JPWO2007083763A1 - Method for preparing emulsion preparation of bioactive peptide and kit for preparing the preparation - Google Patents

Abstract

An object of the present invention is to provide a method that can be carried out very simply and that can prepare a very fine emulsion and a kit that can be used in the method. The method for preparing a bioactive peptide emulsion preparation according to the present invention comprises preparing a mixed solution containing at least a bioactive peptide aqueous solution and an oil adjuvant, and the content ratio of the oil adjuvant to the bioactive peptide aqueous solution is 0.5 to 5 times volume. The mixed liquid is moved between two syringes connected by a connector.

Description

  The present invention relates to a method for producing an emulsion preparation of a physiologically active peptide, and an emulsion preparation preparation kit that can be used in the method.

  In the treatment of malignant tumors such as cancer, attempts have been made to enhance specific immunity against malignant tumors by administering a vaccine containing a peptide as an antigen. In addition, an adjuvant such as an adjuvant may be administered to further enhance the immunity enhancing action. In this case, since the adjuvant is an oily component such as liquid paraffin, the peptide and the adjuvant are mixed with water to form an emulsion, which is administered as an injection.

  However, if the emulsion contained in the preparation contains a large diameter, the living body may recognize this as a foreign substance, and various side reactions such as formation of sarcoma may occur. Therefore, in the emulsion preparation, it is very important to reduce the emulsion diameter.

  Various techniques for producing a fine emulsion have been studied. For example, Japanese Patent Application Laid-Open No. 2005-152740 discloses a method for producing an emulsion characterized in that the flow rate is drastically reduced immediately after a dispersed phase such as soybean oil and a continuous phase such as an aqueous sodium dodecyl sulfate solution are combined, and a method thereof. An apparatus for performing is described.

  Japanese Patent Application Laid-Open No. 2005-186026 discloses a device for preparing an emulsion, in which two syringes are connected by a connector, and a porous body made of shirasu porous glass film is sandwiched between them. .

  As described above, various techniques have been conventionally known for preparing a fine emulsion. However, when an emulsion is used as a preparation, the diameter of the emulsion obtained by the prior art may not always be satisfactory. For example, the emulsion diameter in the emulsion preparation is preferably about 1 μm, but the diameter of the emulsion prepared by the technique of JP-A-2005-152740 is 16, 12 and 8.9 μm.

  On the other hand, according to the technique of JP-A-2005-186026, an emulsion of about 2 μm is also obtained. However, Patent Document 2 describes only an emulsion as a W / O type emulsion, and does not describe at all what raw materials should be used for preparing a special pharmaceutical peptide emulsion formulation.

  In addition, emulsion formulations are poor in stability, and when shipped after production, there is a problem that the emulsions are fused to increase the diameter and increase the risk. Therefore, since it is preferable to prepare an emulsion preparation immediately before use, a technique for preparing an emulsion preparation as easily as possible is required.

  Thus, the problem to be solved by the present invention is to provide a method that can be carried out very simply and that can prepare a very fine emulsion, and a kit that can be used in the method. is there.

  In order to solve the above-mentioned problems, the present inventor has conducted intensive research on conditions under which a fine emulsion can be easily obtained. As a result, the inventors have found that the above object can be achieved by repeatedly moving a mixed liquid of oil and water between syringes via a connector, and completed the present invention.

A method for preparing an emulsion preparation of a physiologically active peptide according to the present invention includes:
Preparing a liquid mixture containing at least a physiologically active peptide aqueous solution and an oil adjuvant, wherein the content ratio of the oil adjuvant to the physiologically active peptide aqueous solution is 0.5 to 5 times volume;
The mixed liquid is moved between two syringes connected by a connector.

The emulsion preparation preparation kit according to the present invention comprises:
One or more physiologically active peptide compositions obtained by lyophilizing a solution in which the physiologically active peptide is dissolved in physiological saline or an aqueous 1-6 w / v% alkali metal bicarbonate solution;
By dissolving the physiologically active peptide composition, an amount of water that is isotonic or nearly isotonic with plasma can be obtained in the same amount of water as that of the physiologically active peptide composition; and the same amount of oil adjuvant as that of the physiologically active peptide composition. ;
It is characterized by containing.

It is a schematic diagram which shows an example of the syringe used by this invention. In the figure, 1 indicates a plunger, 2 indicates a flange of a syringe barrel, 3 indicates a gasket, 4 indicates a nozzle (knock type), and 5 indicates a mixed solution containing a bioactive peptide aqueous solution and an oil adjuvant. . It is a schematic diagram which shows an example of the connector used by this invention. In the figure, 6 indicates a syringe mounting site, 7 indicates a liquid passing portion, 8 indicates air bubble removal, and 9 indicates a cock. It is a schematic diagram which shows a part of apparatus which performs the pumping based on this invention method. In the figure, 10 indicates a pumping unit, and 11 indicates a fixed base. It is a schematic diagram which shows an example of the connector used by this invention.

The method for preparing a physiologically active peptide emulsion preparation according to the present invention comprises:
Preparing a liquid mixture containing at least a physiologically active peptide aqueous solution and an oil adjuvant, wherein the content ratio of the oil adjuvant to the physiologically active peptide aqueous solution is 0.5 to 5 times volume;
The mixed liquid is moved between two syringes connected by a connector.

  The type of the physiologically active peptide used in the present invention is not particularly limited, and examples thereof include those that can be used as antigens used for preventing or treating diseases of humans and animals. For example, vaccines that increase specific immunity of patients against malignant tumors such as cancer, vaccines that prevent specific infections caused by viruses and bacteria, and vaccines that increase specific immunity to viruses and bacteria in patients with persistent infection, or desensitization that suppresses allergic reactions. Examples include antigens for therapeutic therapy.

  Furthermore, although there is no restriction | limiting in particular in the magnitude | size of the peptide used for this invention, Preferably it is 20 or less in the number of amino acids which comprise a peptide, More preferably, it is 4-20, More preferably, it is 8-20, More preferably, it is 8-15. , Most preferably 9 or 10. This is because if the number of amino acids constituting the peptide is too large, the solubility may be easily deteriorated.

  The oil adjuvant used in the present invention is not particularly limited as long as it is an oil-based immune adjuvant that is acceptable as a component of a pharmaceutical preparation. Examples thereof include liquid paraffin, lanolin, and Freund.

  As the water used in the present invention, those permitted as constituents of pharmaceutical preparations such as pure water, purified water, distilled water and sterilized water can be used. However, when a physiologically active peptide is dissolved, its osmotic pressure needs to be equivalent to that of plasma. Therefore, if the peptide is lyophilized with salts, etc., adjust the amount of sterilized water or the like so that the solution is isotonic or substantially isotonic with plasma, Add salt. The isotonicity may be any concentration as long as the salt concentration in the aqueous solution is acceptable for administration to humans, but the salt concentration in the aqueous solution is preferably 0.5 to 6 w / v%. When the peptide does not contain salts or the like, physiological saline or aqueous glucose solution may be used. Finally, the pH may be adjusted with a pharmacologically acceptable acid or base.

  In the present invention, a bioactive peptide, an oil adjuvant and water are mixed to form a mixed solution. Preferably, the bioactive peptide is first dissolved in water and then an oil adjuvant is added. The mixing ratio at this time is 0.5 to 5 times the content ratio of the oil adjuvant to the physiologically active peptide aqueous solution in order to facilitate the formation of the emulsion.

  In the present invention, in preparing the above mixed solution, it is preferable to stir the mixture of the physiologically active peptide aqueous solution and the oil adjuvant to eliminate the separated layer composed of the aqueous phase and the oil phase. Usually, when a physiologically active peptide aqueous solution that is an aqueous phase and an oil adjuvant that is an oil phase are simply mixed, the aqueous phase and the oil phase are separated into two layers. However, if the two-layer separation in the mixed solution is eliminated, the efficiency of the subsequent emulsion preparation process can be further increased. Here, eliminating a separated layer composed of an aqueous phase and an oil phase means that the formation of at least two layers by the aqueous phase and the oil phase cannot be observed with the naked eye by stirring the liquid separated into the two layers. It shall be said. When the interface between the two layers is eliminated, an emulsion having a relatively large diameter is formed, and the mixed solution becomes cloudy.

  For example, the bioactive peptide aqueous solution is added little by little while stirring the oil adjuvant, or a part of the bioactive peptide aqueous solution is added to the oil adjuvant and stirred. Also, insert the whole amount of the bioactive peptide aqueous solution and the oil adjuvant into one syringe and shake, or insert a part of the bioactive peptide aqueous solution and the oil adjuvant into one syringe and shake and shake. By repeating this operation, the separated layer composed of the aqueous phase and the oil phase may be eliminated in the syringe. More preferably, an operation of adding half the amount of the physiologically active peptide to the oil adjuvant and stirring for about 5 seconds to 1 minute is performed twice.

  In addition, if the liquid mixture from which the separated layer consisting of the aqueous phase and the oil phase has been dissolved is allowed to stand, phase separation may occur again. After eliminating these two layers, proceed to the next step as soon as possible. It is preferable to migrate.

  Next, in the method of the present invention, the mixed liquid is repeatedly moved between two syringes connected by a connector. That is, a fine emulsion having a diameter of about 1 μm can be easily prepared by vigorously stirring the mixed solution by repeatedly moving from a syringe cylinder having a large cross-sectional area to a connector having a small cross-sectional area and from the connector to the syringe. Can do.

  The cross-sectional area of the syringe barrel used in this step is 2 to 150 times, preferably 2 to 50 times the cross-sectional area of the connector. If the cross-sectional area of the syringe cylinder is less than twice the cross-sectional area of the connector, the above-described effects may not be sufficiently exhibited. On the other hand, if the cross-sectional area exceeds 150 times, the burden on the syringe or the device may become too large. is there.

More specifically, the cross-sectional area of the cylindrical portion of the syringe may be a ² order of 50 to 500 mm, the cross-sectional area of the connectors may be set to ² about 0.5 to 30 mm. In addition, although the shape of the cross section of the syringe barrel or the connector is not particularly limited, a circular shape, an elliptical shape, or a substantially circular shape that is a polygonal shape is preferable to prevent a burden from being locally applied, and a circular shape is particularly preferable. Moreover, the cross-sectional area in the said prescription | regulation shall be the cross-sectional area in the hollow part of the cylinder part of a syringe, and the cross-sectional area of the hollow part in the narrowest part among the paths currently formed in the connector. In other words, since the connector used in the present invention needs to be in close contact with the syringe at the end, the end may be expanded conically. In this case, the sectional area of the hollow portion of the central passage portion is used as a reference.

  When the cross-sectional shape of the flow path of the connector is circular, the internal diameter of the flow path is preferably 0.8 to 1.7 mm, and more preferably 0.9 mm or more and 1.5 mm or less. . This is because a fine emulsion can be stably prepared. In addition, when the thickness of a flow path is not uniform, the internal diameter of a flow path shall mean the internal diameter in the narrowest part.

  The length of the connector channel is preferably 10 to 15 mm. When the length of the flow path is less than 10 mm, the resistance decreases, and thus the efficiency of emulsion preparation may be deteriorated. On the other hand, if it is longer than 15 mm, the connector strength may not be sufficiently secured.

  The connector of the present invention may be a three-way cock having a discharge port for extracting bubbles at the center, or any of a straight tubular connector having no discharge port. However, from the viewpoint of preventing liquid leakage and the like, it is preferable to use a straight tubular connector.

  The movement of the mixed liquid between the syringes is preferably repeated 30 times or more. This is because as the number of times increases, it becomes easier to form a fine emulsion, and when it is 30 times or more, an emulsion suitable as a preparation is considered to be formed. On the other hand, the upper limit is not particularly limited, but even if the movement is repeated excessively, the emulsion does not become infinitely fine. Therefore, the movement may be appropriately stopped while visually confirming, for example. Usually, about 150 times is sufficient.

  Moreover, it is also a suitable aspect to repeat the movement of the mixed liquid between the syringes 20 times or more, and then leave it for 0.5 to 10 minutes, and repeat the movement once more or more, more preferably 10 times or more. This aspect can exert an effect particularly when an emulsion is difficult to be formed, and the formation of the emulsion is further facilitated.

  In the following description, the period from when one of the syringes passes through the connector to the other syringe is referred to as one pumping. That is, one movement of the mixed solution between the syringes is one pumping.

  Furthermore, in the present invention, when the mixed solution is stirred in advance and the separated layer composed of the aqueous phase and the oil phase is eliminated, an emulsion can be prepared with a smaller number of pumping operations. In this case, the pumping is performed at least 3 times and 50 times or less, more preferably 10 to 50 times.

  In addition, the pumping speed is not particularly limited, but if the pumping speed is too slow or too fast, it may be difficult to form an emulsion. Therefore, the pumping speed is preferably 30 to 90 times per minute, more preferably 60 to 90 times.

  The kit for preparing an emulsion preparation according to the present invention contains one or more physiologically active peptide compositions and the same number of water and oil adjuvants.

  The one or more physiologically active peptide compositions in the kit of the present invention are obtained by lyophilizing a solution dissolved in physiological saline or 1 to 6 w / v% aqueous solution of alkali metal hydrogencarbonate. Here, the term “one or more” is used in consideration of the fact that a plurality of physiologically active peptides may be administered depending on the patient, particularly in the peptide emulsion preparation for enhancing the immune activity of the patient. Preferably two or more, more preferably two are used. In addition, as the physiologically active peptide here, the same peptide as described for the method of the present invention can be used.

  As a solvent for dissolving the physiologically active peptide, physiological saline or a 1 to 6 w / v% aqueous solution of alkali metal bicarbonate is used. Here, you may mix and use the physiological saline and the said alkali metal hydrogencarbonate aqueous solution.

  The peptide composition contained in the kit for preparing an emulsion preparation of the present invention is produced by dispensing a bioactive peptide once dissolved in a solvent into a vial and then lyophilizing it. This is because the solution state is better from the viewpoint of convenience, but there is a problem that the stability is deteriorated, and the lyophilized powder improves the stability. Although physiological saline is suitable as a solvent, some physiologically active peptides are difficult to dissolve in physiological saline. In addition, since the type of physiologically active peptide to be used is often different depending on the patient, if the solvent is examined one by one, the productivity of the kit is lowered. Therefore, in the present invention, a 1 to 6 w / v% aqueous alkali metal bicarbonate solution is more preferably used as a solvent for dissolving the physiologically active peptide. Since the solvent exhibits solubility in a peptide having low solubility in physiological saline, the productivity of the kit can be improved.

  The peptide composition of the present invention may be one obtained by dissolving two or more types of peptides in a solvent and lyophilized, or one obtained by dissolving one type of peptide and lyophilizing it. It is preferable that each type is individually dissolved and freeze-dried and a plurality of peptides are not mixed. This is because if the lyophilized powder in which two or more kinds of peptides are mixed is stored in a lyophilized powder, a problem may occur when used.

  Examples of the alkali metal hydrogen carbonate include lithium hydrogen carbonate, sodium hydrogen carbonate, and potassium hydrogen carbonate. Sodium hydrogen carbonate is preferable. Moreover, the alkali metal hydrogen carbonate aqueous solution concentration is set to 1 to 6 w / v%. If the concentration is less than 1 w / v%, the peptide dissolution effect may not be sufficiently exerted, and if it exceeds 6 w / v%. This is because the volume of water increases in order to adjust the salt concentration to an appropriate level for administration, which may result in an inappropriate state for administration.

  The amount of water in the kit of the present invention is such that a physiologically active peptide solution that is isotonic or substantially isotonic with plasma can be obtained by dissolving the physiologically active peptide composition. Here, “substantially isotonic” has the same meaning as described above.

  In producing the physiologically active peptide composition contained in the kit of the present invention, the amount of physiological saline or aqueous bicarbonate solution is not particularly limited as long as the peptide can be uniformly dissolved. However, the ratio of the peptide to physiological saline or alkali metal bicarbonate aqueous solution is such that the peptide concentration of the obtained peptide solution is 0.4% by mass or less, preferably 0.1 to 0.4% by mass, more preferably It is preferable to be 0.3 to 0.4% by mass.

As the kit of the present invention, one containing two syringes having a cross-sectional area of the cylinder part of 50 to 500 mm ² and one containing the syringe connector are suitable. As the connector, the cross-sectional area is 0.02 to 0.5 times the cylindrical cross-sectional area of the syringe, the cross-sectional shape of the flow path is a circle having an inner diameter of 0.8 to 1.7 mm, The thing whose length of a flow path is 10-15 mm is suitable. In the emulsion preparation, the peptide to be used is often different depending on the patient. Therefore, it is necessary to replace the syringe and the connector for producing the emulsion one by one. Therefore, the kit provided with these syringes and connectors is highly convenient.

  The syringe included in the kit of the present invention may be a general one as shown in FIG. However, in the present invention, since a mixture liquid containing an oily adjuvant is inserted and pumping is repeated, the syringe barrel and gasket material is preferably highly durable such as a surface-coated one.

  The connector for a syringe included in the kit of the present invention is for connecting two syringes. For example, the connector shown in FIGS. 2 and 4 can be used. The connector shown in FIG. 2 has a three-way cock having a discharge port for removing air bubbles. Moreover, FIG. 4 is a straight tubular connector and has a connection part with a syringe on the right and left of a pipe | tube.

  In order to prepare the emulsion preparation according to the present invention, the bioactive peptide composition of the kit of the present invention dissolved in water is inserted into one of syringes connected by a connector and pumped. As shown in FIG. 3, this pumping is performed by at least a pumping device having a fixing base 11 for detachably fixing a syringe connected by a connector and an interlocking mechanism for reciprocating each plunger of the syringe in the same direction. be able to.

  The said fixing stand fixes the syringe demonstrated in the said method or kit so that attachment or detachment is possible. The fixing base needs to be fixed so that the cylindrical portion does not move as the plunger of the syringe is pushed and pulled. For example, in FIG. 3, the flange of the syringe cylinder is hooked on the fixing base 11 and the cover is fixed from above. In addition, each time a preparation to be administered to an individual patient is prepared, or each time a different physiologically active peptide is used, it may be necessary to replace the syringe or connector. Need to be.

  The pumping device has an interlocking mechanism for reciprocating the plungers of the syringe fixed on the fixed base in the same direction. As this interlocking mechanism, for example, a mechanism that moves the pumping unit 10 by a cam that changes the rotational motion of the motor into a horizontal motion and reciprocates each plunger in the same direction can be considered. In addition, the interlocking mechanism may be anything that pushes and pulls each plunger when the fixing base itself is fixed so as not to move, and when the plunger is fixed so that it does not move. Any device that reciprocates the fixed table may be used.

  The pumping device preferably has means for adjusting the reciprocating speed of the plunger. Examples of the means include a transmission that changes the rotation speed of the motor, and a device that controls the rotation of the motor itself.

  According to the method for preparing a physiologically active peptide emulsion preparation according to the present invention, a peptide emulsion preparation having an extremely fine emulsion diameter can be easily prepared. Therefore, if it takes a long time to prepare the emulsion preparation and use it, the emulsions may be fused and the diameter may increase, causing unexpected side effects. It can be administered immediately after preparation.

  In addition, the kit for preparing an emulsion preparation of the present invention can be used in the method of the present invention, and by using a specific solvent, the peptide can be efficiently dispensed and is easy to manufacture.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited by the following examples, but may be appropriately modified within a range that can meet the purpose described above and below. It is also possible to implement, and they are all included in the technical scope of the present invention.

Example 1
A physiologically active peptide (4 mg) for treating prostate cancer or brain tumor shown in Table 1 was dissolved in a 4 w / v% aqueous sodium hydrogen carbonate solution (1 mL), and further ISA51VG (1 mL) manufactured by SEPPIC was added as an oil adjuvant. A mixed solution was obtained. Two syringes (manufactured by Bee Brown, 2 mL syringe, cross-sectional area: 66.3 mm ² ) were connected with a connector having a cross-sectional area: 2.43 mm ² , and the obtained mixed solution was inserted into one syringe. Here, the cylinder cross-sectional area of the syringe is approximately 27 times the connector cross-sectional area. This syringe was installed in a pumping apparatus, and pumping was repeated 100 times at a rate of 100 times / minute. As a result, as shown in Table 1, no. Except 1, 5, and 14, emulsions could be formed. Next, no. 1, 5 and 14, the same treatment was performed except that the syringe was changed to a 5 mL volume (cross-sectional area: 111.63 mm ² , syringe cylinder cross-sectional area / connector cross-sectional area = about 46). . In No. 1, an emulsion could be formed. 5 and 14 could not be formed. Then, using a 5 mL syringe in Examples 1 to 16, pumping was repeated 50 times at a rate of 100 times / minute, and then allowed to stand for 5 minutes, and further pumping was repeated 50 times at the same rate. In the example an emulsion could be formed.

  About emulsion formed by repeating pumping 50 times through standing for 5 minutes, using a particle size distribution meter (manufactured by Nikkiso Co., Ltd., Microtrac UPA150-EX), after completion of pumping, and when standing for another day The particle size distribution of the emulsion was measured by a dynamic light scattering method. Table 2 shows the median diameter of each emulsion.

  As a result, it was demonstrated that the method and apparatus of the present invention can easily obtain a peptide emulsion having an extremely fine emulsion diameter of about 1 μm.

Example 2
In consideration of the case where the emulsion preparation preparation kit of the present invention is manufactured, the No. 1 used in Example 1 is related to various solvents. The solubility of 1-12 peptides was examined. Specifically, after returning the peptide stored in a cool dark place to room temperature, about 5 mg was weighed in a low humidity chamber (temperature: 23.0 ° C., humidity: 10% RH) and put into a transparent container. 200 μL of the solvent was added thereto, and it was visually confirmed whether or not it was dissolved by gentle stirring. When dissolution could not be confirmed, the operation of adding 200 μL of solvent and stirring was repeated, and when dissolution did not occur even with a solvent amount of 1.25 mL, it was judged as “insoluble”. The results are shown in Table 3.

  In the table, ◎ is dissolved at 5 mg / 200 μL, ○ is dissolved at least up to 5 mg / 1.25 mL, × is not dissolved even at 5 mg / 1.25 mL,-is not conducted, * is 1% It shows that acetic acid was used.

  As described above, first, when physiological saline widely used for injection preparations was used, half of the peptides could be dissolved, but 8 peptides did not show solubility. Therefore, when 4 w / v% sodium bicarbonate was used, all peptides showed solubility. No. Similar experiments were performed with 1, 2, 4, 6, 7, and 9 peptides with 10% or 1% acetic acid, but 3 peptides were insoluble.

  In producing an emulsion preparation preparation kit, productivity is higher when the types of solvents for dissolving peptides are unified as much as possible to reduce the number. This Example 2 revealed that the aqueous sodium hydrogen carbonate solution exhibits high solubility for the physiologically active peptide. In addition, although not shown as a result, when it experimented also with 1M sodium hydroxide, high solubility was shown. However, sodium hydroxide is not suitable for injection. On the other hand, sodium bicarbonate has no particular problem as a component of an injection.

  From the above results, it was demonstrated that an aqueous sodium hydrogen carbonate solution is suitable as a solvent for dissolving the physiologically active peptide once in producing the emulsion preparation preparation kit of the present invention.

Example 3
A peptide solution was obtained by mixing the physiologically active peptide (No. 1, 3, 6) used in Example 1 above and physiological saline or 4 w / v% sodium bicarbonate aqueous solution in the volumes shown in Table 4. Later, the peptide solution was filled into a vial and freeze-dried to obtain a physiologically active peptide composition.

  The solubility and stability of the physiologically active peptide composition were evaluated as follows. First, 1 mL of water was added to one vial of the bioactive peptide composition, and after gently mixing and stirring for 1 minute, the state was visually confirmed. The results are shown in Table 5.

  As shown in Table 5, it was demonstrated that a bioactive peptide composition obtained by dissolving a bioactive peptide in physiological saline or a sodium hydrogen carbonate aqueous solution and then lyophilizing it can be dissolved in water very easily.

Example 4
The bioactive peptide composition produced in Example 3 above, the oil adjuvant and water used in Example 1 were each packaged in separate vials, and a 5 mL syringe (cylinder cross-sectional area: 111.63 mm ² ) and its connector (Cross sectional area: 2.43 mm ² ) was added to prepare an emulsion preparation preparation kit shown in Table 6.

  Using the emulsion preparation preparation kit, the bioactive peptide composition was dissolved in water in the same manner as in Example 1, and after adding an oil adjuvant, it was inserted into one of 5 mL syringes connected by a connector and pumped intermittently. As a result of repeating the above, a uniform and fine emulsion could be prepared.

  As described above, it was demonstrated that the emulsion preparation preparation kit of the present invention is extremely useful as an emulsion preparation that can be easily prepared at the time of use.

Example 5
In order to examine the inner diameter of the connector, an emulsion formulation was prepared as follows using physiological saline (1 mL) and ISA51VG (1 mL) manufactured by SEPPIC as an oil adjuvant. First, prepare ^two syringes (5 mL volume (cross-sectional area: 111.63 mm ² ) manufactured by Bea Brown Co., Ltd.) in which these physiological saline and oil adjuvant are mixed. The whole amount was collected and connected with one of the connectors in Table 7, and pumping was performed five times manually. Subsequently, this syringe was installed in the pumping apparatus and pumped under the conditions described in Table 7.

  Moreover, the drop test was done about the obtained emulsion as follows. Specifically, one drop of the obtained emulsion dispersion was dropped onto the water surface, and the case where the dispersion did not disperse immediately in water was defined as “good (◯)”, and the case where it was immediately dispersed was defined as “bad (×)”. . In addition, if the emulsion is appropriately formed, the dispersion does not immediately disperse even when dropped in water. However, if the emulsion is not properly formed, it is immediately dispersed. The results are shown in Table 7.

  From the results of Table 7, when the inner diameter of the connector flow path is 0.8 mm, the emulsion may not be stably obtained. However, an emulsion could be obtained by slowing down the pumping speed. Further, it was found that when a connector having an inner diameter of 1.0 to 1.4 mm is used, a fine emulsion can be prepared more stably.

Example 6
The preparation method of the emulsion and the length of the connector were examined as follows. First, N0.14 peptide (4 mg) described in Table 1 was dissolved in 4 W / V% aqueous sodium hydrogen carbonate solution (1 mL). Then, two 5 mL syringes were connected with straight tubular connectors described in Table 8. An aqueous physiologically active peptide solution and oil adjuvant (1 mL) were added to one syringe, and an emulsion was prepared under the conditions described in Table 8. The results are shown in Table 8.

  From the results of Table 8, in the mixing step, it is more reproducible to prepare an emulsion with better reproducibility by pumping after removing the two layers consisting of an aqueous phase and an oil phase by shaking and mixing the bioactive peptide aqueous solution by half with oil adjuvant. I knew it could be. Further, it was found that the emulsion having a connector length of 15 mm can be prepared with a smaller number of pumping times than that of 10 mm.

Example 7
No. described in Table 1 1-No. 6, no. 8, N0.9 and no. 11-No. For 16 peptides, an emulsion was prepared in the same manner as in Example 6 using the straight tubular connector having an inner diameter of 1 mm and a flow path length of 10 mm used in Example 6. The results are shown in Tables 9 and 10.

  From the results of Tables 9 and 10, if the two layers consisting of the aqueous phase and the oil phase of the mixed solution are eliminated before pumping, a fine emulsion formulation can be prepared more efficiently with various bioactive peptides. I understood it.

Claims (13)

A method for preparing an emulsion formulation of a bioactive peptide comprising the steps of:
Preparing a liquid mixture containing at least a physiologically active peptide aqueous solution and an oil adjuvant, wherein the content ratio of the oil adjuvant to the physiologically active peptide aqueous solution is 0.5 to 5 times volume;
A method for preparing a bioactive peptide emulsion preparation, wherein the mixed liquid is moved between two syringes connected by a connector.   The preparation method according to claim 1, wherein in the step of preparing the mixed solution, the mixture of the physiologically active peptide aqueous solution and the oil adjuvant is stirred to eliminate the separated layer composed of the aqueous phase and the oil phase.   The preparation method of Claim 1 or 2 which makes the cross-sectional area of a syringe cylinder part 2-50 times the cross-sectional area of a connector.   The preparation method according to any one of claims 1 to 3, wherein a cross-sectional shape of the flow path of the connector is circular, and an inner diameter of the flow path is 0.8 to 1.5 mm.   The preparation method according to any one of claims 1 to 4, wherein the length of the flow path of the connector is 10 to 15 mm.   The preparation method according to any one of claims 1 to 5, wherein the movement of the mixed liquid between the syringes is repeated 30 times or more.   The preparation method according to any one of claims 1 to 6, wherein the movement of the mixed solution between the syringes is repeated 20 times or more, and then left still for 0.5 to 10 minutes, and the movement is repeated once or more again. A kit for preparing an emulsion formulation of a bioactive peptide,
One or more physiologically active peptide compositions obtained by lyophilizing a solution in which the physiologically active peptide is dissolved in physiological saline or an aqueous 1-6 w / v% alkali metal bicarbonate solution;
By dissolving the bioactive peptide composition, the same amount of water as that of the bioactive peptide composition is obtained in the same amount as that of the bioactive peptide composition; and the amount of water adjuvant is the same as that of the bioactive peptide composition. ;
A kit for preparing an emulsion preparation, comprising:   The preparation of an emulsion preparation according to claim 8, comprising a physiologically active peptide composition obtained by freeze-drying a solution obtained by dissolving a physiologically active peptide in an aqueous solution of 1 to 6 w / v alkali metal bicarbonate. kit. Furthermore, the kit for emulsion formulation preparation of Claim 8 or 9 containing two syringes whose cross-sectional area of a cylinder part is 50-500 mm < ² >.   Furthermore, it is a connector for said syringes, Comprising: The cross-sectional shape of a flow path contains the connector with an internal diameter of 0.8-1.5 mm, The connector for emulsion formulation preparation in any one of Claim 8 to 10 kit.   The emulsion preparation preparation kit according to claim 11, wherein the length of the channel of the connector is 10 to 15 mm.   The kit for preparing an emulsion preparation according to claim 11 or 12, wherein the cross-sectional area of the connector is 0.02 to 0.5 times the cylindrical cross-sectional area of the syringe.

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