the method described in Example 1, a large number of experiments are carried out on the ratio of the drug to the phospholipid, the ratio of sodium oleate to the phospholipid, the ratio of the external aqueous phase and the organic phase, and Table 2 shows the experimental results:

Table 2

The Z-GP-Dox mixed micelle preparations prepared according to the prescription in Table 1 are all red clear suspensions, and the average particle size, ξ potential and 12-month stability test of the Z-GP-Dox mixed micelles The results are shown in Table 3:

table 3

As can be seen from the experimental results in Table 2, the particle size distribution of the Z-GP-Dox mixed micelles prepared by the method of the present invention is relatively uniform, and they are all red and clear suspensions. The results of the stability test for 12 months all show the effect of the present invention Stable, strong operability, controllable and adjustable prescription and process, ensuring the reliability of the quality of the preparation and the safety of clinical medication.

Claims (7)

1. a kind of load Z-GP-Dox mixed micelle preparation, it is characterised in that its each component consisted of is made：0.10% ~0.40% Z-GP-Dox, 0.62%~2.50% phosphatide, 0.06%~0.50% surfactant, the injection of surplus Solvent, the percentage of above each component is calculated by mass volume ratio；

Wherein, the phosphatide is egg yolk lecithin, soybean lecithin, hydrogenated yolk lecithin, hydrogenated soya phosphatide, cephalin, flesh Alcohol phosphatide, serinephosphatide or Polyene Phosphatidylcholine；

The surfactant is enuatrol；

The injection solvent is water for injection, injection physiological saline, the D/W of injection 5% or pH7.4~8.2 One kind in phosphate buffer.

2. Z-GP-Dox mixed micelle preparation is carried according to claim 1, it is characterised in that the phosphatide is phosphatidyl courage Alkali content is more than 97% soybean lecithin.

3. the preparation method of Z-GP-Dox mixed micelle preparation is carried described in claim 1 or 2, it is characterised in that including following Step：

S1. Z-GP-Dox, phosphatide, surfactant are completely dissolved in organic solvent, form solution A；

S2. S1 resulting solutions A is slowly dropped in the injection solvent of high-speed stirred, forms solution B；

S3. it is evaporated under reduced pressure and removes the organic solvent remained in S2 resulting solutions B and be concentrated to appropriate volume, obtains and carry Z-GP- Dox mixed micelle preparation.

4. the preparation method of Z-GP-Dox mixed micelle preparation is carried according to claim 3, it is characterised in that phosphorus described in S1 Fat is egg yolk lecithin, soybean lecithin, hydrogenated yolk lecithin, hydrogenated soya phosphatide, cephalin, lipositol, serine phosphorus Fat or Polyene Phosphatidylcholine；The surfactant is enuatrol；

Injection solvent described in S2 be water for injection, injection physiological saline, the D/W of injection 5% or pH 7.4~ One kind in 8.2 phosphate buffer.

5. the preparation method of Z-GP-Dox mixed micelle preparation is carried according to claim 3, it is characterised in that described in S1 Organic solvent is one kind in methanol, ethanol, ethyl acetate, acetone or chloroform.

6. the preparation method of Z-GP-Dox mixed micelle preparation is carried according to claim 5, it is characterised in that described in S1 Organic solvent is ethanol；

Injection solvent described in S2 is water for injection, injection physiological saline, the D/W of injection 5% or pH 7.4 One kind in~8.2 phosphate buffer.

7. a kind of intravenous formulations of the Z-GP-Dox mixed micelles prepared containing preparation method described in claim 3.