WO2011016435A1 - Method for producing freeze-dried composition containing polylactic acid particles - Google Patents

Abstract

Provided are a method for preventing the formation of coarse particles in the process of freeze-drying an aqueous suspension of fine polylactic acid particles, and a method for producing polylactic acid particles for use as an agent for rendering the transparent tissue of the eye visible. The method for suppressing the formation of coarse particles when an aqueous suspension of fine polylactic acid particles is freeze-dried includes: (a) a step in which an aqueous suspension of fine polylactic acid particles containing a dispersing agent and from 3.5% to 10% mannitol is prepared by (i) adding, while stirring, a solution of polylactic acid dissolved in a water miscible organic solvent to a dispersing agent aqueous solution and then adding mannitol, or (ii) adding, while stirring, the solution of polylactic acid dissolved in said solvent to a dispersing agent/mannitol aqueous solution containing a dispersing agent and mannitol; and (b) a step in which said suspension is freeze-dried in a glass container. The method for producing polylactic acid particles for use as an agent for rendering transparent tissue visible includes the steps (a) and (b).

Description

Method for producing lyophilized composition containing polylactic acid particles

The present invention relates to a method for suppressing the generation of aggregates of polylactic acid particles obtained by freeze-drying an aqueous suspension of polylactic acid fine particles.

眼 Transparent tissues such as the vitreous body, lens and cornea are gathered in the eye. Cataract surgery removes the nucleus and cortex in the capsular bag, but the lens cortex remaining near the posterior capsule becomes cloudy and is known to cause secondary cataract.

Also, in vitreous body removal surgery, it is required to remove as much as possible the vitreous tissue that has adhered to the retina and the vitreous body that will serve as a scaffold for growth. However, the vitreous body is a transparent tissue, and complete removal of the vitreous body is not easy. As a solution, a transparent vitreous body is visualized by injecting a steroid suspension such as a triamcinolone preparation (Kenacort-A: registered trademark) into the vitreous cavity using a syringe during a vitreous removal operation. (See Non-Patent Document 1). By adopting this method, the surgical procedure can be facilitated and the vitreous can be removed reliably. However, in the use of the triamcinolone preparation for the treatment of the vitreous body, increased intraocular pressure and progression of cataract have been reported as side effects (see Non-Patent Documents 2 and 3). Therefore, a method of improving the visibility of the vitreous body by injecting a suspension of particles made of polylactic acid or other high molecular compound having no pharmacological action into the vitreous cavity, and a transparent tissue visualization agent (eye) used therefor A drug for improving the visibility of the transparent tissue of the eye at the time of surgery has been proposed (Patent Document 1).

In the above method, the suspension of fine particles is extremely supplied in the form of a freeze-dried suspension for reasons such as avoiding stability problems such as precipitation separation due to long-term storage in liquid form. preferable. However, some polymer compounds, such as polylactic acid, form agglomerates and form coarse particles when the suspension is lyophilized. Since the polymer compound particles are used in the form of being injected into the eye by a syringe, the particles must be of a particle size that can be passed without clogging the injection needle. In Patent Document 1, although the particle diameter is disclosed as an average value, the maximum particle diameter is not mentioned, and there is no clear description regarding the preparation conditions for controlling the particle diameter. Further, Patent Document 2 discloses a method for improving the visibility of ocular tissue by adding a divalent or trivalent metal salt to a polylactic acid dispersion. There is no description regarding prevention of the formation of coarse particles. Further, Patent Document 3 discloses a method for producing an injectable implant by forming 20-120 μm polylactic acid microparticles and freeze-drying in an aqueous carrier containing glycolic acid. In the preparation of pharmaceuticals, taking into account the possibility of chlorinated organic solvent remaining in the formulation, including the steps of dissolving and precipitating in the chlorinated organic solvent, manufacturing pharmaceutical formulations, especially formulations that are injected into the eye It is not preferable as a method. In addition, Patent Documents 4 and 5 discuss technical conditions for freeze-drying emulsions and suspensions, but prevent the formation of coarse particles due to particle aggregation during freeze-drying of polylactic acid suspensions. There is no description about what to do.

International Publication No. 2005/115411 International Publication No. 2008/126720 International Publication No. 2003/007782 International Publication No. 2001/051032 International Publication No. 2004/112754

Sakamoto, T. et al., Graefe ’s Archive for Clinical andExperimental Ophthalmology, 240: p.423 (2002). Challa, J.K. et al., Australian and New Zealand Journal ofOphthalmology, 26: p.277 (1998). Wingate, R.J. et al., Australian and New Zealand Journal ofOphthalmology, 27: p.431 (1999).

In the above background, an object of the present invention is to provide a method for producing polylactic acid particles by freeze-drying from an aqueous suspension of polylactic acid microparticles, which can prevent generation of coarse particles in the freeze-drying process. That is.
Another object of the present invention is to provide a method for producing a transparent tissue visualization agent which is a preparation for improving the visibility of transparent tissues of the eye by applying to the transparent tissues of the eye, particularly vitreous body and lens. It is to be.

As a result of the study for the above purpose, the present inventor added polylactic acid dissolved in a mixture of acetone and ethanol to an aqueous solution containing the dispersant dissolved therein or an aqueous solution containing the dispersant and mannitol dissolved. By stirring, an aqueous suspension of polylactic acid fine particles having an average particle size of 500 to 1000 nm (maximum particle size of 5 μm or less) as primary particles (referring to individual particles in an unaggregated state) is obtained. By adding an appropriate amount of mannitol to the suspension during freeze-drying, the aggregation of polylactic acid particles that occurs during freeze-drying is suppressed, and coarse particles (particularly, the minor axis of the particles is 0.3 mm or more, It was found that the production of 0.2 mm or more) can be prevented. The present invention has been completed based on the above findings and further studies. That is, the present invention provides the following.

1. A method for suppressing the formation of coarse particles of polylactic acid during freeze-drying of a polylactic acid microparticle aqueous suspension,
(a) preparing an aqueous polylactic acid microparticle suspension containing a dispersant and 3.5-10% mannitol, comprising:
(i) A polylactic acid solution prepared by dissolving polylactic acid in a water-miscible organic solvent is added to an aqueous dispersant solution containing a dispersing agent after stirring, and then mannitol is added, or (ii) polylactic acid is added. Adding a polylactic acid solution dissolved in a water-miscible organic solvent to a dispersant / mannitol-containing dispersant / mannitol aqueous solution containing a dispersant and mannitol under stirring;
(b) subjecting the polylactic acid microparticle aqueous suspension to lyophilization in a glass container;
Comprising a method.
2. The method according to 1 above, wherein the concentration of the dispersing agent in the dispersing agent aqueous solution or the dispersing agent / mannitol aqueous solution is 0.11 to 2.2 W / V%.
3. The method according to 1 or 2 above, wherein the dispersant is polyvinyl pyrrolidone or polyvinyl alcohol.
4). 4. The method according to any one of 1 to 3 above, wherein the concentration of mannitol in the aqueous polylactic acid microparticle suspension is 5 to 7 W / V%.
5. 5. The method according to any one of 1 to 4 above, wherein the concentration of the polylactic acid in the polylactic acid solution is 1 to 50 W / V%.
6). 6. The method according to any one of 1 to 5 above, wherein the water-miscible organic solvent is an acetone / ethanol mixture.
7). 6. The method according to 6 above, wherein the mixing ratio of acetone and ethanol in the acetone / ethanol mixed solution is acetone: ethanol = 3: 7 to 5: 5 by volume.
8). The mixing ratio of the aqueous dispersant solution or the aqueous dispersant / mannitol solution to the polylactic acid solution is, by volume, the aqueous dispersant solution or the aqueous solution containing the dispersant / mannitol: polylactic acid solution = 85: 15 to 95: 5. The method according to any one of 1 to 7 above.
9. 9. The method according to any one of 1 to 8 above, wherein the glass container is a glass vial.
10. In step (b), the glass vial containing the polylactic acid fine particle aqueous suspension is placed in a freeze dryer chamber, and the cooling rate in the chamber when the polylactic acid fine particle aqueous suspension is frozen. 9. The method according to 9 above, wherein is adjusted to 0.1 to 1 ° C./min.
11. A method of producing polylactic acid particles for visualizing a transparent tissue of the eye,
(a) preparing an aqueous polylactic acid microparticle suspension containing a dispersant and 3.5-10% mannitol, comprising:
(i) A polylactic acid solution prepared by dissolving polylactic acid in a water-miscible organic solvent is added to an aqueous dispersant solution containing a dispersing agent after stirring, and then mannitol is added, or (ii) polylactic acid is added. Adding a polylactic acid solution dissolved in a water-miscible organic solvent to a dispersant / mannitol-containing dispersant / mannitol aqueous solution containing a dispersant and mannitol under stirring;
(b) subjecting the polylactic acid microparticle aqueous suspension to lyophilization in a glass container;
A production method comprising:
12 12. The production method as described in 11 above, wherein the concentration of the dispersing agent in the aqueous dispersing agent solution or the aqueous solution containing the dispersing agent / mannitol is 0.11 to 2.2 W / V%.
13. 13. The method according to 11 or 12 above, wherein the dispersant is polyvinyl pyrrolidone or polyvinyl alcohol.
14 14. The production method according to any one of 11 to 13 above, wherein the concentration of mannitol in the aqueous polylactic acid microparticle suspension is 5 to 7 W / V%.
15. 15. The method according to any one of 11 to 14 above, wherein the concentration of the polylactic acid in the polylactic acid solution is 1 to 50 W / V%.
16. 16. The production method according to any one of 11 to 15 above, wherein the water-miscible organic solvent is an acetone / ethanol mixed solution.
17. 16. The production method as described in 16 above, wherein the mixing ratio of acetone and ethanol in the acetone / ethanol mixed solution is acetone: ethanol = 3: 7 to 5: 5 by volume.
18. The mixing ratio of the aqueous dispersant solution or the aqueous solution containing the dispersant / mannitol and the polylactic acid solution is, by volume, the aqueous dispersant solution or the aqueous solution containing the dispersant / mannitol: polylactic acid solution = 85: 15 to The manufacturing method according to any one of 11 to 17, which is 95: 5.
19. 19. The production method according to any one of 11 to 18 above, wherein the glass container is a glass vial.
20. In step (b), the glass vial containing the polylactic acid fine particle aqueous suspension is placed in a freeze dryer chamber, and the cooling rate in the chamber when the polylactic acid fine particle aqueous suspension is frozen. 20. The production method according to any one of the above 11 to 19, wherein is adjusted to 0.1 to 1 ° C./min.

The present invention having the above structure suppresses the aggregation of the polylactic acid particles during freeze-drying of the polylactic acid fine particle aqueous suspension, and is a coarse aggregate (particularly, the minor axis of the particles is 0.3 mm or more, especially 0.2 mm or more). Generation) can be prevented. For this reason, according to the present invention, when injecting an aqueous suspension of polylactic acid particles into the eye for visualizing a transparent tissue, the polylactic acid particles have a minor axis of 0.3 mm or more, particularly 0.2 mm or more. By substantially eliminating agglomerates, it is possible to smoothly inject the suspended particles into the eye so that the injection needle generally used in ophthalmic surgery is not clogged with particles and smooth injection is possible. To do.

The polylactic acid particles produced according to the present invention are not spherical, but individually take various irregular shapes, but such irregularly shaped polylactic acid particles are more vitreous and crystalline than the spherical particles. When it comes into contact with a transparent tissue such as the eye, it adheres well to the tissue and does not easily fall out of the tissue due to the flow of intraocular perfusate during surgery. For this reason, irregular-shaped polylactic acid particles are excellent as a transparent tissue visualization agent.

Since the polylactic acid used in the study is DL-polylactic acid, D-polylactic acid, L-polylactic acid, and DL-polylactic acid are not distinguished, and appropriate mixtures thereof are also used in the present invention. can do.

In this specification, the term “transparent tissue of the eye” refers to a transparent tissue in the ocular tissue such as the vitreous body, the vitreous membrane, the lens and the cornea.

In this specification, the term “transparent tissue visualization agent” refers to a preparation in which polylactic acid particles obtained by freeze-drying are dispersed in a dispersion.

In the present specification, the term “polylactic acid fine particles” means polylactic acid particles having a particle diameter of 5 μm or less.

In this specification, the term “polylactic acid microparticle aqueous suspension” is a liquid containing polylactic acid microparticles in a suspended state, and the dispersion medium is an aqueous medium, that is, organic in a mixed state. What is water which contains a solvent in part.

In the present specification, the term “dispersant aqueous solution” refers to an aqueous solution obtained by adding a dispersant to water and dissolving it, and the term “dispersant / mannitol aqueous solution” refers to a dispersant and D-mannitol. An aqueous solution obtained by adding and dissolving in water.

In the present invention, the water-miscible organic solvent for dissolving polylactic acid is preferably an acetone / ethanol mixed solution. The volume ratio of acetone and ethanol used to prepare this may be appropriate, but it is usually preferably in the range of 3: 7 to 5: 5. Both of these solvents are safer for body tissues than chlorinated organic solvents, and, unlike chlorinated organic solvents, are miscible with water. It is easy to shift from the water phase to the water phase, it is quickly removed by lyophilization, and does not substantially remain in the final lyophilized composition.

The concentration of polylactic acid dissolved in a water-miscible organic solvent such as the acetone / ethanol mixture is usually in the range of 1 to 50 W / V%, preferably 2 to 30 W / V%, and 5 to 20 W / V. % Is more preferable.

In the present invention, the amount of mannitol is preferably not less than 3.5 times by weight, more preferably not less than 5 times the amount of polylactic acid contained in the aqueous polylactic acid microparticle suspension. Is more preferable. This is because if the amount of mannitol is too small, some of the fine particles may aggregate to form coarse particles during lyophilization. There is no clear upper limit for the content of mannitol, but if it is too much, it takes time for lyophilization, which is not only disadvantageous, but also has a large effect on the osmotic pressure of the liquid when mixed with a dispersion medium during use of the lyophilizate. It becomes difficult to handle. Accordingly, the content of mannitol in the polylactic acid fine particle aqueous suspension is preferably 10 times or less in terms of weight ratio with respect to the amount of polylactic acid contained in the polylactic acid solution added thereto. It is more preferable to set it to double or less. Since the polylactic acid fine particle aqueous suspension is prepared by adding the polylactic acid solution to the dispersant aqueous solution or the dispersant / mannitol aqueous solution, the concentration of mannitol in the dispersant aqueous solution or the dispersant / mannitol aqueous solution is usually 3 It is preferably set to 0.85 to 11 W / V%, more preferably 4.5 to 10 W / V%, and still more preferably 5.5 to 8 W / V%.

The dispersant contained in the dispersant aqueous solution or the dispersant / mannitol aqueous solution is a step of adding polylactic acid solution dissolved in a water-miscible organic solvent to the aqueous solution containing the dispersant to precipitate polylactic acid fine particles. In addition, it is useful for the suppression of particle growth, and also when maintaining a uniform suspension state of polylactic acid fine particles before lyophilization and preparing a transparent tissue visualization agent by mixing a lyophilized composition with a dispersion medium. This is useful for rapidly suspending polylactic acid particles and maintaining a stable suspended state. As the dispersant, a surfactant or a water-soluble polymer having surface activity can be used. A water-soluble polymer that is solid at room temperature and immediately soluble in water is preferably used, and vinyl polymers such as polyvinylpyrrolidone and polyvinyl alcohol are particularly preferably used. When polyvinylpyrrolidone or polyvinyl alcohol is used, the content of the compound in the aqueous dispersant solution or the dispersant / mannitol aqueous solution is preferably 0.11 to 2.2 W / V%, preferably 0.22 to 1.1 W / V V% is more preferable, and 0.44 to 0.66 W / V% is more preferable. In the case of using polyvinylpyrrolidone, the content of the compound in the aqueous dispersant solution or the dispersant / mannitol aqueous solution is preferably 0.55 to 2.2 W / V%, and 0.22 in the case of using polyvinyl alcohol. It is preferably set to 0.55 W / V%.

In the above, the volume ratio of the dispersant aqueous solution or the dispersant / mannitol aqueous solution to the polylactic acid solution added thereto is usually preferably 95: 5 to 85:15, particularly preferably about 90:10. When a polylactic acid solution is added to the aqueous solution at such a ratio and stirred, an aqueous suspension of polylactic acid fine particles having an average primary particle size of 500 to 1000 nm and a maximum particle size of 5 μm or less can be easily obtained. . The primary particles may be partially agglomerated during the preparation of the aqueous suspension, but there is no problem if the particles applied to the primary suspension are removed by passing the aqueous suspension through a sieve having a diameter of 106 μm after the preparation.

In the present invention, the content of polylactic acid in the aqueous polylactic acid microparticle suspension subjected to freeze-drying is usually 0.05 to 7.5 W / V%, preferably 0.1 to 5.0 W / V%, more preferably 0.5 to 2.5 W / V%. The polylactic acid content in the aqueous polylactic acid microparticle suspension may be adjusted by adjusting the concentration of polylactic acid contained in the acetone / ethanol mixture in advance. You may carry out by adjusting the addition ratio of a solution. When the polylactic acid content in the aqueous polylactic acid microparticle suspension falls within these ranges, aggregation of the polylactic acid particles is prevented in combination with the presence of mannitol during freeze-drying of the aqueous polylactic acid microparticle suspension. it can. This method can be applied even when the polylactic acid content is less than these ranges.

The mannitol contained in the polylactic acid fine particle aqueous suspension is useful for preventing aggregation of the polylactic acid particles when the polylactic acid fine particle aqueous suspension is freeze-dried. The concentration of mannitol is preferably 3.5 to 10 W / V%, more preferably 5 to 7 W / V%.

In this specification, the term “average molecular weight” for polylactic acid refers to the weight average molecular weight. The average molecular weight of the polylactic acid used in the present invention is usually 500 or more, preferably 1000 or more, more preferably 3000 or more, particularly preferably 4000 or more, and usually 20000 or less, preferably 15000 or less, more preferably It is 10,000 or less, more preferably 8000 or less, and particularly preferably 6000 or less.

The lyophilized composition containing polylactic acid particles produced according to the present invention is mixed with an aqueous dispersion medium and prepared in the form of an aqueous suspension of polylactic acid particles, and used as a transparent tissue visualization agent. As the aqueous dispersion medium to be used, an aqueous solution having a composition that can be injected into the eye as a medicine can be appropriately used. Various compositions are well known to those skilled in the art as intraocular perfusion / cleaning solutions, and they can be appropriately selected and used as a dispersion medium. In addition, pharmaceuticals already sold as intraocular perfusion / cleaning solution may be used as a dispersion medium. Examples of those particularly suitable as such pharmaceuticals include Opegard MA (registered trademark, manufactured by Senju Pharmaceutical Co., Ltd.) and Opegard Neo Kit (registered trademark, manufactured by Senju Pharmaceutical Co., Ltd.).

In the present invention, an aqueous suspension of polylactic acid fine particles to be lyophilized includes pharmaceutically acceptable additives such as isotonic agents (salts such as sodium chloride and potassium chloride: saccharides such as glycerin, sorbitol and glucose: Polyhydric alcohols such as glycerin and propylene glycol: boric acid, borax, etc.), buffer (phosphate buffer, acetate buffer, borate buffer, carbonate buffer, citrate buffer, Tris buffer, etc.) , Thickener (hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose sodium, polyvinyl alcohol, polyethylene glycol, sodium alginate, etc.), stabilizer (sodium bisulfite, sodium thiosulfate, sodium edetate, sodium citrate) Ascorbic acid, dibutyl hydroxytoluene, magnesium chloride, calcium chloride and the like), pH adjusting agents (hydrochloric acid, sodium hydroxide, can be added phosphoric acid, such as acetic acid) and the like as appropriate. In that case, these additives may be added in advance to the aqueous dispersion or dispersion / aqueous mannitol solution before the polylactic acid solution is added, and after the preparation of the aqueous polylactic acid microparticle suspension, In addition, a part of the aqueous solution may be added in advance to the aqueous dispersant solution or the dispersant / mannitol aqueous solution before the polylactic acid solution is added, and the rest after the preparation of the aqueous solution containing the polylactic acid microparticles. You may add to this.

In the present invention, the pH of the aqueous polylactic acid microparticle suspension subjected to freeze-drying is usually adjusted to 4.0 to 8.0, preferably about 5.0 to 7.5.

In the freeze-drying step of the polylactic acid fine particle aqueous suspension obtained above, the suspension is usually preferably freeze-dried in a glass container. A particularly preferred glass container is a glass vial. It is preferable to freeze the suspension in a glass vial gradually. For example, a glass vial into which an aqueous suspension of polylactic acid fine particles is dispensed is placed in a freeze dryer chamber, and the cooling rate in the chamber is 0 when freezing (at the start of ice crystal formation in the suspension). It can be adjusted to 1 to 1 ° C./min. By doing so, the generation of coarse particles can be prevented more reliably.

The polylactic acid particles produced according to the present invention are not spherical, but have various irregular shapes, but such irregular shapes are more likely to occur in eyes such as the vitreous body and the lens than the spherical particles. When in contact with transparent tissue, it adheres well to the tissue and does not easily fall out of the tissue due to the flow of intraocular perfusate during surgery. For this reason, it is excellent as a transparent tissue visualization agent.

Hereinafter, the present invention will be described more specifically with reference to examples. However, it is not intended that the present invention be limited to the examples.

[Examples 1 and 2, Comparative Examples 1 to 3]
Polyvinylpyrrolidone (Povidone K-30, manufactured by BASF Japan, Japanese Pharmacopoeia) in purified water to a concentration of 0.55 W / V%, and D-mannitol in Comparative Examples 1 to 3 in Table 1 and Examples Each solution was dissolved so as to be 1.1 times the amount specified in 1-2, and the solution was filtered through a 0.22 μm aqueous filter to obtain solutions A-1 to A-5, respectively.

 

 

PLA0005 (DL-polylactic acid, weight average molecular weight 5000, manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in an acetone-ethanol mixture (acetone: ethanol = 4: 6 by volume) to a concentration of 10 W / V%. The resulting solution was filtered through a 0.22 μm non-aqueous filter to obtain solution B.

The solutions A-1 to A-5 and the solution B were mixed at a ratio of 9: 1 as follows. While stirring each solution of A-1 to A-5 in a 200 mL beaker with a stirrer (medium speed 700 to 800 rpm) using a stirrer (diameter 6 mm, length 20 mm), a micropipette tip (Eppendolf ep TIPS Using a tube pump (PERISTALTIC PUMP PST-100 IWAKI) with a silicon tube (outer diameter 6 mm, inner diameter 4 mm) with a standard 2-200 μL) attached to the discharge side, the B solution was A- Liquids 1 to A-5 were fed to deposit PLA0005 fine particles. At this time, the tip of the micropipette tip as the discharge port was completely immersed in the liquid. This was stirred for about 30 minutes to obtain suspensions D-1 to D-5, respectively. The amounts of polylactic acid, polyvinylpyrrolidone and D-mannitol contained in the suspensions D-1 to D-5 are as shown in Table 1.

The suspensions D-1 to D-5 obtained above were passed through a sieve (caliber 106 μm) to remove agglomerates. The suspensions D-1 to D-5 after sieving were filled in 1.5 mL glass vials (18Φ, height 33 mm) and freeze-dried under the following conditions. ~ E-5 was obtained.

(Freeze drying conditions)
a) Test equipment: Triomaster-A04 (Nissei Corporation)
b) Tray: Pull-out tray Frame 290W * 445L, 1 sheet used c) Vial: 18φ * 33H (glass)
d) Dispensing volume: 1.5 mL / vial e) Number of freeze-dried bottles: A vial of actual liquid or dummy liquid (2.5% D-mannitol aqueous solution) was laid to fill one tray.
f) Setting of freeze-drying program <preliminary freezing>
Vials were placed in a room temperature shelf, and the shelf was cooled to freeze the drug solution. The shelf temperature was cooled from + 20 ° C. to −40 ° C. over 2 hours (average cooling rate of 0.5 ° C./min).
<Primary drying and secondary drying>
The suspensions D-1 to D-5 were subjected to primary drying and secondary drying according to the following freeze-drying program.
In the primary drying, the shelf temperature was increased from −45 ° C. or 40 ° C. to −10 ° C. over 1 hour, and then set at −10 ° C. for 19 hours. The vacuum control value at this time was set to 0.1 Torr. Secondary drying was ramped from −10 ° C. to + 20 ° C. over 1 hour and then set at + 20 ° C. for 12 hours. The degree of vacuum at this time was assumed to be a success. After the secondary drying was completed, it was sealed with a rubber stopper.

[Evaluation of freeze-dried composition]
Suspensions were prepared by dispersing lyophilized compositions E-1 to E-5 in dispersion medium C having the composition shown in Table 2 so that the polylactic acid content was 1 W / V%.

 

 

Evaluation method: Transfer the entire amount of the suspension prepared above to a 12-well plate (12.5 cm × 8 cm, inner diameter 22 mm, IWAKI) and observe the size of the particles with a digital microscope (model number: VHX-500, Keyence) did. At that time, the magnification was set so that an almost whole image of one hole could be captured. Photographs were taken, and the number of aggregates having a minor axis of 0.3 and 0.2 mm or more was measured. The results are shown in Table 3 below.

 

 

As can be seen in Table 3, no coarse particles were found in the suspensions of lyophilized compositions E-4 and E-5 (corresponding to suspensions A-4 and A-5 before lyophilization, respectively). It was confirmed that it was not seen.

Formulation Examples [Formulation Examples 1-5]
Based on Table 4, polyvinyl pyrrolidone (Povidone K-30) or polyvinyl alcohol and D-mannitol were dissolved in purified water to 1.1 times the amounts specified for H-1 to H-4, Filter the solution through a 0.22 μm aqueous filter to make F-1 to F-4 solutions, respectively. In addition, polyvinyl pyrrolidone (Povidone K-30) is dissolved in purified water to 1.1 times the amount specified for H-5, and the solution is filtered through a 0.22 μm aqueous filter to obtain F-5 solution. To do.

 

 

PLA0005 (DL-polylactic acid, weight average molecular weight 5000, manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in an acetone-ethanol mixture (acetone: ethanol = 4: 6 by volume) to a concentration of 10 W / V%. The resulting solution is filtered through a 0.22 μm non-aqueous filter to give solution G. The same process is performed, and PLA0005 is mixed with acetone / ethanol so as to have a concentration of 30 W / V%.

Mix each liquid of F-1 to F-4 and G liquid, and F-5 liquid and G-5 liquid at a ratio of 9: 1 as follows. While stirring each solution of F-1 to F-5 in a 200 mL beaker with a stirrer (medium speed 700 to 800 rpm) using a stirrer (diameter 6 mm, length 20 mm), a micropipette tip (Eppendolf ep TIPS Using a tube pump (PERISTALTIC PUMP PST-100 IWAKI) fitted with a silicon tube (outer diameter 6 mm, inner diameter 4 mm) with a standard 2-200 μL) on the discharge side, about 1 mL / min of G solution or G-5 solution The liquid is fed into each of the liquids F-1 to F-5 at a speed of 1 to precipitate PLA0005 fine particles. At this time, the tip of the micropipette tip which is the discharge port is completely immersed in the liquid. As for F-5, after the fine particles of PLA0005 are precipitated, a prescribed amount of D-mannitol is added and dissolved. These F-1 to F-5 are stirred for about 30 minutes to obtain suspensions H-1 to H-5, respectively. The amounts of polylactic acid, polyvinylpyrrolidone and D-mannitol contained in the suspensions H-1 to H-5 are as shown in Table 4.

The suspensions H-1 to H-5 obtained above are passed through a sieve (caliber 106 μm) to remove aggregates. The suspensions H-1 to H-5 after sieving were filled in 1.5 mL glass vials (18Φ, height 33 mm) and lyophilized under the following conditions. Obtain ~ I-5.

(Freeze drying conditions)
a) Test equipment: Triomaster-A04 (Nissei Corporation)
b) Tray: Pull-out tray Frame 290W * 445L, 1 sheet used c) Vial: 18φ * 33H (glass)
d) Dispensing amount: 1.5 mL / vial e) Number of freeze-dried: A vial of actual liquid or dummy liquid (2.5% D-mannitol aqueous solution) is spread so that one tray is filled.
f) Setting of freeze-drying program <preliminary freezing>
Place vials in a room temperature shelf, cool the shelf and freeze the drug solution. Cool the shelf temperature from + 20 ° C. to −40 ° C. over 2 hours (average cooling rate 1 ° C./min).
<Primary drying and secondary drying>
For I-1 to I-5, primary drying and secondary drying are performed using the following freeze-drying program.
In primary drying, the shelf temperature is raised from −45 ° C. or 40 ° C. to −10 ° C. over 1 hour, and then set at −10 ° C. for 19 hours. The vacuum control value at this time is 0.1 Torr. In secondary drying, the shelf temperature is raised from −10 ° C. to + 20 ° C. over 1 hour, and then set at + 20 ° C. for 12 hours. The degree of vacuum at this time is assumed to be good. After secondary drying, seal with a rubber stopper.

[Examples 3 to 6]
Based on Table 5, polyvinyl pyrrolidone (Povidone K-30) or polyvinyl alcohol and D-mannitol were dissolved in purified water to 1.1 times the amounts specified for D-6 to D-9, The solution was filtered through a 0.22 μm aqueous filter to obtain A-6 to A-9 solutions, respectively.

 

 

PLA0005 (DL-polylactic acid, weight average molecular weight 5000, manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in an acetone-ethanol mixture (acetone: ethanol = 4: 6 by volume) to a concentration of 10 W / V%. The resulting solution was filtered through a 0.22 μm non-aqueous filter to obtain solution B.

The liquids A-6 to A-9 and the liquid B were mixed at a ratio of 9: 1 as follows. While stirring each solution of A-6 to A-9 in a 200 mL beaker with a stirrer (diameter 6 mm, length 20 mm) with a stirrer (medium speed 700 to 800 rpm), a micropipette tip (Eppendolf ep TIPS Using a tube pump (PERISTALTIC PUMP PST-100 IWAKI) with a silicon tube (outer diameter 6 mm, inner diameter 4 mm) with a standard 2-200 μL) attached to the discharge side, the B solution was A- Liquids 6 to A-9 were fed to deposit PLA0005 fine particles. At this time, the tip of the micropipette tip as the discharge port was completely immersed in the liquid. These A-6 to A-9 were stirred for about 30 minutes to obtain suspensions D-6 to D-9, respectively. The amounts of polylactic acid, polyvinylpyrrolidone and D-mannitol contained in the suspensions D-6 to D-9 are as shown in Table 5.

The suspensions D-6 to D-9 obtained above were passed through a sieve (caliber 106 μm) to remove agglomerates. The suspensions D-6 to D-9 after sieving were filled in 1.5 mL glass vials (18Φ, height 33 mm) and lyophilized under the following conditions. To E-9.

(Freeze drying conditions)
a) Test equipment: Triomaster-A04 (Nissei Corporation)
b) Tray: Pull-out tray Frame 290W * 445L, 1 sheet used c) Vial: 18φ * 33H (glass)
d) Dispensing volume: 1.5 mL / vial e) Number of freeze-dried bottles: A vial of actual liquid or dummy liquid (2.5% D-mannitol aqueous solution) was laid to fill one tray.
f) Setting of freeze-drying program <preliminary freezing>
Vials were placed in a room temperature shelf, and the shelf was cooled to freeze the drug solution. The shelf temperature was cooled from + 20 ° C. to −40 ° C. over 2 hours (average cooling rate of 0.5 ° C./min).
<Primary drying and secondary drying>
D-6 to D-9 were subjected to primary drying and secondary drying by the following freeze-drying program.
In the primary drying, the shelf temperature was raised from −45 ° C. or 40 ° C. to −10 ° C. over 1 hour, and then at −10 ° C. for 19 hours. The vacuum control value at this time was set to 0.1 Torr. In secondary drying, the shelf temperature was increased from −10 ° C. to + 20 ° C. over 1 hour, and then at + 20 ° C. for 12 hours. The degree of vacuum at this time was assumed to be a success. After the secondary drying was completed, it was sealed with a rubber stopper.

[Evaluation of freeze-dried composition]
Suspensions were prepared by dispersing lyophilized compositions E-6 to E-9 in purified water so that the content of polylactic acid was 1 W / V%.

Evaluation method: The particle size distribution of the suspension prepared above was measured using a laser diffraction particle size distribution analyzer (SALD-2100, Shimadzu Corporation).

Results: The maximum particle size of Examples 3, 4 and 6 is 0.287 mm, the maximum particle size of Example 5 is 0.233 mm, and in each of Examples 3 to 6, coarse particles of 0.3 mm or more are detected. There wasn't.

The production method of the present invention provides a redispersible lyophilized composition containing polylactic acid particles that can suppress aggregation of polylactic acid particles during lyophilization and does not contain coarse particles that clog the injection needle. It can be used to produce a transparent tissue visualization agent suitable for use in ophthalmic surgery such as removal surgery.

Claims (20)

A method for suppressing the formation of coarse particles of polylactic acid during freeze-drying of a polylactic acid microparticle aqueous suspension,
(a) preparing an aqueous polylactic acid microparticle suspension containing a dispersant and 3.5-10% mannitol, comprising:
(i) A polylactic acid solution prepared by dissolving polylactic acid in a water-miscible organic solvent is added to an aqueous dispersant solution containing a dispersing agent after stirring, and then mannitol is added, or (ii) polylactic acid is added. Adding a polylactic acid solution dissolved in a water-miscible organic solvent to a dispersant / mannitol-containing dispersant / mannitol aqueous solution containing a dispersant and mannitol under stirring;
(b) subjecting the polylactic acid microparticle aqueous suspension to lyophilization in a glass container;
Comprising a method. The method according to claim 1, wherein the concentration of the dispersant in the dispersant aqueous solution or the dispersant / mannitol aqueous solution is 0.11 to 2.2 W / V%. The method according to claim 1 or 2, wherein the dispersant is polyvinylpyrrolidone or polyvinyl alcohol. The method according to any one of claims 1 to 3, wherein the concentration of mannitol in the aqueous polylactic acid microparticle suspension is 5 to 7 W / V%. The method according to any one of claims 1 to 4, wherein the concentration of the polylactic acid in the polylactic acid solution is 1 to 50 W / V%. The method according to any one of claims 1 to 5, wherein the water-miscible organic solvent is an acetone / ethanol mixed solution. The method according to claim 6, wherein a mixing ratio of acetone and ethanol in the acetone / ethanol mixed solution is acetone: ethanol = 3: 7 to 5: 5 by volume. The mixing ratio of the aqueous dispersant solution or the aqueous dispersant / mannitol solution and the polylactic acid solution is, by volume, the aqueous dispersant solution or the dispersant / mannitol aqueous solution: polylactic acid solution = 85: 15 to 95: 5. A method according to any one of claims 1 to 7. The method according to any one of claims 1 to 8, wherein the glass container is a glass vial. In step (b), the glass vial containing the polylactic acid fine particle aqueous suspension is placed in a freeze dryer chamber, and the cooling rate in the chamber when the polylactic acid fine particle aqueous suspension is frozen. 10. The method of claim 9, wherein is adjusted to 0.1-1 ° C./min. A method for producing polylactic acid particles for visualizing a transparent tissue of the eye,
(a) preparing an aqueous polylactic acid microparticle suspension containing a dispersant and 3.5-10% mannitol, comprising:
(i) A polylactic acid solution prepared by dissolving polylactic acid in a water-miscible organic solvent is added to an aqueous dispersant solution containing a dispersing agent after stirring, and then mannitol is added, or (ii) polylactic acid is added. Adding a polylactic acid solution dissolved in a water-miscible organic solvent to a dispersant / mannitol-containing dispersant / mannitol aqueous solution containing a dispersant and mannitol under stirring;
(b) subjecting the polylactic acid microparticle aqueous suspension to lyophilization in a glass container;
A production method comprising: 12. The production method of claim 11, wherein the concentration of the dispersant in the dispersant aqueous solution or the dispersant / mannitol aqueous solution is 0.11 to 2.2 W / V%. The method according to claim 11 or 12, wherein the dispersant is polyvinyl pyrrolidone or polyvinyl alcohol. The method according to any one of claims 11 to 13, wherein the concentration of mannitol in the aqueous polylactic acid microparticle suspension is 5 to 7 W / V%. The method according to any one of claims 11 to 14, wherein the concentration of the polylactic acid in the polylactic acid solution is 1 to 50 W / V%. The method according to any one of claims 11 to 15, wherein the water-miscible organic solvent is an acetone / ethanol mixed solution. The production method according to claim 16, wherein a mixing ratio of acetone and ethanol in the acetone / ethanol mixed solution is acetone: ethanol = 3: 7 to 5: 5 by volume. The mixing ratio of the aqueous dispersant solution or the aqueous dispersant / mannitol solution and the polylactic acid solution is, by volume, the aqueous dispersant solution or the dispersant / mannitol aqueous solution: polylactic acid solution = 85: 15 to 95: 5. A manufacturing method according to any one of claims 11 to 17. The manufacturing method according to any one of claims 11 to 18, wherein the glass container is a glass vial. In step (b), the glass vial containing the polylactic acid fine particle aqueous suspension is placed in a freeze dryer chamber, and the cooling rate in the chamber when the polylactic acid fine particle aqueous suspension is frozen. The method according to any one of claims 11 to 19, wherein is adjusted to 0.1 to 1 ° C / min.