CN112156170B

CN112156170B - Triptorelin sustained release microspheres for subcutaneous injection and preparation method and application thereof

Info

Publication number: CN112156170B
Application number: CN202011212241.4A
Authority: CN
Inventors: 周晓庆
Original assignee: Beijing Kangxin Donghong Pharmaceutical Technology Co ltd
Current assignee: Beijing Kangxin Donghong Pharmaceutical Technology Co ltd
Priority date: 2020-11-03
Filing date: 2020-11-03
Publication date: 2022-10-21
Anticipated expiration: 2040-11-03
Also published as: CN112156170A

Abstract

The invention relates to a triptorelin sustained release microsphere for subcutaneous injection and a preparation method and application thereof. The triptorelin sustained release microspheres comprise: triptorelin, a lactide-glycolide copolymer with the model number of 75. The triptorelin sustained release microspheres have the characteristics of low auxiliary material dosage, high drug loading rate and stable drug release in animals.

Description

Triptorelin sustained release microspheres for subcutaneous injection and preparation method and application thereof

Technical Field

The invention belongs to the field of pharmaceutical preparations, and relates to a triptorelin sustained release microsphere for subcutaneous injection, a preparation method and application thereof.

Background

Triptorelin is an artificially synthesized decapeptide gonadotropin-releasing hormone analogue and is clinically used for diseases such as prostatic cancer, endometriosis, precocious puberty and the like. The conventional injection triptorelin has short biological half-life, poor preparation stability and frequent clinical medication, and is not suitable for oral administration clinically because the polypeptide structure characteristics of the triptorelin can be subjected to enzymolysis through gastrointestinal tracts.

According to the administration characteristics of triptorelin clinical indications, patients often need to administer triptorelin for a long time, and currently, the triptorelin preparation which is developed and sold on the market is

And the like, the active ingredients in two acid radical forms of acetate and pamoate of triptorelin, a triptorelin acetate injection sustained-release preparation and a triptorelin acetate microsphere sustained-release preparation for injection are on the market, and the clinical application has been for nearly 40 years.

The triptorelin acetate injection of the normal release formulation is generally administered by subcutaneous injection, and the specification dose is 1ml:0.1mg, and auxiliary materials including sodium chloride, acetic acid and water. The triptorelin acetate microspheres for sustained-release injection are generally injected intramuscularly, the specification and dosage are 3.75mg, 11.25mg and 22.5mg, the triptorelin acetate microspheres respectively take effect for 1 month, 3 months and 6 months, the sustained-release materials used are mainly lactide-glycolide copolymers (PLGA), wherein the 3.75mg specification is used for a PLGA model of 50, the 11.25mg specification is used for a PLGA model of 75, and the 22.5mg specification is used for a PLGA model which is not published in the literature.

Some terms are explained as follows:

microsphere: microspheres refer to micro-spherical entities formed by dissolving or dispersing a drug in a carrier vehicle. Microspheres are generally called with a particle size of 1-250 μm, while sub-microspheres with a particle size of 0.1-1 μm and nanospheres with a particle size of 10-100 nm.

Sustained release preparation: the preparation is a preparation which slowly releases the drug at a non-constant speed according to requirements in a specified release medium, reduces the administration frequency by half or a little compared with the corresponding common preparation, and can obviously increase the medication compliance of patients.

In vitro release test: under the condition of simulating digestive tract in vivo (such as temperature, pH value of medium and stirring rate), the medicine release rate of the preparation is measured, and finally a reasonable in vitro medicine release degree standard is formulated so as to monitor the production process of the product and control the quality of the product. In combination with in vivo and in vitro correlation study, the release degree can predict the in vivo behavior of the product to a certain extent. For the evaluation of the reliability and the limit rationality of the release rate method, comprehensive analysis can be carried out by combining in-vivo research data.

Burst release: in the pharmaceutical formulation, the phenomenon of mass release of an injection microsphere sustained-release preparation in the first day after administration is called burst release, the degree of the burst release is generally measured by the accumulated release (%) of the medicament within 24 hours, and the control requirements of different pharmacological action differences of the medicament on the burst release are different.

ICH: for the abbreviation of international harmonization conference in english, chinese is usually translated into "international harmonization conference required for registration of drugs for human use" according to the contents of harmonization conference.

Disclosure of Invention

The invention aims to provide a pharmaceutical composition which is applied less than the existing marketed pharmaceutical excipients and is safer to use clinically, and animal in vivo experiments prove that the pharmaceutical composition is feasible.

Products on the market of triptorelin

The inventor of pharmaceutical corporation of Yipusheng of France introduces similar LHRH aqueous sustained-release composition in patent CN 105579055A, which specifies that the salt formation type of active ingredients and the proportion of the active ingredients in the salt are not less than 95%, the proportion of the active ingredients in the composition is 1.5-22%, and the composition is added with stabilizer, surfactant or antioxidant, uses water for sterilization and injection as solvent, and is not equipped with special solvent. The inventor finds that the use of the 75 type lactide-glycolide copolymer as a slow-release auxiliary material can greatly reduce the dosage of the medicinal auxiliary material and improve the medication safety by researching the copolymer. Furthermore, in some embodiments, the drug release may be delayed for an effective release period of 1 month.

One aspect of the present invention provides a triptorelin sustained release microsphere, which comprises: triptorelin, a lactide-glycolide copolymer with the model number of 75.

The triptorelin may be one or more selected from triptorelin free base, triptorelin acetate, or triptorelin pamoate.

The lactide-glycolide copolymer (7525) is glycolide-lactide copolymer for injection specified by pharmacopoeia, the end capping is-COOH group, and the weight-average molecular weight can be 5000 Da-800000 Da, preferably 10000 Da-300000 Da.

In the triptorelin sustained-release microspheres, the lactide-glycolide copolymer may be used in an amount of 100 to 1500 parts by weight, preferably 300 to 1200 parts by weight, and more preferably 600 to 800 parts by weight, based on 100 parts by weight of triptorelin free base. Within the dosage range, the preparation process can be smoother, the size of the formed emulsion drops is proper, the release rate of the prepared microspheres is suitable for the control requirement of the indication diseases, and the loss of effective components in the process preparation process is less. If the dosage is more than the range, the drug loading rate is low, and the release period is prolonged; the active ingredients in the microsphere inclusion compound are not uniform, so that the quality of the product in the same batch is not uniform; if the dosage is less than the range, the active ingredient triptorelin is excessively lost in the production process, so that the raw material medicine is wasted, and the production cost is too high.

The lyoprotectant can be any lyoprotectant suitable for the freeze-drying process of the microsphere preparation in the field, and can be one or more of mannitol, glycine, glucose, dextran, trehalose and the like, preferably mannitol, in particular low endotoxin injection-grade mannitol. The lyoprotectant may be used in an amount of 10 to 500 parts by weight, preferably 100 to 200 parts by weight, based on 100 parts by weight of triptorelin free base. Within the dosage range, the freeze-drying protection effect can be well played, and the microspheres are prevented from being adhered to the connecting blocks.

The particle size D of the triptorelin sustained-release microspheres ₉₀ Not more than 150 μm, preferably not more than 100 μm, D ₁₀ Not less than 0.7. Mu.m, preferably not less than 1 μm. Furthermore, preferably, D is preferred ₅₀ In the range of 10 to 50 μm, preferably 10 to 40 μm. If the particle size is smaller than the above range, the release amount at the early stage of drug release may be too high, that is, the burst release problem of the microsphere preparation, which may cause too high blood concentration in the body of a patient in a short period of time, and thus the safety of the drug may be affected.

The invention also provides a preparation method of the triptorelin sustained release microspheres, which comprises the following steps:

(1) Dissolving triptorelin in water to prepare an aqueous solution, dissolving a lactide-glycolide copolymer with the model of 75;

(2) Injecting the W/O primary emulsion obtained in the step (1) into a polyvinyl alcohol aqueous solution, and mixing and emulsifying to form a W/O/W multiple emulsion;

(3) Transferring the W/O/W multiple emulsion prepared in the step (2) into a reaction kettle, controlling the temperature in the reaction kettle at 0-40 ℃ and the vacuum degree at 0-minus 0.10MPa, simultaneously introducing dry air with the flow rate less than 30L/min, and performing reduced pressure drying until dichloromethane is completely removed to obtain cured microspheres;

(4) Filtering, collecting microspheres, dispersing in a freeze-drying protective agent aqueous solution, and freeze-drying to obtain the dry microspheres.

In the above method, the descriptions of triptorelin, the lactide-glycolide copolymer and the lyoprotectant are the same as the above description, and are not repeated herein.

In the step (1), the weight percentage concentration of the triptorelin aqueous solution can be 30-80% (w/w), preferably 50-60% (w/w); the concentration of the lactide-glycolide copolymer in methylene dichloride solution can be 5-60% (w/w), preferably 20-35% (w/w). Within the concentration range, the preparation process is smoother, the size of formed emulsion drops is proper, the release rate of the prepared microspheres is suitable for the control requirement of the indication diseases, and the loss of effective components in the process preparation process is less.

In the step (1), the amounts of the triptorelin aqueous solution and the lactide-glycolide copolymer methylene chloride solution used in the two-phase mixing emulsification may be such that the lactide-glycolide copolymer may be 100 to 1500 parts by weight, preferably 300 to 1200 parts by weight, and further preferably 600 to 800 parts by weight, based on 100 parts by weight of triptorelin free base. If the dosage is more than the range, the emulsion drop formation may be not facilitated due to too large viscosity of the liquid in the process preparation process, and if the dosage is less than the range, the drug-loading rate of the prepared microspheres is low, the loss of effective components in the process is large, the production cost is increased, and the industrialization is not facilitated.

In the step (1), a method of mixing and emulsifying the two phases of the triptorelin aqueous solution and the lactide-glycolide copolymer-methylene chloride solution is not particularly limited, and a mixing and emulsifying method which is conventional in the art may be employed. For example, a method of ultrasonic emulsification, a microfluidics technique, or a high-speed shearing method may be employed.

In step (2), the polyvinyl alcohol refers to a pharmaceutical grade polyvinyl alcohol having a degree of hydrolysis of 88%, and the weight average molecular weight may range from 20000Da to 150000Da, preferably from 30000Da to 100000Da. The polyvinyl alcohol may be used in an amount of 100 parts by weight to 2000 parts by weight, preferably 150 parts by weight to 1000 parts by weight, and more preferably 400 parts by weight, based on 100 parts by weight of triptorelin free base, within the above-mentioned range, to stably and effectively disperse the emulsion droplets so that the W/O type emulsion droplets are uniformly dispersed in the aqueous medium. If the amount is more than the above range, the solvent removal process is easy to foam, which is not beneficial to the production process control, and if the amount is less than the above range, the emulsion droplet state before the microsphere molding can not be stabilized, the emulsion droplet has a demulsification risk, and the prepared microsphere is easy to adhere. The concentration of the polyvinyl alcohol aqueous solution may be 0.02 to 2.0% (w/w), preferably 0.05 to 1% (w/w), and more preferably 0.1 to 0.3% (w/w). In the above concentration range, the emulsion droplets can be stably and effectively dispersed, so that the W/O type emulsion droplets are uniformly dispersed in the aqueous medium, if the amount of the emulsion droplets is larger than the above range, the solvent removal process is easy to foam, the production process control is not facilitated, if the amount of the emulsion droplets is smaller than the above range, the state of the emulsion droplets before microsphere forming cannot be stabilized, the emulsion droplets have a demulsification risk, and the prepared microspheres are easy to adhere.

In step (2), there is no particular limitation on the method of mixed emulsification, and a mixed emulsification method conventional in the art may be employed. For example, a shear emulsification method may be employed, in which the shear rotation speed may be 500 to 50000rpm, preferably 2000 to 20000rpm.

The step (3) may be carried out under stirring at a stirring speed of 5 to 50rpm, preferably 15 to 20rpm. The total time of the reduced pressure drying is preferably controlled to be 1 to 12 hours, preferably 3 to 5 hours.

After the step (3), the organic solvent methylene chloride in the emulsion can be removed, and simultaneously the lactide-glycolide copolymer is solidified into microspheres due to the loss of methylene chloride.

In step (4), the polyvinyl alcohol is removed by leaving it in the aqueous solution by filtration.

In the step (4), the concentration of the lyoprotectant aqueous solution is not particularly limited, and may be selected according to actual needs. For example, the lyoprotectant aqueous solution may be a 1% to 20%, preferably 5% to 15%, and especially 10% by weight mannitol aqueous solution.

After freeze-drying, the remaining lactide-glycolide copolymer wraps triptorelin to form drug-loaded microspheres, and the drug-loaded microspheres are dispersed in a freeze-drying protective agent.

In still another aspect, the present invention provides a pharmaceutical composition comprising the above triptorelin sustained release microspheres according to the present invention.

In an embodiment, the pharmaceutical composition according to the present invention may comprise components a and B, wherein component a is the above-mentioned triptorelin sustained release microspheres according to the present invention, and component B is a special vehicle comprising mannitol, polysorbate 80, sodium carboxymethylcellulose and water.

In component B, mannitol 50mg, polysorbate 80 mg, sodium carboxymethylcellulose 5mg and the balance water may be contained based on 1ml of component B.

The amount of component a used may be 3.75mg based on 1ml of component B, in terms of triptorelin free base.

When in use, the component A can be dispersed in the component B and can be administered by subcutaneous or intramuscular injection.

The packaging manner of the component A and the component B is not limited, and for example, the components A and B may be packaged in vials, respectively.

Yet another aspect of the present invention relates to the use of the sustained release microspheres or pharmaceutical composition of triptorelin according to the present invention for the preparation of a medicament for the prevention/treatment of one or more diseases selected from the group consisting of prostate cancer, prostatic hypertrophy, endometriosis, uterine fibroids, precocious puberty, breast cancer, and the like.

The single administration of the pharmaceutical composition according to the present invention can achieve a sustained release for more than 28 days, but is not limited thereto.

All features or conditions defined herein as numerical ranges or percentage ranges are for brevity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be considered to have covered and specifically disclosed all possible subranges and individual numerical values within the ranges, particularly integer numerical values. For example, a description of a range of "1 to 8" should be considered to have specifically disclosed all subranges such as 1 to 7, 2 to 8, 2 to 6, 3 to 6, 4 to 8, 3 to 8, and so on, particularly subranges bounded by all integer values, and to have specifically disclosed individual values within that range such as 1, 2, 3, 4, 5, 6, 7, 8, and so on. Unless otherwise indicated, the foregoing explanatory methods apply to all matters throughout the present invention, whether broad or not.

If an amount or other value or parameter is expressed as a range, preferred range, or a list of upper and lower limits, then it is to be understood that all ranges subsumed therein as either the upper or preferred value for that range and the lower or preferred value for that range are specifically disclosed herein, regardless of whether ranges are separately disclosed. Further, when a range of numerical values is recited herein, unless otherwise stated, the range is intended to include the endpoints thereof, and all integers and fractions within the range.

In this context, numerical values should be understood to have the precision of the number of significant digits of the value, provided that the object of the invention is achieved. For example, the number 40.0 should be understood to cover a range from 39.50 to 40.49.

The above embodiments are only exemplary embodiments of the present invention, and are not intended to limit the present invention, and the scope of the present invention is defined by the claims. Various modifications and equivalents may be made by those skilled in the art within the spirit and scope of the present invention, and such modifications and equivalents should also be considered as falling within the scope of the present invention.

Advantageous effects

The triptorelin sustained-release microspheres which are on the market at present mainly comprise triptorelin sustained-release microspheres obtained by the German Feilang company

And of the Probiotics of France

Two products, the active ingredients of which are triptorelin acetate and triptorelin pamoate, wherein the triptorelin sustained-release microspheres which can continuously release triptorelin for one month are both triptorelin free alkali3.75mg specification.

The specification and the foreign marketing data disclose that each bottle of powder injection contains 3.75mg of triptorelin acetate, 170mg of lactide-glycolide copolymer (50 type 50), 85mg of mannitol, 30mg of sodium carboxymethylcellulose and 80 mg of polysorbate, and is additionally prepared with 2ml of sterile water for injection.

The specification and the foreign marketing data disclose that each bottle contains triptorelin acetate 3.75mg and poly DL-cyclic diester-composite-anhydroglucose; special solvent containing polysorbate 80, dextran 70, sodium dihydrogen phosphate dihydrate, sodium chloride, etc. is additionally prepared, and the pH value is adjusted by sodium hydroxide to 1ml in total.

Compositions and methods of the invention

And

for comparison see table below:

the comparison is illustrated as follows:

1) Has come into the market

For intramuscular injection, there is a risk of damaging the muscle nerve in clinical use;

2) Has come into the market

Although the dextran 70 can be injected intramuscularly or subcutaneously, the dextran 70 used in the prescription is mainly obtained by fermentation or enzyme synthesis, and the risk of anaphylactic reaction exists after the dextran is injected into a human body.

3)

The amount of the biodegradable material lactide-glycolide copolymer used in (a) was 170mg per bottle, whereas only less than 50mg per bottle was used in the composition of the invention.

Thus, the composition of the present invention has the following technical effects:

1) Reduces the dosage of the auxiliary materials for the clinical application of the medicine and improves the safety of the medicine.

2) Subcutaneous injection is compared with intramuscular injection, has the clinical operation of nurse of being convenient for, avoids intramuscular injection to injure the medical accident risk of human nerve.

3) The single administration volume is reduced, and the medication compliance of patients is improved.

Drawings

FIG. 1 is a graph comparing the cumulative release profiles over 0-72 hours for different triptorelin microspheres.

FIG. 2 is a graph comparing the cumulative release profiles over 72-840 hours for different triptorelin microspheres.

Fig. 3 is a graph of blood triptorelin concentration in beagle dogs over time from 0 to 8 hours.

Fig. 4 is a graph of blood triptorelin concentration in beagle dogs over time between 8 and 840 hours.

FIG. 5 is a graph of testosterone plasma levels in a beagle over time from 0 to 840 hours.

Detailed Description

The following examples are intended to illustrate embodiments of the present invention only and not to limit the present invention in any way, and it will be understood by those skilled in the art that various modifications may be made without departing from the spirit and scope of the present invention, and such modifications are within the scope of the present invention.

Example 1:

(1) Dissolving 5g of triptorelin acetate in 8ml of water to prepare a triptorelin acetate aqueous solution, weighing 40.22g of lactide-glycolide copolymer (type 75, model 25) to dissolve in 120.66g of dichloromethane, mixing the two phases, performing ultrasonic emulsification to form a primary emulsion (W/O), cooling to 5-15 ℃, and transferring to a syringe pump.

(2) Weighing 20g of polyvinyl alcohol, adding water to dissolve and dilute the polyvinyl alcohol to prepare 0.2% solution, sterilizing and filtering the solution, starting a high-shear homogenizer at a rotating speed of 3000-4000rpm, injecting the cooled primary emulsion obtained in the step (1) at a constant speed while shearing, continuing homogenizing the mixture for 2-5 minutes after the injection is finished, and cooling the mixture to 10-15 ℃ to form multiple emulsion (W/O/W).

(3) Transferring the multiple emulsion suspension prepared in the step (2) into a 20L reaction kettle, controlling the temperature in the reaction kettle at 0-40 ℃ and the vacuum degree at 0-minus 0.10MPa, simultaneously introducing dry air with the flow rate less than 30L/min, and carrying out in-liquid reduced pressure drying until dichloromethane is completely removed to obtain cured microspheres;

(4) And after the operation is finished, filtering and collecting the microspheres, dispersing the microspheres into a 10% mannitol aqueous solution, and freeze-drying to obtain the dry microspheres QPRL A.

Example 2:

(1) Dissolving 5.8g triptorelin pamoate in 8.5ml water to prepare a hydrophilic drug solution, weighing 40.5g of lactide-glycolide copolymer (75 type 25) to be dissolved in 121.5g dichloromethane, mixing the two phases, shearing and emulsifying at high speed to form a primary emulsion (W/O), cooling to 8-15 ℃, and transferring to a syringe pump.

(2) Weighing 28g of polyvinyl alcohol, adding water to dissolve and dilute the polyvinyl alcohol to prepare 0.2% solution, sterilizing and filtering the solution, starting a high-shear homogenizer at a rotating speed of 3000-4000rpm, injecting the cooled primary emulsion obtained in the step (1) at a constant speed while shearing, continuing homogenizing the mixture for 2-5 minutes after the injection is finished, and cooling the mixture to 10-15 ℃ to form multiple emulsion (W/O/W).

(3) And (3) transferring the multiple emulsion suspension prepared in the step (2) into a 20L reaction kettle, controlling the temperature in the reaction kettle to be 0-40 ℃ and the vacuum degree to be 0-0.10 MPa, simultaneously introducing dry air with the flow rate being less than 30L/min, and carrying out in-liquid reduced pressure drying until dichloromethane is completely removed to obtain the solidified microspheres.

(4) And after the operation is finished, filtering and collecting the microspheres, dispersing the microspheres into a 10% mannitol aqueous solution, and freeze-drying to obtain the dry microspheres QPRL B.

Comparative example 1:

(1) 0.9g of triptorelin acetate is dissolved in 8.5ml of water to prepare a hydrophilic drug solution, 40.5g of lactide-glycolide copolymer (50 type) is additionally weighed and dissolved in 121.5g of dichloromethane, the two phases are mixed and then are sheared and emulsified at high speed to form a primary emulsion (W/O), the temperature is reduced to 8-15 ℃, and the primary emulsion is transferred into a syringe pump.

(2) Weighing 28g of polyvinyl alcohol, adding water to dissolve and dilute the polyvinyl alcohol to prepare 0.2% solution, sterilizing and filtering the solution, starting a high-shear homogenizer at a rotating speed of 3000-4000rpm, injecting the cooled primary emulsion obtained in the step (1) at a constant speed while shearing, continuing to homogenize the emulsion for 2-5 minutes after the injection is finished, and cooling the mixed emulsion to 10-15 ℃ to form multiple emulsion (W/O/W).

(4) And after the operation is finished, filtering and collecting the microspheres, dispersing the microspheres into a 10% mannitol aqueous solution, and freeze-drying to obtain the dry microspheres QPRL C.

Example 3:

weighing 0.50g of polysorbate 80, 2.50g of sodium carboxymethylcellulose and 25.0g of mannitol in sequence, adding water, stirring for dissolving, diluting to 500ml, filtering, filling into colorless glass ampoules according to 1 ml/ampoule, sealing by melting, and sterilizing in a steam sterilizer at 121 ℃ for 30 minutes to obtain the special solvent.

Experimental example 1:

respectively measuring the QPRL A, the QPRL B, the QPRL C and the commercially available QPRL C of the microsphere samples by adopting an HPLC external standard method

The medium drug loading and encapsulation efficiency, 35-day constant-speed release rate of four samples measured by a water bath shaking bed method in a phosphate medium at 37 ℃, and particle size distribution data of the four samples measured by a laser particle sizer are shown in the following table:

a comparison of the cumulative release profiles over 0-72 hours and 72-840 hours for different triptorelin microspheres is shown in FIGS. 1 and 2. From the data, it can be found that when the lactide-glycolide copolymer with the model number of 75 is used for preparing microspheres with the drug loading of about 8.0-8.5%, whether triptorelin acetate or pamoate, the release curve of the microspheres at 37 ℃ is detected by the same release degree method as that of the microspheres with the model number of 50: microspheres with a drug loading of 1.29% prepared from the 50 lactide-glycolide copolymer were almost identical. The triptorelin microsphere QPRL C prepared by adopting the lactide-glycolide copolymer with the model number of 50. Therefore, QPRL A and QPRL B are selected to carry out animal in-vivo PK comparison study with daphne and dabiejia which are on the market and have the release period of 1 month.

Experimental example 2:

the prepared triptorelin acetate microspheres QPRL A or the triptorelin pamoate microspheres QPRL B are subpackaged in 5ml penicillin bottles according to the loading of 3.75mg triptorelin free alkali/bottle, and after 1 triptorelin/bottle of special solvent is mixed with each bottle, the subcutaneous injection is given to the Zhuang-year healthy beagle dog according to the weight of 0.125mg/kg (calculated by the triptorelin free alkali); the control group adopts Daphtalene and Daphnika medicine with specification of 3.75mg (calculated by triptorelin free base) respectively, and is administrated according to the method of the instruction usage and dosage, after 2ml of sterilization injection water is added into each bottle of Daphtalene to be evenly dissolved, the intramuscular injection of the Zhuangnian healthy beagle dog is administrated according to the weight of 0.125mg/kg (calculated by the triptorelin free base), the 1ml of special solvent is added into each bottle of Daphnika, and the subcutaneous injection of the Zhuangnian healthy beagle dog is administrated according to the weight of 0.125mg/kg (calculated by the triptorelin free base).

At 0.25 hr, 0.50 hr, 0.75 hr, 1.0 hr, 2.0 hr, 3.0 hr, 4.0 hr, 5.0 hr, 6.0 hr, 7.0 hr, 8.0 hr, 10 hr, 14 hr, 24 hr (first day), 48 hr (second day), 168 hr (second day), 336 hr (fourth day 14), 504 hr (fourth day 21), 672 hr (fourth day 28), 720 hr (fourth day 30), 792 hr (fourth day 33), and 840 hr (fourth day 35), respectively, after administration. 3.0ml of blood was taken from the median vein of the forelimb, centrifuged at 4 ℃ for 5 minutes (4000 rpm) in a heparinized centrifugation tube, and the plasma was separated and stored at-70 ℃ for testing. A total of 23 spots (with blank blood samples) were collected.

The samples were kept in an ice-water bath during blood collection and transport.

The plasma sample is collected into a centrifuge tube which is stuck with a sample label, and the content on the label comprises a project number (QPRL-DPK-20200201), a sample type (plasma: P), a test number of a tested animal and blood collection time. For example, D1-1-P-2h, represents a plasma sample taken at the 2h sampling point after administration of the animal identified by test number D1-1 in this study. And detecting the concentration of triptorelin and testosterone in the plasma by an LC-MS/MS method.

Test animal groups, numbers and preparation tables

The mean time plots and testosterone blood concentrations versus time plots for 6 male beagle dogs following subcutaneous injection administration of QPRL a and QPRL B, respectively, were determined as shown in figures 3-5.

The results in fig. 3-5 show that the triptorelin acetate microspheres and the triptorelin pamoate microspheres prepared by the pharmaceutical composition meet the quality requirements of the Chinese pharmacopoeia 2020 edition and ICH related technical guidelines on injection microsphere preparations. More surprisingly, the composition uses a smaller amount of the biodegradable sustained-release material lactide glycolide copolymer, triptorelin original drug is always detected in animal blood within 1 month period after animal administration, and the triptorelin original drug is shown to effectively suppress the generation of testosterone in male animals, which indicates that the composition and the composition which is already on the market

And

in contrast, similar testosterone suppression effects were obtained, and the present compositions were compared

And

the blood concentration is more stable in the later period of the administration period, and the testosterone suppression effect is more effective.

It will be understood by those skilled in the art that the above embodiments are specific examples of the implementation of the inventive arrangements and that various changes in form and detail may be made therein without departing from the spirit and scope of the inventive arrangements.

Reference:

research progress of novel injection drug delivery system 2016, 47 (3) P333-339 in journal of Chinese medicine industry

Judy Senior, michael Radomsky, eds., zhengjunmin et al, injectable sustained Release preparation, chemical industry Press.

Claims

1. A triptorelin sustained release microsphere having a release period of one month, comprising: triptorelin, a lactide-glycolide copolymer with a model number of 75, and a freeze-drying protective agent;

the weight average molecular weight range of the lactide-glycolide copolymer is 10000Da to 300000Da;

the lactide-glycolide copolymer is used in an amount of 300 to 1200 parts by weight based on 100 parts by weight of triptorelin free base;

the amount of the cryoprotectant is 100-200 parts by weight based on 100 parts by weight of triptorelin free base;

the particle size D of the triptorelin sustained-release microspheres ₉₀ Not more than 150 μm, D ₁₀ Not less than 0.7 μm, D ₅₀ In the range of 10 to 50 μm;

the preparation method of the triptorelin sustained-release microspheres comprises the following steps:

(1) Dissolving triptorelin in water to prepare an aqueous solution, dissolving a lactide-glycolide copolymer with the model of 75; wherein the weight percentage concentration of the triptorelin aqueous solution is 50% -60%; the weight percentage concentration of the lactide glycolide copolymer dichloromethane solution is 20% -35%;

(2) Injecting the W/O primary emulsion obtained in the step (1) into a polyvinyl alcohol aqueous solution, and mixing and emulsifying to form a W/O/W multiple emulsion; wherein the polyvinyl alcohol is used in an amount of 400 parts by weight based on 100 parts by weight of triptorelin free base; the polyvinyl alcohol is medicinal polyvinyl alcohol with the hydrolysis degree of 88 percent, and the weight average molecular weight range is 30000Da to 100000Da; the weight percentage concentration of the polyvinyl alcohol aqueous solution is 0.1% -0.3%; the mixing emulsification adopts a high-shear emulsification or homogenizing emulsification method, wherein the shearing rotating speed is 2000 to 20000rpm;

(3) Transferring the W/O/W multiple emulsion prepared in the step (2) into a reaction kettle, controlling the temperature in the reaction kettle to be 0 to 40 ℃ and the vacuum degree to be 0 to-0.10 MPa, simultaneously introducing dry air with the flow rate being less than 30L/min, and drying under reduced pressure until dichloromethane is completely removed to obtain cured microspheres;

2. The triptorelin sustained release microspheres of claim 1, wherein the triptorelin is one or more selected from the group consisting of triptorelin free base, triptorelin acetate, and triptorelin pamoate.

3. A triptorelin sustained release microsphere according to claim 1, wherein the amount of the poly (lactide-co-glycolide) is 600 to 800 parts by weight based on 100 parts by weight of the triptorelin free base.

4. The triptorelin sustained release microspheres of claim 1, wherein the particle size D of the triptorelin sustained release microspheres ₉₀ Not more than 100 μm, D ₁₀ Not less than 1 μm, D ₅₀ Within the range of 10 to 40 μm.

5. A method for preparing the triptorelin sustained release microspheres of any one of claims 1-4, the method comprising:

(2) Injecting the W/O primary emulsion obtained in the step (1) into a polyvinyl alcohol aqueous solution, and mixing and emulsifying to form a W/O/W multiple emulsion; wherein the polyvinyl alcohol is used in an amount of 400 parts by weight based on 100 parts by weight of triptorelin free base; the polyvinyl alcohol is medicinal polyvinyl alcohol with the hydrolysis degree of 88 percent, and the weight average molecular weight range is 30000Da to 100000Da; the weight percentage concentration of the polyvinyl alcohol aqueous solution is 0.1% -0.3%; the mixing emulsification adopts a high-shear emulsification or homogenizing emulsification method, wherein the shear rotation speed is 2000 to 20000rpm;

6. The process according to claim 5, wherein in step (1), the amounts of the aqueous triptorelin solution and the glycolide copolymer methylene chloride solution are such that the glycolide copolymer accounts for 600 to 800 parts by weight based on 100 parts by weight of triptorelin free base.

7. The method according to claim 5, wherein the step (3) is performed under stirring at a speed of 5 to 50rpm; the total time of reduced pressure drying is controlled to be 1h to 12h; and/or

In the step (4), the freeze-drying protective agent aqueous solution is 1-20% by weight of mannitol aqueous solution.

8. The method according to claim 7, wherein the step (3) is performed under stirring at a speed of 5 to 20rpm.

9. The method of claim 7, wherein in the step (3), the total time of the reduced pressure drying is controlled to be 3 to 5 hours.

10. The method according to claim 7, wherein in step (4), the aqueous lyoprotectant solution is a 5-15% by weight aqueous mannitol solution.

11. The method according to claim 10, wherein, in step (4), the lyoprotectant aqueous solution is a 10% by weight mannitol aqueous solution.

12. A pharmaceutical composition comprising triptorelin sustained release microspheres according to any one of claims 1-4 or triptorelin sustained release microspheres prepared according to the method of any one of claims 5-11.

13. The pharmaceutical composition according to claim 12, comprising components A and B, wherein,

the component A is triptorelin slow release microspheres according to any one of claims 1-4 or prepared according to the method of any one of claims 5-11,

the component B is a special solvent which comprises mannitol, polysorbate 80, sodium carboxymethylcellulose and water.

14. The pharmaceutical composition of claim 13, wherein,

in component B, based on 1ml of component B, 50mg of mannitol, 80 mg of polysorbate, 5mg of sodium carboxymethylcellulose and the balance of water are contained; and/or

The amount of component A used was 3.75mg based on 1ml of component B, in terms of triptorelin free base.

15. Use of triptorelin sustained release microspheres according to any one of claims 1-4, or triptorelin sustained release microspheres prepared according to the method of any one of claims 5-11, or the pharmaceutical composition of any one of claims 12-14 in the manufacture of a medicament for the prevention/treatment of one or more diseases selected from the group consisting of prostate cancer, prostatic hypertrophy, endometriosis, uterine fibroids, precocious puberty and breast cancer.