CN118267362A

CN118267362A - Long-acting slow-release injection of alidenafil, its preparation and use

Info

Publication number: CN118267362A
Application number: CN202410359256.5A
Authority: CN
Inventors: 于飞; 宋更申; 欧阳旭; 苏颖
Original assignee: Beijing Youcare Kechuang Pharmaceutical Technology Co ltd
Current assignee: Beijing Youcare Kechuang Pharmaceutical Technology Co ltd
Priority date: 2024-03-27
Filing date: 2024-03-27
Publication date: 2024-07-02

Abstract

The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to a long-acting sustained-release injection of alidenafil, a preparation method and application thereof, wherein the long-acting sustained-release injection can treat Alzheimer's Disease (AD), pulmonary arterial hypertension (pulmonary hypertension, PH) and erectile dysfunction (erectile dysfunction, ED), and can realize one subcutaneous injection for 4 weeks to months, thereby greatly reducing the treatment burden of patients, improving the medication compliance, reducing the treatment cost, and simultaneously, the results of in vitro release research and animal experiments prove that the long-acting sustained-release injection of alidenafil can slowly release medicines in vitro and in vivo for a long time.

Description

Long-acting slow-release injection of alidenafil, its preparation and use

Technical Field

The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to a long-acting sustained-release injection of alidenafil, a preparation method thereof and application of the injection in treating diseases such as Alzheimer disease, pulmonary hypertension, erectile dysfunction and the like.

Background

Alidenafil citrate tablet, approval document: national drug standard H20210051, trade name: alishi, english name AILDENAFIL CITRATE Tablets, belongs to chemicals, and its main component is alidenafil citrate, which is mainly used for treating male erectile dysfunction.

Alidenafil, compared with sildenafil, tadalafil, etc., belongs to the PDE5 inhibitor family, but is a brand new class and has a brand new drug molecular structure. As a new class of PDE5 inhibitors which are independently developed in China, a test about enzymatic activity test of the 'Aidinafei and other PDE marketed inhibitors' shows that the molecular docking energy value of Aidinafei is-10.1833 for PDE5A, namely a main target for treating erectile dysfunction; molecule and IC50: nM up to 0.66: and +/-0.15, and has high pharmaceutical activity and enzyme specific selectivity in all the similar products tested.

The alidenafil belongs to a new class of PDE5 inhibitors, can be applied to different applicable diseases, and is clinically applicable to the application treatment of pulmonary arterial hypertension (pulmonary hypertension, PH), alzheimer's Disease (AD) and other aspects besides the treatment of erectile dysfunction (erectile dysfunction, ED).

However, the dosage form of the currently marketed alidenafil preparation is a common quick-release tablet, the specification is 30mg, the oral bioavailability is low, the half-life period is 3.5 hours (the half-life period is short), and the treatment needs to be taken for 2 times or 3 times in 1 day. Therefore, for the purposes of pulmonary arterial hypertension (PH), alzheimer's Disease (AD) and the like other than Erectile Dysfunction (ED), there is a urgent need in clinic to develop a long-acting therapeutic pharmaceutical formulation for once a month or even several months, which is simple in process, easy to apply clinically, so as to improve patient compliance and reduce treatment costs and patient burden.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide a long-acting injection of alidenafil and a preparation method thereof, which enable alidenafil to be released over a long period of time, for example, several weeks to 6 months, preferably over at least 4 weeks, or substantially release all active ingredients, by embedding alidenafil in a biocompatible pharmacologically acceptable polymer and suspending in a suitable carrier, and a long-acting microsphere pharmaceutical formulation which is simple in process and easy to be applied in a bed, so as to improve patient compliance and reduce treatment costs and reduce patient burden.

The invention provides a long-acting sustained-release injection of alidenafil, which comprises a solid component and a solvent component; the solid component comprises, by mass, solid components: 0.7 to 15 weight percent of active ingredient calculated by alidenafil, 30 to 80 weight percent of biodegradable polymer matrix, 1 to 10 weight percent of amino acid, and the balance of pH value regulator and freeze-drying protective agent; the active ingredient is one or more of alidenafil, pharmaceutically acceptable salt and pharmaceutically acceptable ester thereof.

In some preferred embodiments provided herein, the pH of the solid component is preferably 7 to 10; the invention puts the active components, especially the alinafil citrate, in the alkaline microenvironment (pH 7.0-10.0), reduces the dissociation degree of the medicine to the maximum extent, makes the medicine deviate to the free alkali state, and reduces the solubility of the medicine; the active components, especially the alidenafil citrate, are released in the microsphere preparation in an alkaline environment all the time, so that the diffusion of the medicine into an external water phase can be reduced. In some embodiments provided herein, the pH of the solid component is specifically 7, 8 or 10.

In some preferred embodiments provided herein, the solid component comprises a microsphere formulation; the particle diameter D90 of the microsphere preparation is preferably 10 to 130. Mu.m, more preferably 20 to 90. Mu.m, still more preferably 20 to 70. Mu.m, most preferably 50 to 70. Mu.m.

In some preferred embodiments provided herein, the solid component comprises a microsphere formulation and a lyoprotectant.

In some preferred embodiments provided herein, the active ingredient is embedded with the amino acid in a biodegradable polymer matrix; further specifically, the active ingredient and the amino acid are embedded in a biodegradable polymer matrix to form a microsphere preparation. The active ingredient is embedded in a biodegradable polymer matrix to prepare a long-acting sustained-release injection, the main dosage form is microsphere preparation for injection, suspension is added before use, intramuscular injection can be carried out, compared with an alinafil oral preparation, the effect of the alinafil long-acting sustained-release injection is obviously prolonged, the administration frequency is reduced (the preparation can be prepared for 4 weeks), and the patient compliance is obviously improved.

In some preferred embodiments provided herein, the amount of active ingredient, in terms of alidenafil, in the solid component is preferably from 0.71 to 14.5wt%, more preferably from 0.71 to 14.28wt%, based on the mass of the solid component; further specifically, it may be 0.71wt%, 3.57wt% or 14.5wt%.

In some preferred embodiments provided herein, the active ingredient is one or more of alidenafil, a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable ester thereof, preferably alidenafil citrate.

In some preferred embodiments provided herein, the solid component comprises, by mass of the solid component: 1 to 20 weight percent of sildenafil citrate, 30 to 80 weight percent of biodegradable polymer matrix, 1 to 10 weight percent of amino acid, and the balance of pH value regulator and freeze-drying protective agent.

In some preferred embodiments provided herein, the biodegradable polymer matrix is present in the solid component in an amount of 40 to 80wt%, preferably 50 to 80wt%, more preferably 60 to 80wt%; the biodegradable polymer matrix in the solid component in some embodiments provided herein is specifically present in an amount of 30wt%, 67.3wt% or 80wt%.

In some preferred embodiments provided herein, the biodegradable polymer matrix comprises a polyester-based polymer; more specifically, the polyester polymer is one or more of polylactic acid-glycolic acid copolymer (PLGA), polylactic acid (PLA), polyglycolic acid (PGA), polyvinyl acid, polyhydroxybutyrate, polycaprolactone (PCL), polyalkylene oxalate, polyalkylene glycol ester, a block copolymer of polylactic acid-glycolic acid copolymer polyethylene glycol derivative, a block copolymer of polylactic acid polyethylene glycol derivative, a block copolymer of polyglycolic acid polyethylene glycol derivative, a block copolymer of polyhydroxybutyrate polyethylene glycol derivative, a block copolymer of polycaprolactone polyethylene glycol derivative, a block copolymer of polyalkylene oxalate polyethylene glycol derivative and a block copolymer of polyalkylene glycol ester polyethylene glycol derivative.

In some preferred embodiments provided herein, the molecular weight of the polyethylene glycol block in the block copolymer is preferably 4000 to 8000, more preferably 4000 to 6000, still more preferably 5000.

In some preferred embodiments provided herein, the polyester-based polymer preferably has a molecular weight of 10,000 ～ 300,000 daltons, more preferably 10,000 ～ 100,000 daltons, even more preferably 30,000 ～ 100,000 daltons, and most preferably 30,000 ～ 35,000 daltons.

In some preferred embodiments provided herein, the polyester-based polymer is a polylactic acid-glycolic acid copolymer.

In some preferred embodiments provided herein, the polylactic acid-glycolic acid copolymer has a molecular weight ranging from 10000 to 300000 daltons, preferably from 30000 to 100000 daltons.

In some preferred embodiments provided herein, the polylactic acid-glycolic acid copolymer comprises lactide monomer units and glycolide monomer units; the molar ratio of lactide monomer units to glycolide monomer units was 75: 25-25: 75, preferably 50:50; in some embodiments provided herein, the molar ratio of lactide monomer units to glycolide monomer units is specifically 75: 25. 25: 75. 40: 60. 60:40 or 50:50.

In some preferred embodiments provided by the present invention, the amino acid content of the solid component is 2 to 10wt%; in some embodiments provided herein, the amino acid content of the solid component is specifically 1wt%, 2wt%, or 10wt%.

In some preferred embodiments provided herein, the amino acid is a neutral amino acid and/or a basic amino acid, preferably a basic amino acid; the addition of the neutral and basic amino acids in the microspheres can not only raise the pH of the microenvironment of the solid component so as to reduce the solubility of the alidenafil, especially the alidenafil citrate, but also can lead to the rapid self-repair and pore closure of the polymer due to the hydrogen bond formed between the alidenafil citrate and the polymer, thereby inhibiting the burst effect of the drug. Therefore, the invention can reduce the low encapsulation efficiency and the burst release phenomenon of the alidenafil, especially the alidenafil citrate long-acting microsphere preparation to the greatest extent through the synergism of the pH value of the solid component and the neutral and alkaline amino acids.

Further specifically, the amino acid is one or more of L-arginine, lysine, histidine, serine, threonine, tyrosine, asparagine, glutamine, alanine, glycine and cysteine.

In some preferred embodiments provided herein, the biodegradable polymer matrix is a polyester-based polymer: polylactic-co-glycolic acid (PLGA) with molecular weight 10000 ～ 300,000 daltons; wherein the molar ratio of lactide monomer units to glycolide monomer units is 75:25 to 25:75; the amino acid is a basic amino acid; the pH value of the solid component is 7.0-10.0.

In some preferred embodiments provided by the invention, the content of the pH regulator in the solid component is to regulate the pH value of the solid component to 7-10, and particularly regulate the pH value of the microsphere preparation in the solid component to 7-10.

In some preferred embodiments provided herein, the pH adjustor is one or more of trisodium phosphate, sodium phosphate dibasic-sodium phosphate monobasic buffer solute, sodium carbonate, sodium hydroxide, and arginine, preferably sodium hydroxide.

In some preferred embodiments provided herein, the lyoprotectant is one or more of mannitol, glucose, dextran, and sucrose.

In some preferred embodiments provided herein, the ratio of the solid component to the solvent component is from 10 to 1800mg:0.5 to 20mL, preferably 100 to 1800mg:0.5 to 20mL, more preferably 500 to 1800mg:1 to 20mL, more preferably 1000 to 1800mg: 5-20 mL, most preferably 1000-1200 mg: 5-10 mL; in some embodiments provided herein, the ratio of the solid component to the solvent component is specifically 1115mg:5mL.

In some preferred embodiments provided herein, the solvent component comprises, by mass, 0.01 to 1% of a surfactant, 0.1 to 5% of a suspending agent, 0.5 to 5% of an osmotic pressure regulator, and the balance water for injection.

In some preferred embodiments provided herein, the surfactant is present in the solvent component in an amount of from 0.05 to 0.5wt%, preferably from 0.05 to 0.3wt%, more preferably from 0.1 to 0.2wt%.

In some preferred embodiments provided herein, the surfactant comprises a nonionic surfactant; further specifically, the surfactant is one or more of poloxamer, polysorbate and span.

In some preferred embodiments provided herein, the suspending agent is preferably present in the solvent component in an amount of 1 to 5wt%, preferably 2 to 4wt%, more preferably 3 to 3.5wt%, and even more preferably 3.2wt%.

In some preferred embodiments provided herein, the suspending agent is one or more of sodium carboxymethyl cellulose, sorbitol, polyvinylpyrrolidone, and aluminum monostearate.

In some preferred embodiments provided herein, the osmolality adjusting agent is present in the solvent component in an amount of 0.5 to 3 wt.%, preferably 0.5 to 2 wt.%, more preferably 0.5 to 1 wt.%, still more preferably 0.9 wt.%.

In some preferred embodiments provided herein, the osmolality adjusting agent is sodium chloride.

The invention also provides a preparation method of the long-acting sustained-release injection of the alidenafil, which adopts a W/O/W emulsification-solidification method and comprises the following steps:

S1) mixing a biodegradable polymer matrix with an organic solvent to obtain an oil phase;

mixing the active ingredients, amino acid and water, and regulating the pH value to 7-10 by adopting a pH value regulator to obtain an inner water phase;

Mixing water or aqueous solution with an emulsifier to obtain an external water phase;

S2) adding the oil phase into an inner water phase to obtain colostrum;

S3) adding the colostrum into an external water phase to obtain compound emulsion;

s4) solidifying the compound emulsion to remove the organic solvent, collecting the solid, and washing the solid with a buffer solution to remove the emulsifier, thereby obtaining microspheres;

S5) mixing the microspheres, the freeze-drying protective agent and water, and freeze-drying to obtain a solid component;

The solid component and the solvent component form the long-acting sustained-release injection of the alinafil.

Wherein, the preparation sequence of the oil phase, the inner water phase and the outer water phase in the step S1) is not separated.

In some preferred embodiments provided herein, the organic solvent in step S1) is one or more of dichloromethane, chloroform, ethyl acetate and hexafluoroisopropanol, preferably dichloromethane.

In some preferred embodiments provided by the present invention, the mass concentration of the biodegradable polymer matrix in the oil phase in step S1) is preferably 10% to 20%, more preferably 15%.

In some preferred embodiments provided by the present invention, the mass concentration of the active ingredient in the inner aqueous phase in the step S1) is preferably 0.1% to 5%.

In some preferred embodiments provided herein, the emulsifier in step S1) is one or more of polyvinyl alcohol, gelatin, polyvinylpyrrolidone and aluminum monostearate, preferably polyvinyl alcohol.

In some preferred embodiments provided herein, the aqueous solution in step S1) is a buffer, preferably a phosphate buffer; the pH value of the phosphate buffer solution is preferably 8-8.5.

In some preferred embodiments provided by the present invention, the mass concentration of the emulsifier in the external aqueous phase in the step S1) is preferably 0.1% to 5%, more preferably 0.5% to 2%, still more preferably 0.5% to 1%.

In some preferred embodiments provided by the present invention, the oil phase in step S2) is added into the inner water phase, and then sheared at a high speed, and fully mixed to obtain colostrum; the rotation speed of the high-speed shearing is preferably 800-1500 rpm, more preferably 800-1200 rpm, and still more preferably 1000rpm; the time of the high-speed shearing is preferably 2 to 5 minutes.

In some preferred embodiments provided by the present invention, the volume ratio of the colostrum to the external water phase in step S3) is preferably 1: (3 to 8), more preferably 1: (4 to 6), and more preferably 1:5.

In some preferred embodiments provided by the present invention, the colostrum in step S3) is added into the external water phase to be sheared at high speed, so as to obtain the multiple emulsion; the rotation speed of the high-speed shearing is preferably 3000-8000 rpm, more preferably 4000-6000 rpm, and still more preferably 5000rpm; the time of the high-speed shearing is preferably 2 to 5 minutes.

In some preferred embodiments provided herein, the organic solvent is removed in step S4) by heating in a water bath; the temperature of the water bath heating is preferably 30-50 ℃.

In some preferred embodiments provided herein, the buffer in step S4) is preferably a phosphate buffer; the pH value of the phosphate buffer solution is preferably 8-8.5.

In some preferred embodiments provided in the present invention, the freeze-drying in step S5) is specifically: pre-freezing at-30 to-45 ℃, primary sublimation drying at-5 to 10 ℃ in a gradient way, vacuum pressure of 10 to 11Pa, and secondary drying at 20 to 30 ℃; the preferred specific examples are: the pre-freezing temperature is-40 ℃ to-45 ℃, the primary sublimation drying is carried out at the gradient of-5 ℃ to 10 ℃, the vacuum pressure is 10 Pa to 11Pa, and the secondary drying temperature is 25 ℃.

In the invention, the water is water for injection unless otherwise specified.

The invention also provides application of the long-acting sustained-release injection of the alidenafil in preparing one or more of medicines for treating erectile dysfunction, medicines for treating Alzheimer's disease and medicines for treating pulmonary arterial hypertension.

In some preferred embodiments provided herein, a long-acting injection is prepared for intramuscular injection once for 2 weeks to 6 months, preferably 1 time for 4 weeks.

The beneficial effects of the invention are as follows:

The long-acting sustained-release injection of the alidenafil can be used for erectile dysfunction (erectile dysfunction, ED), alzheimer's Disease (AD), or pulmonary arterial hypertension (pulmonary hypertension, PH), the long-acting sustained-release of the prepared injection can be up to more than 4 weeks by using specific long-acting sustained-release components, the burst release risk can be greatly reduced (more than 80% of burst release does not occur within 14 days), the encapsulation efficiency is obviously increased (the encapsulation efficiency exceeds 80%), the granularity is smaller (D90 is in the range of 10-130 mu m), and the stability is good; the sustained and slow release in the body is carried out for more than 21 days, the relatively high blood concentration of more than 100ng/ml can still be maintained in 21 days, the complete elimination is basically carried out in 43 days, and the long-term and slow release in the body can be achieved.

The method comprises the following steps:

I) The invention adopts specific slow release polymer materials, in particular to the dosage of polylactic acid-glycolic acid copolymer (PLGA) and lactide: the screening of the proportion and molecular weight of glycolide ensures long in vitro release time (the release time is more than 80 percent and more than 21 days), high encapsulation efficiency (the encapsulation efficiency is more than 80 percent) and small granularity (D90 is in the range of 10-130 mu m), thereby playing the role of long-acting drug release and having good slow release effect.

1. Amounts of polylactic-co-glycolic acid (PLGA)

When the dosage of polylactic acid-glycolic acid copolymer (PLGA) is 30% -80%, the time point for releasing more than 80% is 21-28 days; the encapsulation rate is 82.2% -92.5%; the particle size D90 is 58.2-95.6 μm respectively. The encapsulation efficiency of the long-acting injection in the range of 30% -85% of PLGA dosage meets the requirement (more than 80%), the granularity D90 meets the requirement of 10-130 mu m, the in vitro release time is longer, and the release time is more than 21 days.

2. Lactide: glycolide ratio

Lactide: when the glycolide proportion is 75:25-25:75, the time point for releasing more than 80% is 21-35 days; the encapsulation rate is 81.3% -92.3%; the particle diameter D90 of the microspheres is 58.2-69.2 mu m. Lactide: the encapsulation rate of the long-acting injection with the glycolide ratio within the range of 75:25-25:75 meets the requirements (more than 80%), the granularity D90 meets the requirements within the range of 10-130 mu m, the in vitro release time is longer, and the release time is longer than 80% and is longer than 21 days.

3. Polylactic-co-glycolic acid (PLGA) molecular weight

When the molecular weight of polylactic acid-glycolic acid copolymer (PLGA) is 10,000 ～ 300,000 daltons, the time point of releasing more than 80% is 21-35 days; the encapsulation rate is 81.1 to 92.3 percent; the particle diameter D90 is 52.4-126.9 mu m. The encapsulation efficiency of the long-acting injection with the molecular weight of the polylactic acid-glycolic acid copolymer (PLGA) within the range of 10,000 ～ 300,000 daltons meets the requirement (more than 80 percent), the granularity D90 meets the requirement of 10-130 mu m, the in vitro release time is longer, and the release time is longer than 80 percent and is longer than 21 days.

II) the invention adopts a specific pH range (pH 7.0-10.0), can keep an alkaline environment in the microsphere microenvironment, can prevent the sudden release phenomenon to the greatest extent, can delay the release of the medicine, has the release degree and the encapsulation rate which meet the requirements, and has better stability.

When the pH value is regulated within the range of 7.0-10.0, the time point of releasing more than 80% is 28 days, no sudden release phenomenon (releasing more than 80% in 14 days) is generated, and the result shows that the pH value is regulated within the range of 7.0-10.0, the alkaline environment can be kept in the microsphere microenvironment, the sudden release phenomenon can be prevented to the greatest extent, and the release of the medicine can be delayed. At the same time, after accelerating for 3 months, the properties, the content, the related substances and the dissolution curve have no significant change and have excellent stability compared with 0 day; conversely, the amounts of substances of interest with a pH of less than 7.0 (example 7 method 1) or more than 10.0 (example 7 method 4) increased significantly and the stability decreased significantly.

III) the invention creatively adds the medium basic amino acid into the microsphere, which not only can raise the pH of the microenvironment so as to reduce the solubility of the alidenafil citrate, but also can lead the polymer to be quickly self-repaired and closed by hydrogen bonds formed between the polymer, thereby inhibiting the burst effect of the drug. Therefore, the synergistic effect of the two can furthest reduce the phenomena of low encapsulation efficiency and abrupt release of the long-acting microsphere preparation of the alidenafil citrate.

When the amino acid is a neutral basic amino acid such as lysine, arginine, cysteine, etc., the time point of releasing 80% or more is 28 days, and no burst release phenomenon (releasing 80% or more in 14 days) occurs; the encapsulation rate is 85.1% -92.3%; the particle size distribution D90 is 54.2-64.2 μm. The result shows that the addition of the medium-alkaline amino acid ensures that the encapsulation efficiency meets the requirement (more than 80%), the granularity D90 meets the requirement of 10-130 mu m, the in vitro release time is longer, and the release time of more than 80% is more than 21 days.

When the dosage of the amino acid is 1% -12%, the time point of releasing more than 80% is 21-28 days, and no burst release phenomenon (releasing more than 80% in 14 days) occurs; the encapsulation rate is respectively 81.2% -92.3%; the particle size distribution D90 is 58.2-67.4 mu m respectively; the results show that when the dosage of the added amino acid is 1% -12%, the encapsulation efficiency meets the requirement (more than 80%), the granularity D90 meets the requirement of 10-130 mu m, the in vitro release time is longer, and the release time is longer than 80% and more than 21 days.

IV) in vivo long-acting slow release contrast, the invention can be continuously and slowly released in vivo for more than 21 days, and can achieve the effect of long-term slow release in vivo.

After the common quick-release injection is injected, the peak is reached rapidly in vivo within 0.25 hour, and the injection is eliminated within 8 hours. After the long-acting injection is injected, the long-acting injection is continuously and slowly released in a body for more than 21 days, the relatively high blood concentration of more than 100ng/ml can still be maintained in 21 days, the long-acting injection is basically completely eliminated in 43 days, and the long-acting slow release in the body can be achieved.

In a word, the long-acting sustained-release injection of alidenafil can be used for erectile dysfunction (erectile dysfunction, ED), alzheimer's Disease (AD), or pulmonary arterial hypertension (pulmonary hypertension, PH), the long-acting sustained-release of the prepared injection can reach more than 4 weeks by using specific long-acting sustained-release components, the burst release risk can be greatly reduced (more than 80% of burst release does not occur within 14 days), the encapsulation efficiency is obviously increased (the encapsulation efficiency exceeds 80%), the granularity is smaller (D90 is in the range of 10-130 mu m), and the injection has good stability; the sustained and slow release in the body is carried out for more than 21 days, the relatively high blood concentration of more than 100ng/ml can still be maintained in 21 days, the complete elimination is basically carried out in 43 days, and the long-term and slow release in the body can be achieved.

Drawings

FIG. 1 is a graph showing the in vivo blood concentration of example 1 (long-acting microspheres) of the present invention;

FIG. 2 is a graph showing the blood concentration in the body of comparative example 2 (ordinary injection) according to the present invention.

Detailed Description

The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The reagents used in the examples below are all commercially available.

Example 1

1-1. Prescription:

Note [ ]. The molecular formula of the sildenafil citrate is C ₂₃H₃₂N₆O₄S·C₆H₈O₇, the molecular weight is 680.74, the molecular formula of the sildenafil citrate is C ₂₃H₃₂N₆O₄ S, the molecular weight is 488.74, and the conversion ratio is about 1.4:1.

1-2, Preparation process:

(1) Preparation of the oil phase

And stirring and dissolving the prepared polylactic acid-glycolic acid copolymer (PLGA) in methylene dichloride (the mass concentration of the polymer is 15%) under a sealing condition, and stirring and fully mixing to obtain an organic internal phase.

(2) Preparation of inner aqueous phase

Adding sildenafil citrate and amino acid into water for injection (0.1% of sildenafil citrate by mass concentration), adding pH regulator to adjust pH to 8.0, stirring and dissolving to obtain internal aqueous phase solution.

(3) Preparation of colostrum

Slowly adding the prepared oil phase into the internal water phase, shearing at high speed (1000 rpm, 2-5 min), and fully and uniformly mixing to obtain the colostrum.

(4) Preparation of the external aqueous phase

Polyvinyl alcohol (PVA 04-88, the amount of which is 8 times that of the polymer) is dissolved in the aqueous solution to obtain an external aqueous phase solution of polyvinyl alcohol (1% polyvinyl alcohol).

(5) Preparation of multiple emulsion

Adding the colostrum into an external water phase (v/v is approximately equal to 1:5.5), and shearing at high speed (5000 rpm, shearing for 2-5 min) to prepare the compound emulsion.

(6) Curing

Placing the compound emulsion in a water bath with the temperature of 30-50 ℃ and stirring to volatilize dichloromethane.

(7) Centrifuging and washing

Centrifuging to collect microspheres, adding buffer solution (pH value of phosphate buffer solution is preferably 8-8.5), washing for 3-5 times, and removing polyvinyl alcohol to obtain the Aidenafil microspheres.

(8) Freeze-drying

Sterilizing the microsphere by gamma radiation, adding water for injection to the prescription amount, adding mannitol, stirring for dissolving, packaging into penicillin bottles, freeze-drying (pre-freezing at-45 deg.C, primary sublimation drying at-5 deg.C to 10 deg.C gradient, vacuum pressure of 11Pa, secondary drying at 25 deg.C), removing water and residual dichloromethane.

(9) Preparation of special solvent

Adding sodium carboxymethylcellulose, polysorbate 80 and sodium chloride into injectable water, stirring for dissolving, fixing volume or weight to full amount, sterilizing, filtering, and packaging.

Example 2: changing the dosage of the alidenafil

Example 1 prescription the proportion of sildenafil citrate 210mg (containing sildenafil 150 mg) in the prescription was 9.4% (corresponding to sildenafil ratio 6.71%), the mass proportions in the prescription of sildenafil citrate were respectively adjusted to 1%, 5% and 20% (corresponding to sildenafil ratio 0.71%, 3.57% and 14.28%), the dosage of freeze-dried excipient was adjusted, the other ingredients were unchanged, and the specific steps are as follows:

Method 1: 22.3mg of alidenafil citrate (1% of the prescription), 665.7mg of mannitol;

Method 2: 111.5mg of alidenafil citrate (5% of the prescription), 573.5mg of mannitol;

Method 3: sildenafil citrate 446mg (20% in the recipe), mannitol 239mg;

the preparation process was identical to example 1.

Example 3: polyester polymer type

The polylactic acid-glycolic acid copolymer (PLGA) in example 1 was modified as follows:

Method 1: polyglycolic acid (PGA, molecular weight 30,000 daltons)

Method 2: polylactic acid (PLA, molecular weight 30,000 daltons)

Method 3: polycaprolactone (PCL, molecular weight 30,000 daltons)

Method 4: polylactic acid-glycolic acid copolymer polyethylene glycol co-block derivative (PLGA 30,000-PEG 5000, wherein the ratio of lactide to glycolide of PLGA is 50:50)

Other ingredients in the recipe and preparation process were identical to example 1.

Example 4: the amount of polyester-based polymer used

The amount of the polylactic acid-glycolic acid copolymer in example 1 was changed, and the prescription ratio of 67.3% was changed to the following:

Method 1: the proportion of polylactic acid-glycolic acid copolymer is 20%, and the proportion of mannitol is 68.6%;

method 2: the proportion of polylactic acid-glycolic acid copolymer is 30%, and the proportion of mannitol is 58.6%;

Method 3: the ratio of polylactic acid-glycolic acid copolymer is 80%, and the ratio of mannitol is 8.6%;

method 4: the ratio of polylactic acid-glycolic acid copolymer is 85%, and the ratio of mannitol is 3.6%;

Example 5: lactide: glycolide ratio

Polylactic acid-glycolic acid copolymer (PLGA) -lactide in example 1: glycolide molar ratio 50:50, replaced with the following ratio:

Method 1: polylactic-co-glycolic acid (PLGA) -lactide: glycolide molar ratio 75:25, a step of selecting a specific type of material;

method 2: polylactic-co-glycolic acid (PLGA) -lactide: glycolide molar ratio 25:75;

Method 3: polylactic-co-glycolic acid (PLGA) -lactide: glycolide molar ratio 40:60;

Method 4: polylactic-co-glycolic acid (PLGA) -lactide: glycolide molar ratio 60:40, a step of performing a;

Example 6: molecular weight

Example 1 the polylactic acid-glycolic acid copolymer has a molecular weight of 30,000 daltons, and polylactic acid-glycolic acid copolymers of different molecular weights (lactide: glycolide ratio 50:50) were chosen to replace the polylactic acid-glycolic acid copolymer of example 1, as follows:

Method 1: polylactic acid-glycolic acid copolymer with molecular weight of 5000 dalton;

method 2: polylactic acid-glycolic acid copolymer with molecular weight of 10,000 daltons;

method 3: polylactic acid-glycolic acid copolymer with molecular weight of 100,000 daltons;

Method 4: polylactic acid-glycolic acid copolymer with molecular weight of 300,000 daltons;

method 5: polylactic acid-glycolic acid copolymer with molecular weight of 500,000 daltons;

Example 7: pH range

The amount of sodium hydroxide used in the formulation of example 1 was varied to adjust the pH as follows:

Method 1: the pH was 6.0;

Method 2: the pH was 7.0;

method 3: the pH was 10.0;

Method 4: the pH was 11.0;

Example 8: amino acid species

The amino acid type in the recipe of example 1 was L-arginine substituted for the following:

method 1: lysine

Method 2: cysteine (S)

Method 3: aspartic acid

Example 9: amino acid dosage

The amino acid-arginine ratio in the formulation of example 1 was varied as follows:

method 1: the ratio of amino acid-arginine was 0% and the mannitol ratio was 23.2%;

method 2: the ratio of amino acid-arginine was 1% and the mannitol ratio was 22.2%;

Method 3: the ratio of amino acid-arginine is 10%, and the ratio of mannitol is 13.2%;

Method 4: the ratio of amino acid-arginine is 12%, and the ratio of mannitol is 11.2%;

Example 10: oil phase solvent

Preparation of oil phase from preparation Process (1) of example 1"1-2: the methylene chloride in the "stirring and dissolving in methylene chloride solution" of the prepared polylactic acid-glycolic acid copolymer (PLGA) was replaced with the following under the sealing condition:

Method 1: chloroform;

Method 2: ethyl acetate;

the recipe, other ingredients in the preparation process and the procedure were as in example 1.

Example 11: type of external aqueous phase emulsifier

Preparation of the external aqueous phase from preparation Process (4) of example 1"1-2: polyvinyl alcohol (PVA 04-88) 30,000 Da) dissolved in an aqueous solution "was replaced with the following:

method 1: gelatin;

method 2: hydroxypropyl cellulose (model) HPC-JF, molecular weight 140,000 Da);

Method 3: hydroxyethyl cellulose (model) HEC-M, molecular weight 720,000 Da);

Method 4: polyvinylpyrrolidone (model) PVP _K-12, molecular weight 4000 Da);

Example 12: type of external aqueous buffer solution

Preparation of the external aqueous phase from preparation Process (4) of example 1"1-2: the aqueous solution in which polyvinyl alcohol was dissolved in the aqueous solution "was replaced with the following:

method 1: sodium acetate solution of polyvinyl alcohol (ph 4.5);

method 2: phosphate buffer of polyvinyl alcohol (pH 8.0-8.5);

Example 13: lyoprotectant species

The lyoprotectant mannitol in the formulation of example 1 was replaced with the following:

method 1: sodium chloride;

method 2: glucose;

Method 3: dextran;

Method 4: sucrose;

Example 14: application of alidenafil citrate injection

Patent CN115054585A describes that a pharmaceutical tablet containing alidenafil citrate can be used for the treatment of alzheimer's disease. Therefore, the alinafil citrate injection prepared by the invention is used for treating Alzheimer's disease and has good treatment effect.

Patent CN116270553a describes that an orolytic film agent containing alidenafil citrate can be used for treating erectile dysfunction ED. Therefore, the alinafil citrate injection prepared by the invention is used for treating erectile dysfunction ED, and has good treatment effect.

Patent CN114762692a states that sildenafil citrate can be used to treat and/or prevent pulmonary hypertension. Therefore, the alinafil citrate injection prepared by the invention is used for treating pulmonary hypertension and has good treatment effect.

Comparative example 1:

The long-acting microsphere preparation of the alidenafil citrate is prepared by a prescription process in a 'long-acting microsphere injection of the liraglutide and a preparation method thereof' disclosed by reference to patent CN102085355A, wherein the prescription process is as follows:

1-1. Prescription:

1-2, preparation process:

1) The prescription amount of the alidenafil citrate is weighed and added into 80mL of water for injection, so that the medicine is completely dissolved and is used as an internal water phase.

2) PLGA was weighed and added to 100mL of acetone and stirred to dissolve completely as the organic phase.

3) Adding the organic phase in the step 1 into the inner water phase in the step 1, mixing the inner water phase and the organic phase, heating in a water bath at 40 ℃, performing ultrasonic emulsification, and cooling to 13 ℃ to obtain colostrum;

4) According to the volume of the colostrum and polyvinyl alcohol solution 1:10, adding the colostrum into 2% polyvinyl alcohol solution, stirring for 2min at high speed 6000r/min to obtain compound emulsion.

5) Mixing the multiple emulsion with physiological saline 1:50 proportion, heating in 30 ℃ water, stirring for 3 hours at a low speed of 60r/min, volatilizing acetone, centrifugally collecting microspheres at 2500rpm, and washing 3 times with water for injection to obtain the microspheres.

6) Dissolving mannitol and Tween 80 in water for injection, and performing sterile filtration to obtain microsphere suspension;

7) Freeze-drying the microsphere suspension for 14 hours, vacuum-drying at 25 ℃ for 24 hours, and sub-packaging into penicillin bottles under aseptic conditions.

Comparative example 2: common injection

The alidenafil citrate is prepared into common injection with the specification of 30mg, and the preparation process is as follows: dissolving alidenafil citrate in a proper amount of water for injection, adding a proper amount of sodium hydroxide to adjust the pH to 6-7, fixing the volume to 10mL, filtering with a 0.22 mu mPES filter membrane, sealing in a 20mL ampoule bottle in a melting way, and performing hot-press sterilization at 121 ℃ for 15min to obtain the sterile injection.

Experimental example 1: in vitro release

The dissolution conditions were as follows using a CE7smart flow cell dissolution instrument (SOTAX, switzerland): the system device is closed by adopting a flow cell method, the long-acting microsphere injection of the alinafil is placed in a flow cell with a conical part filled with 1mm glass beads, 1000mL of phosphate buffer (pH 7.4 phosphate buffer containing 0.02% sodium azide (NaN ₃)) is used as a dissolution medium, the temperature is 37 ℃, the flow rate is 8mL/min, 5mL of the dissolution liquid is sampled at sampling points of 1 day, 7 days, 14 days, 21 days, 28 days and 35 days respectively, the content of active ingredients in the dissolution liquid is detected through a liquid phase after the sampled dissolution liquid passes through a glass fiber filter membrane of 0.7 mu m on line, and the cumulative release (%) at different time points is calculated, so that the results are shown in Table 1.

Table 1: in vitro Release evaluation results

Experimental example 2: encapsulation efficiency

The encapsulation efficiency is an important index for examining whether the drug is wrapped in the polymer material or not, is an important detection index for examining whether the microsphere preparation is successful, and is specified in Chinese pharmacopoeia, and the encapsulation efficiency is not lower than 80%. The encapsulation efficiency is calculated as follows:

encapsulation efficiency = amount encapsulated in system/total amount of encapsulated and unencapsulated in system x 100%.

Table 2: encapsulation efficiency results (%)

Experimental example 3: particle size distribution

An appropriate amount of the Aidinafop microspheres were weighed into a 5mL test tube, and 2mL of 0.1% polysorbate 20 was added to disperse the microspheres uniformly. Adding a proper amount of dispersed microsphere suspension into a Markov 3000 granularity detector, collecting data, and recording the following data: meansize (average particle size); d10 (equivalent diameter of the largest particle at 10% cumulative distribution in the sample particle size distribution curve); d50 (equivalent diameter of the largest particle at 50% cumulative distribution in the sample particle size distribution curve); d90 (equivalent diameter of the largest particle at 90% cumulative distribution in the sample particle size distribution curve), and the results are shown in Table 3. The particle size D90 of the microspheres is required to be less than 130. Mu.m, more preferably D90 is less than 70. Mu.m.

Table 3: evaluation results of particle size distribution

Experimental example 4: stability test (solid component after freeze-drying)

Accelerated test was conducted at 25.+ -. 2 ℃ and 60%.+ -. 5% relative humidity for 3 months, and samples were taken at the end of 0 and 3 months, respectively, and the changes in the respective indexes were examined, and the results were shown in Table 4.

Table 4: stability evaluation results

Experimental example 5: animal pharmacokinetic PK test

After 8 beagle dogs, namely, a male and a female half, are subjected to intramuscular injection of the long-acting injection of the example 1 and the common injection of the comparative example 2, the blood concentration of each beagle dog group is measured for different time periods by taking blood before and after the administration for 0.083 days, 0.25 days, 0.5 days, 1 day, 3.5 days, 7 days, 14 days, 17.5 days, 21 days, 24.5 days, 28 days, 35 days and 43 days, and the main data of each beagle dog group are as follows:

Table 5: in vivo evaluation results-blood concentration data

Conclusion analysis 1:

Examples 1-2 examine the effect of different amounts of alinafil citrate on microsphere quality, specific ratios are shown in table 6, in vitro release, encapsulation efficiency and particle size distribution are examined, and test results are shown in tables 7 and 8.

Table 6: proportion of sildenafil citrate in the prescription

Object(s)	Proportion of alidenafil citrate (%)
		Example 1	9.4
Example 2 method 1	1
		Example 2 method 2	5
Example 2 method 3	20

Table 7: results of Release degree

Table 8: results of encapsulation Rate and particle size distribution

Microspheres prepared with sildenafil citrate in the formulation at a ratio of 9.4% (example 1), 1% (example 2 method 1), 5% (example 2 method 2) and 20% (example 2 method 3) and released in phosphate buffer ph7.4 for 28 days, 35 days and 28 days, respectively; the encapsulation rates are 92.3%, 90.1%, 91.2% and 84.1%, respectively, which are all more than 80% (the pharmacopoeia requirement is not less than 80%), and the particle size distribution D90 is 58.2, 65.2, 68.1 and 64.2 μm, respectively, which are far less than the required particle size of 130 μm.

Conclusion: microspheres prepared by the use amount of the sildenafil citrate within the range of 1-20% can keep the microspheres released slowly, and the microspheres are released completely basically within 35 days; the encapsulation rate is 84.1-92.3%, and the granularity distribution is 58.2-68.1 μm. Wherein, the proportion of the citric acid alidenafil in the prescription is 9.4 percent (the microsphere prepared in the example 1), the release rate exceeds 92.3 percent in 28 days, the encapsulation efficiency is highest (92.5 percent), and the particle size distribution is smallest (58.2 mu m).

Conclusion analysis 2:

Example 3 in order to examine the effect of the commonly used microsphere polymer types on the product, polyglycolic acid (PGA), polylactic acid (PLA), polycaprolactone (PCL) and polylactic acid-glycolic acid copolymer polyethylene glycol co-block derivatives were examined, respectively, and specific types are shown in table 9. The in vitro release, encapsulation efficiency and particle size distribution are affected as shown in tables 10 and 11.

Table 9: the polymer species employed

Object(s)	Polymer species
		Example 1	Polylactic acid-glycolic acid copolymer (PLGA)
Example 3 method 1	Polyglycolic acid (PGA)
		Example 3 method 2	Polylactic acid (PLA)
Example 3 method 3	Polycaprolactone (PCL)
		Example 3 method 4	Polylactic acid-glycolic acid copolymer polyethylene glycol co-block derivative

Table 10: results of Release degree

Table 11: results of encapsulation Rate and particle size distribution

1) In vitro release: as is clear from Table 10, the release rates of example 3 method 1 (polylactic acid-PGA), example 3 method 2 (polylactic acid-PLA), example 3 method 3 (polycaprolactone-PCL), example 3 method 4 (polylactic acid-glycolic acid copolymer polyethylene glycol co-block derivative) and example 1 (polylactic acid-glycolic acid copolymer (PLGA) were respectively 21 days, 35 days, 21 days, 28 days and 28 days, respectively, and the release rate results showed that the release rate of polylactic acid-glycolic acid copolymer (PLGA) was significantly accelerated by changing to polylactic acid (PGA), the release rate of polylactic acid (PLA) was significantly slowed down, the release of Polycaprolactone (PCL) was significantly accelerated by changing to polylactic acid-glycolic acid copolymer polyethylene glycol co-block derivative, and the release rate of polylactic acid-glycolic acid copolymer polyethylene glycol co-block derivative was slightly accelerated.

2) Encapsulation efficiency and particle size distribution: as can be seen from Table 11, the encapsulation ratios of the samples of example 3 method 1 (polylactic acid-PGA), example 3 method 2 (polylactic acid-PLA), example 3 method 3 (polycaprolactone-PCL), example 3 method 4 (polylactic acid-glycolic acid copolymer polyethylene glycol co-block derivative) and example 1 (polylactic acid-glycolic acid copolymer (PLGA)) were 82.1%, 83.1%, 84.1%, 88.2%, 92.3%, respectively, and were all greater than 80%, respectively, and the particle sizes D90 of the microspheres of each sample were 56.2, 123.3, 56.2, 67.2, and 58.2. Mu.m, respectively, all satisfying the requirements of less than 130. Mu.m.

Conclusion: the microsphere prepared by taking one or more of polylactic acid-glycolic acid copolymer (PLGA) and polylactic acid (PLA), polyglycolic acid (PGA) or block copolymers of polyethylene glycol derivatives thereof as polymers can meet the requirement of long-acting microsphere release in release degree, encapsulation rate and particle size distribution.

Conclusion analysis 3:

Examples 1 and 4 to 6 in order to examine the effect of the commonly used microsphere polymer-polylactic acid-glycolic acid copolymer (PLGA), example 4 examined the effect of the amount of PLGA used; example 5 examine lactide: the effect of different molecular weights of PLGA was examined in example 6 with different proportions of glycolide, the specific differences being shown in tables 12 to 14, and the results of in vitro release, encapsulation efficiency, particle size distribution, stability testing are shown in tables 15 to 17.

Table 12: the amount of PLGA used

Table 13: by using the ratio of lactide to glycolide

Object(s)	Lactide: glycolide ratio
		Example 1	50：50
Example 5 method 1	75：25
		Example 5 method 2	25：75
Example 5 method 3	40：60
		EXAMPLE 5 method 4	60：40

Table 14: PLGA molecular weight

Object(s)	PLGA molecular weight
		Example 1	30,000 Daltons
Example 6 method 1	5000 Daltons
		Example 6 method 2	10,000 Daltons
EXAMPLE 6 method 3	100,000 Daltons
		EXAMPLE 6 method 4	300,000 Daltons
Example 6 method 5	500,000 Daltons

Table 15: results of Release degree

Table 16: encapsulation efficiency and particle size distribution test results

Table 17: stability test results

1) In vitro release

As is clear from the release results of examples 1 and examples 4 methods 1 to 4, which were different amounts of PLGA, the example 4 method 1 (PLGA: 20%) exhibited a burst phenomenon within 7 days, the example 4 method 2 (PLGA: 30%), the example 1 (PLGA: 67.3%), and the example 4 method 3 (PLGA: 80%) did not exhibit a burst phenomenon, and the release time points at 80% or more were 21 days, 28 days, and 28 days, respectively, and the release tended to slow down as the amount of PLGA (PLGA: 30% -80%) was increased. Example 4 method 4 (PLGA 85% ratio) released 65% only for 35 days, not released more than 80% and not completely.

Lactide: the time points for which glycolide release 80% or more of example 5 method 1 (75:25), example 5 method 2 (25:75), example 5 method 3 (40:60), example 5 method 4 (60:40) and example 1 (50:50) in different proportions are 35 days, 21 days, 28 days, respectively; the results demonstrate that lactide: the slow release of the microsphere can be ensured by the glycolide proportion of 75:25-25:75, and the degradation speed is accelerated along with the increase of the glycolide proportion.

From the results of the release rates of example 1 and example 6 methods 1 to 4, which differ in PLGA molecular weight, the burst phenomenon occurs in example 6 method 1 (molecular weight of 5000 daltons), and the release is substantially complete within 1 day. Whereas no burst occurred at molecular weights above 10,000 daltons, example 6 method 2 (molecular weight 10,000 daltons), example 1 (molecular weight 30,000 daltons), example 6 method 3 (molecular weight 100,000 daltons), example 6 method 4 (molecular weight 300,000 daltons) showed no burst, and release was slowed down at 80% or more time points of 21 days, 28 days, 35 days, respectively, with increasing PLGA molecular weight. Example 6 method 5 (molecular weight 500,000 daltons) released 56% only for 35 days, not released more than 80% and the release was incomplete.

2) Encapsulation efficiency and particle size distribution

Different amounts of PLGA, example 4 method 1 (PLGA 20% ratio) had an encapsulation of 45.2% and an encapsulation of less than 80%, which was not satisfactory, and the microsphere particle size D90 was 52.3. Mu.m. The encapsulation efficiency of example 4 method 2 (PLGA: 30%), example 1 (PLGA: 67.3%), example 4 method 3 (PLGA: 80%) and example 4 method 4 (PLGA: 85%) were 82.2%, 92.5%, 86.5% and 87.1%, respectively, and the particle size D90 of the microspheres was 61.5, 58.2, 95.6 and 151.2 μm, respectively, and the encapsulation efficiency of the microspheres was satisfactory with increasing PLGA usage (30% -85%), but the particle size D90 tended to increase. Example 4 method 4 (PLGA 85% ratio) particle size D90 was greater than 130 μm, which is undesirable.

Lactide: the encapsulation rates of glycolide in different proportions of example 5 method 1 (75:25), example 5 method 2 (25:75), example 5 method 3 (40:60), example 5 method 4 (60:40) and example 1 (50:50) were 81.3%, 82.5%, 90.1%, 87.2% and 92.3%, respectively, and the particle diameters D90 of the microspheres were 64.1, 69.2, 62.4, 63.5 and 58.2 μm, respectively, without significant differences.

PLGA molecular weights were varied, and the encapsulation efficiency of example 6 method 1 (molecular weight 5000 daltons) was 60.6%, less than 80%, which was undesirable, with a microsphere particle size D90 of 44.1 μm. The encapsulation efficiency of example 6 method 2 (molecular weight 10,000 daltons), example 1 (molecular weight 30,000 daltons), example 6 method 4 (molecular weight 100,000 daltons), example 6 method 4 (molecular weight 300,000 daltons) was 81.1%, 92.3%, 89.2%, 87.1%, respectively, the particle size D90 of the microspheres was 52.4, 58.2, 94.2, 126.9 μm, respectively, the increase in encapsulation efficiency was insignificant with increasing molecular weight, but the particle size was also significantly increased with increasing molecular weight, but all met the requirements that D90 be less than 130 μm. When the molecular weight of PLGA is increased to 500,000 daltons, as in example 6 method 5, the encapsulation efficiency is 64.2%, less than 80% encapsulation efficiency is required, the particle size D90 is 184 μm, and the requirement that D90 be less than 130 μm is not met.

3) Stability of

The microspheres prepared in example 1 have no significant changes in properties, contents, related substances and dissolution curves compared with 0 day under the conditions of an accelerated high temperature of 25+/-2 ℃ and a relative humidity of 60% +/-5%3 months, and remain stable.

From the stability results of example 4 methods 2 to 4 with different amounts of PLGA, the similarity factor of the release profile under acceleration of example 4 method 2 (PLGA 0%) was 50.1, which was correspondingly reduced. As the PLGA ratio increased above 30%, the properties, content, relevant substances and dissolution profile of example 1 (PLGA ratio 67.3%), example 4 method 3 (PLGA ratio 80%) and example 4 method 4 (PLGA ratio 85%) were not significantly changed and remained stable as compared to 0 day after 3 months of acceleration.

Lactide from PLGA: the stability results of examples 1 to 4 of example 5, which show different proportions of glycolide, show that lactide: the characteristics, the content and the related substances of the glycolide are not obviously changed in the range of 75:25-25:75, but the similar factors of the release profile are relatively reduced as the ratio of the glycolide is increased. Example 5 method 1 (75:25), example 5 method 2 (25:75), example 5 method 3 (40:60), example 5 method 4 (60:40) and example 1 (50:50) have similarity factors of 70.1, 52.1, 62.3, 65.4, 71.2, respectively, with increasing glycolide ratio, increasing degradation tendency and decreasing similarity factor.

From the stability results of examples 6 methods 2 to 4 (10,000 ～ 300,000 daltons) and example 1 (30,000 daltons) with different PLGA molecular weights, it was found that the properties, content, related substances and dissolution profile were not significantly changed and remained stable after acceleration for 3 months at 10,000 ～ 300,000 daltons, compared with 0 day.

Conclusion:

The dosage of PLGA accounts for 30-80% of the prescription, the molar ratio of lactide to glycolide is 75:25-25:75, and the molecular weight is 10,000 ～ 300,000, and can meet the related requirements of the release degree, the encapsulation efficiency, the particle size distribution and the stability of the long-acting microsphere preparation.

Other conditions, such as PLGA having a ratio of less than 30% in the formulation (example 4, method 1), release may exhibit burst and low encapsulation efficiency (< 80%). When the proportion of PLGA in the formulation is greater than 80% (example 4 method 4), its release is incomplete and the cumulative release over 35 days is not more than 80%.

For another example, the PLGA molecular weight is less than 10,000 (example 6, method 1) and the release is burst, the release is basically complete within 1d, the encapsulation efficiency is less than 80%, and the encapsulation efficiency is not satisfactory. And above 300,000 (example 6, method 5) the encapsulation efficiency was less than 80% and the particle size D90 was also not as good as less than 130 μm.

Conclusion analysis 4:

Example 7 the in vitro release, encapsulation efficiency, particle size distribution, stability test results are shown in tables 19 to 21, looking at different pH values, in particular as shown in Table 18.

Table 18: different pH

Examples	pH
		Example 1	8.0
EXAMPLE 7 method 1	6.0
		EXAMPLE 7 method 2	7.0
EXAMPLE 7 method 3	10.0
		EXAMPLE 7 method 4	11.0

Table 19: in vitro Release evaluation results

Table 20: encapsulation efficiency and particle size distribution test results

Table 21: stability test results

1) In vitro release

As is clear from the results of the release rates of the methods 1 to 4 of the example 7 in which the pH was adjusted with sodium hydroxide, the solubility of the sildenafil citrate of the method 1 of the example 7 (pH 6.0) was greatly increased, and there was a risk of burst release, and the time point of release was 14 days at 80% or more. The time points at which 80% or more of the samples of example 7 method 2 (pH 7.0), example 1 (pH 8.0), example 7 method 3 (pH 10.0) and example 7 method 4 (pH 11.0) were released were 28 days, 28 days and 28 days, respectively. The results show that the pH is regulated within the range of 7.0-11.0, the alkaline environment can be maintained in the microsphere microenvironment, the burst release phenomenon can be prevented to the greatest extent, and the release of the medicine can be delayed.

2) Encapsulation efficiency and particle size distribution

In examples of different pH ranges, the encapsulation efficiency is greater than 80% in examples 7 method 1 (pH 6.0), example 7 method 2 (pH 7.0), example 1 (pH 8.0), example 7 method 3 (pH 10.0) and example 7 method 4 (pH 11.0) respectively, and is 83.2%, 80.1%, 92.3%, 88.2% and 90.2%, which meet the requirements; the particle size distribution D90 is 85.1, 88.1, 58.2, 62.3 and 67.2 μm respectively, and no significant difference exists.

3) Stability of

From the stability results of examples 7 methods 1 to 4, which differ in pH, it is understood that the similarity factor of the release profile under accelerated conditions was 32.1 in example 7 method 1 (pH 6.0), and accordingly, the release profile was decreased, the substances were significantly increased, and the phenomenon of lump collapse was also observed in the properties. The properties, contents, substances and elution profiles of example 7 method 2 (pH 7.0), example 1 (pH 8.0) and example 7 method 3 (pH 10.0) were accelerated for 3 months, and were not significantly changed and were stable as compared with 0 day. Example 7 method 4 (ph 11.0) increased significantly with respect to the substances after 3 months of acceleration, from 0.26% on day 0 to 0.95% on month 3, with poor stability.

Conclusion:

The pH is regulated within the range of 7.0-10.0 (the method 2 and the method 3 in the embodiment 1 and the embodiment 7), the alkaline environment can be kept in the microsphere microenvironment, the burst release phenomenon can be prevented to the greatest extent, the release of the medicine can be delayed, the release degree and the encapsulation efficiency of the medicine are both in accordance with the requirements, and the properties, the content, the related substances and the dissolution curve of the medicine are not obviously changed compared with those of 0 day after the medicine is accelerated for 3 months, so that the medicine has excellent stability;

Conversely, the amounts of substances of interest with a pH of less than 7.0 (example 7 method 1) or more than 10.0 (example 7 method 4) increased significantly and the stability decreased significantly.

Conclusion analysis 5:

Examples 8 and 9 are for examining the influence of the type and amount of amino acid on the quality of microspheres, the type of amino acid is shown in Table 22, the amount of amino acid is shown in Table 23, and the main examination indexes are release degree, encapsulation efficiency, particle size distribution and stability, and the test results are shown in tables 24 to 26.

Table 22: amino acid species

Examples	Amino acids
		Example 1	Arginine (Arg)
Example 8 method 1	Lysine
		Example 8 method 2	Cysteine (S)
Example 8 method 3	Aspartic acid

Table 23: amino acid dosage

Examples	Amino acid ratio in the prescription
		Example 1	2％
Example 9 method 1	0％
		Example 9 method 2	1％
Example 9 method 3	10％
		Example 9 method 4	12％

Table 24: in vitro Release evaluation results

Table 25: results of encapsulation Rate and particle size distribution

Table 26: results of stability

1) In vitro release

As is clear from the results of the release rates of the methods 1 to 3 of example 8 for different amino acid types, the amino acid used in the method 3 of example 8 was aspartic acid, and the burst release occurred, and the time point at which 80% or more of the release occurred was 14 days. The time points at which 80% or more of the release of example 8 method 1 (lysine), example 1 (arginine) and example 8 method 2 (cysteine) was performed were 28 days, 28 days and 28 days, respectively. The results show that the amino acid is a medium-alkaline amino acid, and can keep an alkaline environment in the microsphere microenvironment, and hydrogen bonds formed between the amino acid and the polymer can cause the polymer to rapidly self-repair and close pores, so that the burst effect of the drug is inhibited. Thus, the burst release phenomenon can be prevented to the greatest extent, and the release of the medicine can be delayed.

As is clear from the results of the release rates of the methods 1 to 4 of example 9 with different amounts of amino acids, the method 1 of example 9 (no amino acid added) had burst release, and the time point at which 80% or more of the release was 14 days. The time points at which 80% or more of the composition of example 9 method 2 (1%), example 1 (2%), example 9 method 3 (10%), and example 9 method 4 (12%) was released were 21 days, 28 days, and 28 days, respectively. The results show that the dosage of amino acid in the prescription is 1-12%, which can inhibit the burst release of microsphere and delay the release of drug.

2) Encapsulation efficiency and particle size distribution

Example 8 method 3 amino acid is aspartic acid, is acidic amino acid, has encapsulation efficiency of 67.3%, is less than 80%, and is not satisfactory. The particle size distribution D90 is 74.2 μm and is more than 130 μm. The encapsulation rates of the method 1 (lysine), the method 1 (arginine) and the method 2 (cysteine) of the example 8 are respectively 89.1 percent, 92.3 percent and 85.1 percent, and the encapsulation rates are all more than 80 percent, which meets the requirements; the particle size distribution D90 is 54.2, 58.2 and 64.2 mu m respectively, which is less than 130 mu m, meets the requirements and has no significant difference. The result shows that the addition of the medium-alkaline amino acid greatly increases the encapsulation rate of the microsphere, and the granularity meets the requirements.

Example 9 method 1 (no amino acid added) has an encapsulation efficiency of 51.3%, less than 80% and is undesirable. The encapsulation rates of the method 2 (1%), the method 1 (2%), the method 3 (10%) and the method 4 (12%) of the example 9 are respectively 81.2%, 92.3%, 90.1% and 82.3%, and the encapsulation rates are all more than 80%, so that the encapsulation rates meet the requirements; the particle size distribution D90 of the composite material is 67.4, 58.2 and 65.4 mu m respectively, and the composite material meets the requirement that the D90 is smaller than 130 mu m without obvious difference. The result shows that when the dosage of the added amino acid is 1-12%, the encapsulation rate of the microsphere is greatly increased, and the granularity meets the requirement.

3) Stability of

As is clear from the stability results of the methods 1 to 3 of the method 8 of the example 8 of different amino acid types, the amino acid of the method 3 of the example 8 is aspartic acid, is acidic amino acid, and after 3 months of acceleration, the related substances and the dissolution curve are all changed remarkably compared with 0 day, and the total impurities of the related substances are increased from 0.21% to 0.61% in 0 day and increased by 0.4%; the dissolution profile has a similarity factor of 45.2 to day 0 of less than 50, which is undesirable. The properties, contents, substances and dissolution profiles of example 8 method 1 (lysine), example 1 (arginine) and example 8 method 2 (cysteine) were accelerated for 3 months, and were not significantly changed as compared with 0 day, and were stable.

As can be seen from the stability results of the methods 1 to 4 of the example 9 with different amino acid usage amounts, the related substances and the dissolution curves of the method 1 (without amino acid) of the example 9 are significantly changed compared with the method of 0 day, and the total impurities of the related substances are increased from 0.26% to 0.66% in the day of 0, and are increased by 0.4%; the dissolution profile has a similarity factor of 35.2 to day 0 of less than 50, which is undesirable. The properties, contents, related substances and dissolution curves of example 9 method 2 (1%), example 1 (2%) and example 9 method 3 (10%) were not significantly changed and were stable as compared with 0 day after acceleration for 3 months. Example 9 method 4 (12%) showed a significant increase in the relative material after 3 months of acceleration compared to day 0, from 0.26% on day 0 to 0.69% on month 3, with poor relative stability.

Conclusion:

A. The amino acid is controlled to be medium-alkaline amino acid such as lysine, arginine and cysteine, and besides the alkaline environment can be kept in the microsphere microenvironment, hydrogen bonds formed between the amino acid and the polymer can cause the rapid self-repair and pore closure of the polymer, so that the burst effect of the drug is inhibited, the release of the drug can be delayed, and the release degree, the encapsulation rate, the particle size distribution and the stability of a sample meet the requirements.

B. Meanwhile, the amino acid is used in an amount which is within the range of 1-10% of the prescription (the method 2 and the method 3 in the embodiment 1 and the embodiment 9), the burst release of the microspheres can be inhibited, the release of the medicine can be delayed, the release degree meets the requirements, in addition, the encapsulation efficiency of the microspheres is greatly increased, and the particle size distribution meets the requirements. After 3 months of acceleration, the properties, the content, the related substances and the dissolution curve of the composition have no significant change and have excellent stability compared with those of 0 day;

in contrast, the microspheres prepared with amino acids at a ratio of less than 1% (example 9 method 1) or greater than 10% (example 9 method 4) in the formulation showed a significant increase in the relevant substances after 3 months of acceleration compared to 0 day, from 0.26% to 0.69%, with poor stability.

Conclusion analysis 6:

example 10 the effect of the oil phase of a conventional microsphere polymer was examined, the type of oil phase solvent used was shown in table 27, the main index was encapsulation efficiency and particle size distribution, and the test results were shown in table 28.

Table 27: type of oil phase solvent

Examples	Type of oil phase solvent
		Example 1	Dichloromethane (dichloromethane)
Example 10 method 1	Chloroform (chloroform)
		Example 10 method 2	Acetic acid ethyl ester

Table 28: results of encapsulation Rate and particle size distribution

Conclusion:

The above results show that the encapsulation efficiency profiles of example 10 method 1 (chloroform as the oil phase solvent), example 10 method 2 (ethyl acetate as the oil phase solvent) and example 1 (methylene chloride) for the different oil phase solvents were 91.2%, 89.1% and 92.3% satisfactory without significant differences. The particle sizes D90 of the microspheres are 97.1, 65.4 and 58.2 mu m respectively, which meet the requirements. Example 10 method 1 (solvent chloroform) had slightly larger particle sizes with no significant difference in other solvent particle sizes.

Conclusion analysis 7:

Example 11 the effect of the external aqueous phase stabilizer of the conventional microsphere polymer was examined, the external aqueous phase stabilizer used is shown in Table 29, the main index of examination is the encapsulation efficiency and the particle size distribution, and the test results are shown in Table 31.

Table 29: type of external aqueous phase stabilizer

Examples	Type of external aqueous phase stabilizer
		Example 1	Polyvinyl alcohol
Example 11 method 1	Gelatin
		EXAMPLE 11 method 2	Hydroxypropyl cellulose
EXAMPLE 11 method 3	Hydroxyethyl cellulose
		EXAMPLE 11 method 4	Polyvinylpyrrolidone

Example 12 the effect of the external aqueous buffer solution of the conventional microsphere polymer was examined, the buffer solution used is shown in table 30, the main index of examination is encapsulation efficiency and particle size distribution, and the test results are shown in table 31.

Table 30: type of external aqueous buffer solution

Examples	Kind of buffer solution
		Example 1	Water and its preparation method
Example 12 method 1	Acetic acid sodium salt
		Example 12 method 2	Phosphate buffer (pH 8.0-8.5)

Table 31: encapsulation efficiency and particle size distribution test results

The encapsulation efficiency profile for example 11, method 2 (the external aqueous phase stabilizer being hydroxypropyl cellulose) and example 11, method 3 (the external aqueous phase stabilizer being hydroxyethyl cellulose) was 54.2%, 57.3%, both less than 80%, unsatisfactory, and the particle size D90 was 136.9, 163.5 μm, respectively, both greater than 130 μm, both unsatisfactory. The encapsulation efficiency of example 11 method 1 (external water phase stabilizer is gelatin), example 11 method 4 (external water phase stabilizer is polyvinylpyrrolidone) and example 1 (external water phase stabilizer is polyvinyl alcohol) are respectively 84.2%, 88.2% and 92.3%, which are all more than 80%; the particle sizes D90 are 63.5, 68.1 and 58.2 mu m respectively, less than 130 mu m, meet the requirements and have no significant difference.

Example 12 method 1 (the external aqueous buffer solution is sodium acetate) has an encapsulation rate of 59.1%, less than 80% and unsatisfactory; the granularity D90 is 62.3 mu m, which meets the requirement. The encapsulation rates of the method 2 (the external water phase buffer solution is phosphate buffer solution with pH of 8.0-8.5) and the method 1 (the external water phase buffer solution is water) of the embodiment 12 are 91.1 percent and 92.3 percent respectively, and are more than 80 percent, thus meeting the requirements; the particle sizes D90 are 66.5 and 58.2 mu m respectively, and no significant difference exists.

Conclusion:

The polyvinyl alcohol, gelatin and polyvinylpyrrolidone are microspheres of an external water phase stabilizer, and the external water phase buffer solution is microspheres of phosphate buffer solution pH 8.0-8.5 or water medium, so that the encapsulation efficiency and the particle size distribution meet the requirements.

Conclusion analysis 8:

example 13 the effect of lyoprotectant on stability was examined, the lyoprotectant used was shown in table 32, the main index was stability, and the test results were shown in table 33.

Table 32: lyoprotectant species

Examples	Lyoprotectant species
		Example 1	Mannitol (mannitol)
EXAMPLE 13 method 1	Sodium chloride
		EXAMPLE 13 method 2	Glucose
EXAMPLE 13 method 3	Dextran
		EXAMPLE 13 method 4	Sucrose

Table 33: stability evaluation results

As is clear from Table 33, in example 13, method 1 (the lyoprotectant is sodium chloride), the product was not molded, was a white powder in 0 day, and after 3 months of acceleration, it was a white powder and a lump, which were unsatisfactory, and the related substances were significantly increased to 0.81%, and the related substances were increased by 0.4%. Example 13 method 2 (lyoprotectant is glucose), example 13 method 3 (lyoprotectant is dextran), example 13 method 4 (lyoprotectant is sucrose), example 1 (lyoprotectant is mannitol) all have white blocks after 0 day and 3 months of acceleration, no obvious change exists, and the requirements are met; the relative substances are respectively increased by 0.12%, 0.10%, 0.12% and 0.06%; the similar factors of the dissolution curves after 3 months of acceleration are 65.2, 62.3, 60.2 and 71.2 respectively compared with 0 day, and no significant difference exists.

Conclusion:

the appearance of the sample containing sodium chloride as the freeze-drying protective agent is unqualified, and the appearance of the sample containing other freeze-drying protective agents such as glucose, dextran, sucrose and mannitol meets the requirements, so that the stability of the sample is greatly improved.

Conclusion analysis 9:

differences of comparative examples

Referring to the prescription process of the 'a liraglutide long-acting microsphere injection and a preparation method thereof' disclosed in patent CN102085355A, the prescription is shown in a table 34, comparative research is carried out on the prescription, in-vitro release, encapsulation efficiency, particle size distribution, stability and in-vivo evaluation are examined, and test results are shown in tables 35-38.

Table 34 comparative example recipe

Note that: "/" means not related to

Table 35 release test results

Examples	For 1 day	For 7 days	14 Days	21 Days	For 28 days	For 35 days
							Example 1	17.8	28.9	45.7	67.8	92.3	94.5
Comparative example 1	44.2	84.1	93.5	/	/	/

Table 36 encapsulation efficiency and particle size distribution test results

Table 37 stability test results

Table 38: in vivo evaluation results-blood concentration data

The release of comparative example 1 shows burst release phenomenon, 84.1% release over 7 days, more than 80%; meanwhile, the encapsulation efficiency is lower than 42.3%, and the requirement of 80% encapsulation efficiency is not exceeded; the granularity D90 is 91.2 mu m, which meets the requirement of less than 130 mu m; the stability is poor, the related substances increase from 0.32% to 0.85% in 3 months of acceleration, the increase range is 0.53%, the curve similarity factor is also reduced in comparison with the example 1 (increase range is 0.06%).

After the common injection of comparative example 2 was injected, it reached a peak rapidly in vivo for 0.25 hours and was eliminated within 8 hours. After the long-acting injection of the example 1 is injected, the long-acting injection is continuously and slowly released in the body for more than 21 days, the relatively high blood concentration of more than 100ng/ml can still be maintained in 21 days, the long-acting injection is basically completely eliminated in 43 days, and the long-acting and slow-releasing effect in the body can be achieved (figures 1 and 2).

Conclusion:

The release degree of comparative example 1, in which the pH was adjusted to 7 to 10 without adding amino acid and pH adjustor, did not meet the requirements, burst release occurred, release was failed, and encapsulation efficiency was also failed. Compared with the in vivo pharmacokinetics of comparative example 2 (common injection), the common injection reaches peak very rapidly and rapidly, and has no slow release effect; example 1 was sustained and slow released in vivo for 21 days or more and was expected to have a long-acting slow release.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. The long-acting sustained-release injection of the alidenafil is characterized by comprising a solid component and a solvent component; the solid component comprises, by mass, solid components: 0.7 to 15 weight percent of active ingredient calculated by alidenafil, 30 to 80 weight percent of biodegradable polymer matrix, 1 to 10 weight percent of amino acid, and the balance of pH value regulator and freeze-drying protective agent; the active ingredient is one or more of alidenafil, pharmaceutically acceptable salt and pharmaceutically acceptable ester thereof.

2. The long-acting sustained-release injection of alidenafil according to claim 1, wherein the solid component comprises a microsphere preparation;

And/or the active ingredient and amino acid are embedded in a biodegradable polymer matrix;

and/or the pH value of the solid component is 7-10.

3. The long-acting sustained-release injection of alidenafil according to claim 2, wherein the microsphere preparation has a particle size D90 of 10-130 μm, preferably 20-70 μm.

4. The long-acting sustained-release injection of alidenafil according to claim 1, wherein the active ingredient is selected from alidenafil citrate;

and/or, the biodegradable polymer matrix comprises a polyester-based polymer;

Preferably, the polyester polymer is selected from one or more of polylactic acid-glycolic acid copolymer, polylactic acid, polyglycolic acid, polyethylene acid, polyhydroxybutyrate, polycaprolactone, polyalkylene oxalate, polyalkylene glycol ester, block copolymer of polylactic acid-glycolic acid copolymer polyethylene glycol derivative, block copolymer of polylactic acid polyethylene glycol derivative, block copolymer of polyglycolic acid polyethylene glycol derivative, block copolymer of polyhydroxybutyrate polyethylene glycol derivative, block copolymer of polycaprolactone polyethylene glycol derivative, block copolymer of polyalkylene oxalate polyethylene glycol derivative and block copolymer of polyalkylene glycol ester polyethylene glycol derivative.

5. The long-acting sustained-release injection of alidenafil according to claim 4, wherein the molecular weight of the polylactic acid-glycolic acid copolymer is 10000-300000 dalton, preferably 30000-100000 dalton;

and/or, the polylactic acid-glycolic acid copolymer comprises lactide monomer units and glycolide monomer units; the molar ratio of lactide monomer units to glycolide monomer units was 75: 25-25: 75, preferably 50:50.

6. The long-acting sustained-release injection of alidenafil according to claim 1, wherein the amino acid is selected from neutral amino acid and/or basic amino acid; preferably, the amino acid is selected from one or more of L-arginine, lysine, histidine, serine, threonine, tyrosine, asparagine, glutamine, alanine, glycine and cysteine;

And/or the pH regulator is selected from one or more of trisodium phosphate, sodium hydrogen phosphate-sodium dihydrogen phosphate buffer solute, sodium carbonate, sodium hydroxide and arginine;

and/or the lyoprotectant is selected from one or more of mannitol, glucose, dextran and sucrose.

7. The long-acting sustained-release injection of alidenafil according to claim 1, wherein the solvent component comprises 0.01 to 1wt% of surfactant, 0.1 to 5wt% of suspending agent, 0.5 to 5wt% of osmotic pressure regulator and the balance of water for injection by mass of the solvent component.

8. The long-acting sustained-release injection of alidenafil according to claim 7, wherein the ratio of the solid component to the solvent component is 10-1800 mg: 0.5-20 mL.

9. The long-acting sustained-release injection of alidenafil according to claim 7, wherein the surfactant is selected from nonionic surfactants, preferably selected from one or more of poloxamers, polysorbates and span;

and/or the suspending agent is selected from one or more of sodium carboxymethyl cellulose, sorbitol, polyvinylpyrrolidone and aluminum monostearate;

and/or, the osmolality adjusting agent is selected from sodium chloride.

10. A method for preparing the long-acting sustained-release injection of alidenafil according to claim 1, which is characterized by comprising the following steps:

S2) adding the oil phase into an inner water phase to obtain colostrum;

11. The preparation method according to claim 10, wherein the organic solvent is selected from one or more of dichloromethane, chloroform, ethyl acetate and hexafluoroisopropanol;

and/or the emulsifier is selected from one or more of polyvinyl alcohol, gelatin, polyvinylpyrrolidone and aluminum monostearate.

12. Use of the long-acting sustained-release injection of alidenafil according to any one of claims 1 to 9 or the long-acting sustained-release injection of alidenafil prepared by the preparation method of claim 11 or 12 for one or more of preparation of a medicament for treating erectile dysfunction, preparation of a medicament for treating alzheimer's disease and preparation of a medicament for treating pulmonary arterial hypertension.