JP4878463B2 - Pharmaceutical preparation containing peptide hormone-encapsulated nanoparticles and method for producing the same - Google Patents

Description

  The present invention relates to a pharmaceutical preparation containing peptide hormone-encapsulated nanoparticles formed by encapsulating peptide hormones such as insulin and calcitonin in nanoparticles and a method for producing the same.

In recent years, the number of diabetic patients has increased with lifestyle changes and population growth. Diabetes mellitus is caused by insulin-dependent type I, which develops as a result of insulin secretion dysfunction due to destruction of pancreatic islets of Langerhans due to immune abnormalities and viral infections. Of insulin-independent II caused by relative deficiency
It is a metabolic disease roughly divided into types.

There are various treatments for diabetes, such as diet, exercise, and pharmacotherapy, depending on the patient's symptoms. Most patients with type I diabetes need to control blood sugar levels by taking insulin from outside the body. . Insulin's physiological activity includes the secretion of an amount of insulin corresponding to an increase in blood glucose level after eating, lowering the blood glucose level to keep the blood glucose level normal, as well as promoting amino acid incorporation and protein and glycogen synthesis. It has many effects such as an action and an inhibitory action on decomposition, and is widely used as a therapeutic agent for type I diabetes utilizing its blood glucose lowering action.

  Peptide hormones typified by insulin are susceptible to enzymatic degradation in the gastrointestinal tract, so that the bioavailability is extremely low in highly convenient oral preparations. Therefore, conventionally, insulin administration by subcutaneous injection has been performed. However, when insulin is administered by subcutaneous injection, there is a risk of a heavy load on the human body due to a rapid decrease in blood glucose level, and the bioavailability decreases by passing through the liver that enzymatically degrades insulin (the first pass through the liver) Effect).

  Thus, for example, Patent Documents 1 and 2 disclose sustained-release preparations in which insulin is encapsulated in microcapsules of a metal salt of a biodegradable polymer to suppress the initial excessive release of insulin after administration. However, in order to maintain the insulin level in the blood constant and constantly maintain the blood sugar level at a normal level, it is necessary to perform daily subcutaneous injections (about 1 to 5 times) with the patient's own hand, Development of new administration methods that improve pain and convenience during injection and improve patient compliance has been desired.

Under such circumstances, as routes of insulin administration other than oral and injection, administration routes utilizing the transmucosal membrane of the lungs and nasal cavity are actively studied. Of these pulmonary route for delivery via the lung alveoli, it proteolytic enzyme is smaller than in the gastrointestinal tract, the specific surface area of the alveoli is very broad and about 100 m ^2, comparable to the surface area of the intestinal mucosa (200 meters ²⁾ In addition, there are favorable conditions for obtaining a high bioavailability such as thin epithelial cells, good drug migration into the blood, and avoidance of the first-pass effect of the liver.

  Transpulmonary preparations administered by the pulmonary route include inhaled preparations that have been combined with insulin using a spray-drying technique, or high-molecular weight composites of insulin and polymers that have been formed using the spheroid crystal method. Attempts have been made to put molecular nanoparticles into practical use. However, insulin-encapsulated polymer nanoparticles produced by using the conventional spheroid crystal method represented by the emulsion solvent diffusion method in water have a low bulk density and a practical transpulmonary dose (inhalation amount of 100 mg or less per dose). ), It was necessary to increase the insulin content in the preparation.

  Thus, for example, Patent Document 3 discloses that by binding polyethylene glycol (PEG) to insulin, the encapsulation rate in the polymer nanoparticles is increased and the destruction (burst) of the nanoparticles is suppressed, and at the initial administration stage. A method for suppressing a decrease in blood glucose level is disclosed. However, in the method of Patent Document 3, it is necessary to perform PEG coupling treatment to insulin in advance, and the number of manufacturing steps increases. In addition, it is necessary to develop a certain blood glucose level lowering action even in the initial stage of administration. However, when PEG-bonded insulin is used, it is difficult to control the blood sugar level lowering action.

  In addition, calcitonin, a kind of peptide hormone like insulin, is secreted into the body from thyroid C cells and decreases the calcium concentration in blood together with parathyroid hormone and directly acts on osteoclasts to suppress bone lysis. However, it is more difficult to encapsulate in polymer nanoparticles than insulin, and the development of a high-concentration calcitonin preparation has been desired.

On the other hand, it is suitable for the so-called Drug Delivery System (hereinafter referred to as DDS) by the spray-drying fluidized bed granulation method in which a liquid raw material containing nanoparticles composed of drugs and biocompatible polymers and sugar alcohol is sprayed into a flowing gas. A method for producing drug-containing composite particles having excellent handling properties and applicability to transpulmonary drugs has been proposed (see Patent Document 4). However, in order to encapsulate peptide hormones in nanoparticles and use them to make transpulmonary drugs that satisfy practical pulmonary doses, the improvement of the peptide hormone encapsulation rate in the composite particles is further improved. There was room left.
JP 2001-233788 A JP 2002-114667 A JP-T-2004-534721 JP 2004-262810 A

  In view of the above problems, the present invention provides a pharmaceutical preparation comprising peptide hormone-encapsulated nanoparticles that have both immediate and long-lasting effects on peptide hormones and can be miniaturized by increasing the peptide hormone content. The purpose is to provide. Another object of the present invention is to provide a method for producing a pharmaceutical preparation containing peptide hormone-encapsulated nanoparticles with low cost and low environmental impact.

In order to achieve the above object, the first constitution of the present invention is a biocompatible nanoparticle formed of either polylactic acid, polyglycolic acid, or lactic acid / glycolic acid copolymer, and encapsulating peptide hormones inside with each other are combined by binder consisting of a sugar alcohol or sucrose, a pharmaceutical formulation comprising a peptide hormone encapsulated composite particles formed by encapsulating the peptide hormone on the outer layer to be formed in the binder.

  The second configuration of the present invention is characterized in that, in the pharmaceutical preparation of the above configuration, the peptide hormone is insulin.

  The third configuration of the present invention is characterized in that, in the pharmaceutical preparation of the above configuration, the peptide hormone is calcitonin.

A fourth configuration of the present invention is characterized in that the pharmaceutical preparation having the above configuration is used for transpulmonary administration.

According to a fifth aspect of the present invention, a mixture of at least a peptide hormone solution and a solution in which a biocompatible polymer is dissolved in an organic solvent is added to an aqueous polyvinyl alcohol solution so that the peptide hormone is the biocompatible. A nanoparticle forming step for producing a suspension of peptide hormone-encapsulated nanoparticles encapsulated in a polymer; a solvent evaporating step for distilling off the organic solvent from the suspension of peptide hormone-encapsulated nanoparticles; and having a composite step of complexing the binding agent comprising the peptide hormone containing nanoparticles among which the organic solvent is distilled off from the peptide hormone include sugar alcohols or sucrose, peptides It is a manufacturing method of the pharmaceutical formulation containing a hormone inclusion composite particle .

The sixth configuration of the present invention is characterized in that, in the method for producing a pharmaceutical preparation of the above configuration, the complexing step is performed by lyophilization.

The seventh configuration of the present invention is characterized in that, in the method for producing a pharmaceutical preparation of the above configuration, the complexing step is performed by a spray drying fluidized bed granulation method.

The eighth configuration of the present invention is characterized in that, in the method for producing a pharmaceutical preparation of the above configuration, the polyvinyl alcohol concentration in the aqueous polyvinyl alcohol solution is less than 0.5% by weight.

The ninth configuration of the present invention further includes a removal step of removing polyvinyl alcohol from the suspension of peptide hormone-encapsulated nanoparticles after the solvent distillation step in the method for producing a pharmaceutical preparation of the above configuration. It is characterized by.

The tenth configuration of the present invention is characterized in that, in the method for producing a pharmaceutical preparation of the above configuration, the polyvinyl alcohol concentration in the aqueous polyvinyl alcohol solution is 0.1 wt% or more and 10 wt% or less.

The eleventh configuration of the present invention is characterized in that, in the method for producing a pharmaceutical preparation of the above configuration, the organic solvent contains at least acetone.

According to the first configuration of the present invention, the immediate effect of the preparation can be enhanced by the peptide hormone encapsulated in the outer layer of the composite particle, and the formulation is obtained by gradually releasing the peptide hormone encapsulated inside the nanoparticle. Can increase the sustainability. In addition, since peptide hormone is encapsulated in the outer layer of the composite particle in addition to the inside of the nanoparticle, the content of peptide hormone in the preparation can be increased, and a pharmaceutical preparation capable of miniaturizing the dosage form is provided.

  In addition, according to the second configuration of the present invention, in the pharmaceutical preparation of the first configuration, by using insulin as a peptide hormone, the insulin encapsulated in the outer layer of the nanoparticles has an immediate effect of lowering the blood glucose level. An insulin preparation with enhanced blood glucose level-lowering action by insulin encapsulated inside the nanoparticles is enhanced. In addition, the insulin content in the preparation can be increased, and the size of the dosage form can be reduced. Furthermore, by adjusting the encapsulation ratio of insulin inside and outside the nanoparticles to balance the immediate effect and sustainability of the blood glucose lowering action, insulin that exhibits appropriate action according to the taking scene such as before meals and after meals A formulation can be provided.

  In addition, according to the third configuration of the present invention, in the pharmaceutical preparation of the first configuration, by using calcitonin as a peptide hormone, it is difficult to encapsulate calcitonin which has been difficult to encapsulate in nanoparticles in the past, Provided is a high-concentration calcitonin preparation that can be encapsulated in an outer layer to increase the encapsulation rate and that can be downsized. Moreover, by adjusting the encapsulation ratio of calcitonin inside and outside the nanoparticles, a calcitonin preparation that has both immediate effect and sustainability in reducing the blood calcium concentration is obtained.

Further, according to the fourth configuration of the present invention, the pharmaceutical preparation of any one of the first to third configurations is used for pulmonary administration, thereby increasing the peptide hormone content in the formulation and practical. Transpulmonary doses can be achieved. In particular, in the case of insulin preparations used for the treatment of diabetes, it is possible to improve patient compliance by reducing the bioavailability in oral administration and improving pain and convenience in subcutaneous injection.

In addition, according to the fifth configuration of the present invention, a mixed solution of at least a solution of peptide hormone and a solution in which a biocompatible polymer is dissolved in an organic solvent is added to an aqueous polyvinyl alcohol solution so that the peptide hormone can be a suspension of the peptide hormone encapsulated nanoparticles encapsulated in a compatible polymer, after distilling off the organic solvent from the suspension, the peptide by coupling agent consisting of a sugar alcohol or sucrose containing the peptide hormone Combining hormone-containing nanoparticles with each other to produce a pharmaceutical preparation containing peptide hormone-encapsulated composite particles, the peptide hormone content in the preparation is high, easy to handle when filling into a container, and redispersible when used A pharmaceutical preparation that can be complexed with simple aggregated particles can be produced simply and at low cost.

Further, according to the sixth configuration of the present invention, in the method for producing the pharmaceutical preparation of the fifth configuration, the complexing step is performed by freeze-drying, so that the nanoparticles can be complexed satisfactorily and efficiently. Can do.

Moreover, according to the seventh configuration of the present invention, in the method for producing a pharmaceutical preparation of the fifth configuration, the nanoparticle is weakly primary aggregated by performing the complexing step by the spray-drying fluidized bed granulation method. Composite particles can be produced easily and in a short time.

According to the eighth configuration of the present invention, in the method for producing a pharmaceutical preparation of any one of the fifth to seventh configurations, the polyvinyl alcohol concentration in the polyvinyl alcohol aqueous solution is less than 0.5% by weight. This eliminates the need for a removal step of removing excess polyvinyl alcohol by washing the nanoparticles by centrifugation or the like as in the case of using a high-concentration polyvinyl alcohol aqueous solution, thereby reducing the manufacturing process and time. .

According to the ninth configuration of the present invention, in the method for producing a pharmaceutical preparation of any one of the fifth to eighth configurations, after the solvent distillation step, the peptide hormone-encapsulated nanoparticle suspension is further used. By providing a removal step to remove polyvinyl alcohol, after forming nanoparticles using a high concentration polyvinyl alcohol aqueous solution, excess polyvinyl alcohol can be removed, and peptide hormones enclosed in the nanoparticles are encapsulated The rate can be stabilized.

Moreover, according to the 10th structure of this invention, in the manufacturing method of the pharmaceutical formulation of the said 9th structure, the polyvinyl alcohol concentration in polyvinyl alcohol aqueous solution shall be 0.1 to 10 weight%, The concentration (viscosity) of the polyvinyl alcohol aqueous solution can be maintained in an appropriate range for the diffusion of the organic solvent.

According to the eleventh aspect of the present invention, in the method for producing a pharmaceutical preparation having any one of the fifth to tenth aspects, by using acetone as the organic solvent, the pharmaceutical preparation is less likely to have an adverse effect on the human body. Can be manufactured, and the burden on the environment is also reduced.

  Peptide hormone-encapsulated nanoparticles used in the present invention are obtained by encapsulating peptide hormones in a biocompatible polymer to form nano-sized particles (nanospheres). Since the nanoparticles or peptide hormones released from the nanoparticles, or both, can reach the blood via the mucosa, it can be suitably used as a material for a DDS preparation.

  In the present invention, peptide hormones are encapsulated not only in the interior of the nanoparticles but also in the outer layer. With such a configuration, in addition to the peptide hormone that is released from the inside of the nanoparticle in a controlled manner, the peptide preparation that dissolves from the outer layer of the nanoparticle immediately after administration is allowed to act on the pharmaceutical preparation. Can be granted. Further, in the case of peptide hormones that are difficult to encapsulate inside the nanoparticles, the encapsulation rate can be increased because the total encapsulated amount increases by encapsulating in the outer layer.

  As a method of encapsulating peptide hormones in the outer layer of the nanoparticles, the nanoparticles are compounded into aggregated particles (nanocomposites) that can be redispersed when powdered, and the surface of the nanoparticles is used with the binder used for the outer layer as the outer layer. Further, a method of attaching a peptide hormone to the above can be mentioned. The structure of the nanocomposite is shown in FIG. The nanocomposite 1 is formed by combining biocompatible nanoparticles 2 with a binder 3, and the peptide hormone 4 is encapsulated not only in the interior of the nanoparticles 2 but also in the outer layer of the nanoparticles 2 formed with the binder 3. Has been.

  Examples of the nanoparticle composite method include freeze-drying method, fluidized bed dry granulation method, and dry mechanical particle composite method. The freeze-drying method is preferably used. For example, when the peptide hormone is water-soluble (hydrophilic), once the peptide hormone once encapsulated leaks to the surface of the nanoparticle, it is redissolved in the water existing around it. When this water is removed from the system, the peptide hormone component is reduced by that amount, and the encapsulation rate varies. Therefore, it is preferable to combine organic or inorganic binders so that they can be redispersed, and freeze-dry them with the nanoparticles as they are without removing the water in which the peptide hormone is dissolved. At this time, by adding a peptide hormone together with the binder, a predetermined amount of the peptide hormone can be encapsulated in the outer layer formed of the binder.

  The binding agent forms an outer layer of nanoparticles at the time of complexing, and also has an effect of effectively preventing variation in the encapsulation rate of peptide hormones and enhancing the handleability of the nanoparticles. Examples of such a binder include sugar alcohols such as mannitol, trehalose, sorbitol, erythritol, maltose, and xylitol, sucrose, and the like.

  Alternatively, it may be combined by a fluidized bed dry granulation method or a dry mechanical particle combining method (for example, applying a compressive force and a shearing force using a Mechano-Fusion System AMS (manufactured by Hosokawa Micron Corporation)). In particular, when using a spray-drying fluidized bed granulation method in which a mixture containing a material to be granulated is sprayed into a flowing gas among fluidized bed drying granulation methods, it becomes possible to omit a time-consuming and laborious freeze-drying step, Since composite particles can be produced easily and in a short time, it is advantageous for industrialization.

  Specifically, in the spray-drying fluidized bed granulation method, composite particles (nanocomposites) are obtained by using a spray-drying fluidized bed granulation / coating type powder processing apparatus 5 as shown in FIG. obtain.

  The powder processing apparatus 5 generally connects an upper part, which is a substantially cylindrical space, a lower part having a smaller inner diameter than the upper part, and an upper part and a lower part. Has a fluidized bed space 6 formed by integrating three spaces with a central portion having a substantially trapezoidal cross section, and is provided in the lowermost portion of the casing 7 so as to flow upward. Spray nozzle 8 capable of injecting liquid raw material into layer space 6, two bag filters 9a and 9b projecting downward in the upper part of fluidized bed space 6, liquid raw material supply unit (not shown), and fluidized bed An air supply unit (not shown) for supplying drying / fluidizing air (fluid gas for spray drying) to the space 6 is also provided.

  In the powder processing apparatus 5, a granular raw material is formed by repeating agglomeration and layering granulation of particles by spraying a liquid raw material in an empty fluidized bed space 6. be able to. Therefore, there is an advantage that the apparatus configuration is simple and the manufacturing process can be simplified, and high quality granules can be manufactured.

  In the present invention, using the agglomeration granulation and layering granulation, a biocompatible nanoparticle encapsulating peptide hormone therein and a liquid in which sugar alcohol as a binder added with peptide hormone is dispersed and suspended A raw material (mixture) is injected into the fluidized bed space 6 to form first composite particles (composite particles) 10 as generated particles.

  Specifically, first, as shown in the left diagram of FIG. 2, the liquid material (arrow S in the figure) supplied from the liquid material supply unit is injected into the fluidized bed space 6. Since the spray mist diameter of the liquid material supplied by spraying is a very small droplet of about several μm to several tens of μm, the liquid material is instantly solidified in the process of ascending the fluidized bed space 6, One composite particle 10 is collected in the bag filters 9 a and 9 b above the fluidized bed space 6.

  In the bag filters 9a and 9b, the first composite particles 10 are intermittently dropped to the spray zone 8a below the casing 7 by a pulse jet backwashing method. Since this spray zone 8a is a region where the liquid material is ejected from the spray nozzle 8, the liquid material adheres to the surface of the first composite particles 10 and grows while agglomerating to form a larger second Composite particles (composite particles) 11 are obtained. This process is agglomeration granulation.

  At the same time as the agglomeration and granulation, the liquid raw material adheres directly to the surface of the second composite particle 11 that has grown larger, and is further dried and solidified. Thereby, the second composite particles 11 further grow. This process is layering granulation. By continuing such agglomeration granulation and layering granulation, as shown in the middle diagram of FIG. 2, the fluidized bed (arrow F in the diagram) of the second composite particles 11 is in the fluidized bed space 6. It is formed.

  Almost all of the liquid raw material supplied thereafter adheres to and spreads on the surface of the second composite particles 11, and is further precipitated and dried. That is, layering granulation progresses after the fluidized bed is formed. Therefore, by adjusting the period of this layering granulation, it becomes possible to control the spheroidization and weight increase of the second composite particles 11, and finally, as shown in the right diagram of FIG. Thus, the second composite particle 11 having an appropriate average particle diameter is obtained. Moreover, particle physical properties such as porous particles can be controlled depending on conditions. Further, by applying jet air in a pulsed manner at this portion, the aggregate of the composite particle group can be crushed to control the particle size.

  In the spray-drying fluidized bed granulation method, when a sugar alcohol having low crystallinity is used as a binder, it becomes amorphous at the time of compounding and cannot be formed into particles well. Therefore, it is preferable to use mannitol having strong crystallinity.

  The composite particles obtained as described above are easy-to-handle agglomerated particles in which nanoparticles are collected before use, and sugar alcohol is dissolved and held in sugar alcohol by touching moisture during use. It becomes composite particles that can be restored to peptide hormones and nanoparticles encapsulating peptide hormones.

  The biocompatible polymer used in the present invention is desirably a biocompatible polymer that has low irritation and toxicity to the living body, is biocompatible, and is decomposed and metabolized after administration. Moreover, it is preferable that it is the particle | grains which release the peptide hormone to include continuously and gradually. As such a material, polylactic acid / glycolic acid copolymer (PLGA) can be particularly preferably used. It is known that PLGA can contain a drug and can be stored for a long time while maintaining the efficacy of the drug. Furthermore, it is considered that sustained release in units of several hours to several tens of hours can be achieved by degradation of PLGA by an enzyme in the living body.

  The molecular weight of PLGA is preferably in the range of 5,000 to 200,000, and more preferably in the range of 15,000 to 25,000. The composition ratio of lactic acid and glycolic acid may be 1:99 to 99: 1, but glycolic acid is preferably 0.333 with respect to lactic acid 1. PLGA having a lactic acid and glycolic acid content in the range of 25 wt% to 65 wt% is preferably used because it is amorphous and soluble in an organic solvent such as acetone.

  Other biocompatible polymers include polyglycolic acid (PGA), polylactic acid (PLA), polyaspartic acid, and the like. In addition, aspartic acid / lactic acid copolymer (PAL) and aspartic acid / lactic acid / glycolic acid copolymer (PALG), which are these copolymers, may be used, and charged groups such as amino acids or functionalizable groups may be used. You may have.

  Other biocompatible polymers include polyalkylenes such as polyamide, polycarbonate, polyethylene, polypropylene, polyethylene glycol, polyethylene oxide, polyethylene terephthalate, polyvinyl compounds such as polyvinyl alcohol, polyvinyl ether and polyvinyl ester, acrylic acid and Polymers with methacrylic acid, cellulose and other polysaccharides, and peptides or proteins, or copolymers or mixtures thereof. Further, chitosan for enhancing mucoadhesiveness may be complexed to the surface of the nanoparticle, or phospholipid (lecithin / phosphatidylcholine) may be complexed.

  In addition, it is preferable to modify the surface of PLGA with polyethylene glycol (PEG) because the affinity between the hydrophilic peptide hormone and PLGA is improved and encapsulation becomes easy. In particular, using a block copolymer in which a hydrophobic polymer block and a hydrophilic polymer block are combined as a biocompatible polymer, the hydrophilic polymer block is placed on the outer shell (shell) of the particle surface, and the hydrophobic polymer block is A method of forming a biocompatible nanoparticle having a core-shell structure located inside the particle (core part) and encapsulating the peptide hormone using the shell part as an outer layer can also be used. The structure of such nanoparticles is shown in FIG.

  The biocompatible nanoparticle 2 is formed by agglomerating a large number of block copolymers 14 in which a hydrophobic polymer block 12 and a hydrophilic polymer block 13 are combined, and an emulsion in water using an aqueous phase as a poor solvent. When prepared by the method, as shown in FIG. 3, the hydrophilic polymer block 13 protrudes from the particle surface to form an outer shell portion (shell portion) 15, and the hydrophobic polymer block 12 is located in the particle inner portion (core portion) 16. Forming a core-shell structure.

  Since the hydrophilic polymer block 13 has a high ability to attract and hold water molecules, the shell portion 15 where the hydrophilic polymer block 13 protrudes attracts water molecules in a poor solvent to form a hydrated phase. The hydrophilic peptide hormone 4 diffuses into the poor solvent with the diffusion of the organic solvent (good solvent) during nanoparticle crystallization, but leaks into the poor solvent by interacting with the hydrophilic polymer block 13. It is considered that the encapsulation of the nanoparticles 2 in the shell portion 15 becomes possible.

  In addition, when the hydrophilicity of the peptide hormone 4 is weakened, the interaction with the hydrophilic polymer block 13 is also weakened, and the encapsulation rate into the shell portion 15 is reduced. On the contrary, the core due to the interaction with the hydrophobic polymer block 12 is reduced. Since the encapsulation rate in the portion 16 is improved, even if the peptide hormone 4 is amphiphilic, a high encapsulation rate is ensured as a whole nanoparticle.

  Therefore, nanoparticles with an increased encapsulation rate of hydrophilic peptide hormones, which were difficult to produce by conventional spherical crystallization methods, can be produced simply and at low cost. Further, since the peptide hormone can be encapsulated in the outer layer of the nanoparticles without complexing the nanoparticles, the production process is simplified. Furthermore, by combining the nanoparticles of FIG. 3 having a core-shell structure by the method described above, the peptide hormone 4 is also encapsulated in the binder layer 3 (see FIG. 1) formed further outside the shell portion 15. Therefore, the encapsulation rate of peptide hormone 4 can be further increased.

  The nanoparticles may be prepared by an emulsion-in-oil method using an oil phase as a poor solvent. In that case, the hydrophobic polymer block 12 protrudes from the particle surface to form the shell portion 15, and the hydrophilic polymer block 13 is located in the core portion 16. That is, since it has a core-shell structure opposite to the configuration of FIG. 3, the hydrophilic peptide hormone 4 is mainly enclosed in the core portion 16. In this case, in order to increase the immediate effect, it is desirable that the nanoparticles 2 are combined and the peptide hormone 4 is encapsulated in the binder layer 3 (see FIG. 1).

  The biocompatible block copolymer 14 is preferably a biocompatible one that has low irritation and toxicity to the living body, is biocompatible, and is decomposed and metabolized after administration. Moreover, it is preferable that it is the particle | grains which release the peptide hormone to include continuously and gradually. As such a material, a PEG-PLGA copolymer in which the hydrophobic polymer block 12 is composed of a polylactic acid / glycolic acid copolymer (PLGA) and the hydrophilic polymer block 13 is composed of polyethylene glycol (PEG). Can be suitably used.

  Peptide hormones encapsulated in nanoparticles include insulin, calcitonin, gastrin, prolactin, adrenocorticotropic hormone (ACTH), thyroid stimulating hormone (TSH), luteinizing hormone (LH), follicle stimulating hormone (FSH), oxytocin, Examples include vasopressin (antidiuretic hormone), atrial natriuretic peptide (ANP), atrial natriuretic factor (ANF), growth hormone, somatostatin (growth hormone secretory hormone), interferon and the like.

  Among the above peptide hormones, in particular, insulin used for the treatment of diabetes is suitable as a peptide hormone to be encapsulated in nanoparticles because the effect is improved by combining the preparation with immediate effect and sustainability as described later. is there. In addition, calcitonin used for the prevention and treatment of osteoporosis has heretofore been extremely difficult to encapsulate inside nanoparticles, but by encapsulating in the outer layer of nanoparticles by the above method, it can be used as a material for high-concentration calcitonin preparations. Preferably used. Incidentally, insulin is non-hydrophilic and calcitonin is strongly hydrophilic.

  The higher the encapsulation rate of these peptide hormones in the nanoparticles and in the outer layer, the higher the peptide hormone content in the preparation, which is preferable. However, an increase in the encapsulation rate in the outer layer of nanoparticles becomes a strong immediate effect as a formulation, and an increase in the encapsulation rate in the interior of nanoparticles becomes excellent in sustainability. Both of these should be provided in a well-balanced manner according to the dosage form of the preparation and the scene of taking it. Therefore, the encapsulation rate of the peptide hormone inside the nanoparticle is preferably 0.01% by weight or more and 20% by weight or less, and particularly preferably 0.1% by weight or more and 10% by weight or less with respect to the biocompatible polymer.

  In addition, any one of the above peptide hormones may be encapsulated, but a synergistic effect of each component can be expected if a plurality of peptide hormones having different efficacy and action mechanism are encapsulated. Furthermore, components other than peptide hormones such as vitamin derivatives can be encapsulated together with peptide hormones.

  In addition, the encapsulation ratio of peptide hormones inside and outside the nanoparticles can be appropriately set according to the mechanism of action of the pharmaceutical preparation produced using the nanoparticles, the required immediate effect, the degree of sustainability, etc. . In other words, in the case of a preparation that requires time from drug administration to the arrival of the target site in the blood or a preparation that requires long-lasting effect after administration, the encapsulation rate inside the nanoparticles can be increased. good. On the other hand, in the case of a preparation that reaches a target site in blood or the like in a short time or a preparation that requires an effect from immediately after administration, the encapsulation rate of the nanoparticles in the outer layer may be increased.

  Peptide hormones can be encapsulated by adjusting the amount of peptide hormone added at the time of nanoparticle formation and the amount of peptide hormone complexed with the binder, It can be changed according to the type of biocompatible polymer forming the particles. On the other hand, when the shell part of a nanoparticle having a core-shell structure is encapsulated as an outer layer, the size of the shell part depends on the molecular weight of the hydrophilic polymer block constituting the block copolymer. What is necessary is just to select the molecular weight of a polymer block according to the encapsulation rate of a peptide hormone.

  The peptide hormone-encapsulated nanoparticles used in the present invention are not particularly limited as long as they have an average particle size of less than 1,000 nm, but in order to increase the efficiency of reaching the target site, the average particle size is set to 300 nm or less. It is preferable to do. On the other hand, as described above, the smaller the particle diameter of the nanoparticles, the lower the encapsulation rate. Therefore, the average particle diameter is preferably 30 nm or more.

  The method for producing peptide hormone-encapsulated nanoparticles used in the present invention is not particularly limited as long as the target substance can be processed into particles having a particle diameter of less than 1,000 nm. It is highly preferred to use a crystallization method. Spherical crystallization is a method in which spherical crystal particles can be designed and processed by directly controlling their physical properties by controlling the crystal generation and growth process in the final process of compound synthesis. One of the spherical crystallization methods is an emulsion solvent diffusion method (ESD method).

  The ESD method is a technique for producing nanospheres based on the following principle. In this method, two types of solvents are used: a good solvent that can dissolve PLGA or the like as a base polymer for encapsulating the drug, and a poor solvent that does not dissolve PLGA. As the good solvent, acetone or the like that dissolves PLGA or the like and is miscible with the poor solvent is used. And a polyvinyl alcohol aqueous solution etc. are normally used for a poor solvent.

  As an operation procedure, first, after dissolving PLGA in a good solvent, a drug solution is added and mixed in the good solvent so that the PLGA does not precipitate. Next, when the PLGA / drug mixture is dropped into the poor solvent while stirring, the good solvent (acetone) in the mixture rapidly diffuses and transfers into the poor solvent. As a result, self-emulsification of the good solvent occurs in the poor solvent, and submicron sized good solvent emulsion droplets are formed. Furthermore, due to the mutual diffusion of the good solvent and the poor solvent, acetone, which is a good solvent, continuously diffuses from the emulsion to the poor solvent, so that the solubility of PLGA and the drug in the emulsion droplets is lowered, and finally , PLGA nanospheres of spherical crystal particles containing the drug are produced.

  In the above spherical crystallization method, nanoparticles can be formed by a physicochemical method, and the resulting nanoparticles are substantially spherical. Therefore, it is necessary to consider the problem of catalyst and raw material compound residues from homogeneous nanoparticles. And can be easily formed. Thereafter, the organic solvent which is a good solvent is distilled off under reduced pressure (solvent distillation step) to obtain peptide hormone-encapsulated nanoparticle powder. Then, the obtained powder is composited as it is or by freeze-drying or the like as necessary (compositing step) to form composite particles, which are then filled into a container to obtain a pharmaceutical preparation containing peptide hormone-encapsulated nanoparticles.

  The pharmaceutical preparation thus produced can be used for various applications such as pulmonary administration, subcutaneous and intravenous injection, transdermal and oral administration. In particular, when it is used for pulmonary administration, the dosage form can be miniaturized because it contains a high concentration of peptide hormone, and the preparation can satisfy a practical pulmonary dose. In addition, when used for oral administration, the peptide hormone encapsulated in the outer layer is made into tablets using an excipient such as chitosan or filled into a capsule so that it is difficult to be degraded by digestive enzymes. It is preferable to use an agent.

  The types of good solvent and poor solvent used in the spherical crystallization method and the type of liquid cross-linking agent are determined according to the type of target drug and the like, and are not particularly limited. Since the hormone-encapsulated nanoparticles are used as a raw material for pharmaceutical preparations that are directly administered to the human body, it is necessary to use those that are highly safe for the human body and have a low environmental impact. As such a poor solvent, for example, a polyvinyl alcohol aqueous solution is suitably used, and as a good solvent, for example, only acetone or a mixture of acetone and ethanol is suitably used. In addition, when the excess polyvinyl alcohol remains, the process (removal process) of removing polyvinyl alcohol by centrifugation etc. is provided after a solvent distillation process.

  The concentration of the polyvinyl alcohol aqueous solution, or the mixing ratio of acetone and ethanol, the conditions at the time of crystal precipitation and the method of applying mechanical shearing force are not particularly limited, and the type of the target drug and the spherical granulated crystals It may be determined appropriately according to the particle size (in the case of the present invention, nano-order), etc., but the higher the concentration of the polyvinyl alcohol aqueous solution, the better the adhesion of the polyvinyl alcohol to the nanoparticle surface, and the re-dispersion in water after drying On the other hand, when the concentration of the polyvinyl alcohol aqueous solution exceeds a predetermined level, the viscosity of the poor solvent increases and adversely affects the diffusibility of the good solvent. Therefore, although depending on the polymerization degree and saponification degree of polyvinyl alcohol, when a removal step is provided after the nanoparticle formation step, it is preferably 0.1% by weight or more and 10% by weight or less, more preferably about 2%. In addition, when not providing a removal process, it is preferable to set it as 0.5 weight% or less.

  The present invention is not limited to the above-described embodiments, and various modifications are possible. Embodiments obtained by appropriately combining technical means disclosed in different embodiments are also included in the present invention. Included in the technical scope. Hereinafter, the manufacturing method and action effect of the pharmaceutical preparation containing the peptide hormone-encapsulated nanoparticles of the present invention will be specifically described by taking insulin preparation and calcitonin preparation as examples.

[Preparation of insulin-encapsulated PLGA nanospheres]
100 mg of insulin (derived from bovine, manufactured by SIGMA, 28.0 units / mg) was dissolved in 18 mL of 0.01 mol / L hydrochloric acid. A biocompatible polymer, lactic acid / glycolic acid copolymer (PLGA: molecular weight 20,000, lactic acid / glycolic acid = 75/25, Tg = 45 ° C., PLGA 7520 manufactured by Wako Pure Chemical Industries, Ltd.) 2 g of acetone as its good solvent After dissolving in 100 mL to obtain a polymer solution, the insulin-hydrochloric acid solution is added to obtain a mixed solution. Next, this mixed solution was dropped at a constant rate (4 mL / min) with stirring at 40 ° C. and 400 rpm into 250 mL of a 2 wt% aqueous solution of polyvinyl alcohol (PVA: Poval 403, manufactured by Kuraray) prepared as a poor solvent. A suspension of insulin-encapsulated PLGA nanospheres was obtained by diffusion of the good solvent into the poor solvent.

  Subsequently, after distilling off acetone under reduced pressure, the precipitate of insulin-encapsulated PLGA nanospheres is recovered by centrifugation (centrifugal acceleration 41,000 G), and the precipitate is resuspended in purified water and centrifuged again to remove excess. The polyvinyl alcohol was removed. After removing the supernatant, it was freeze-dried at −45 ° C. and powdered to obtain an insulin-encapsulated PLGA nanosphere powder having good redispersibility in water.

[Preparation of insulin-encapsulated PLGA nanocomposite preparation]
700 mg of trehalose (manufactured by Hayashibara) was dissolved in purified water (pH = 6.8, the same applies hereinafter), and 12.4 mg of insulin (derived from bovine, manufactured by SIGMA) was suspended to make up to 50 mL. This insulin suspension was added to a suspension obtained by resuspending 350 mg of the nanosphere powder obtained in Example 1 in 100 mL of purified water, mixed uniformly, and then freeze-dried at −45 ° C. A PLGA nanocomposite preparation of the present invention in which insulin was encapsulated inside and outside the nanoparticles was obtained.

Comparative Example 1

  In place of the insulin suspension of Example 2, 700 mg of trehalose (manufactured by Hayashibara) was dissolved in purified water to make 50 mL of trehalose aqueous solution, and insulin was enclosed only inside the nanoparticles by the same method as in Example 2. PLGA nanocomposite preparation was obtained.

Comparative Example 2

  In place of the nanosphere powder obtained in Example 1, PLGA nanospheres not encapsulating insulin were used, and a PLGA nanocomposite formulation in which insulin was not encapsulated in the inner and outer layers of the nanoparticles was performed in the same manner as in Comparative Example 1. Obtained.

[Evaluation of insulin content in insulin-filled PLGA nanocomposite preparation]
The composition weight ratio in each PLGA nanocomposite formulation obtained in Example 2 and Comparative Examples 1 and 2 was measured. 30 mg each of the nanocomposite preparations of Example 2 and Comparative Examples 1 and 2 were weighed and each was diluted with 10.0 mL of purified water to dissolve trehalose. The PLGA nanospheres precipitated at this time and the insulin in the outer layer were isolated as solids by a centrifugal separation operation (centrifugal acceleration 41,000 G, −20 ° C., 20 minutes).

  Next, 3.0 mL of acetonitrile was added to the solid content isolated from the preparations of Example 2 and Comparative Example 1 to dissolve PLGA, and insulin in the nanospheres was leaked. The leaked insulin was dissolved in 0.01N hydrochloric acid and made up to 10 mL. After reprecipitation of PLGA was removed by centrifugation, 20 μL of the supernatant was quantified by high performance liquid chromatography (HPLC) to determine the inside of the particles and The amount of insulin encapsulated in the outer layer was measured.

  Here, the HPLC measured value of Comparative Example 1 can be regarded as the insulin content inside the PLGA nanosphere, and this value is also applied to the insulin content inside the nanosphere of Example 2 consisting of nanospheres prepared by crystallization in the same batch. it can. Therefore, using the actually measured HPLC value of Example 2 (total value of the inner and outer layers) and the actually measured HPLC value of Comparative Example 1 (internal value), the inner and outer layers of the nanoparticle in the nanocomposite preparation of Example 2 (total value) -Insulin values) were calculated.

  As HPLC operating conditions, a mobile phase (acetonitrile / 1.2% by volume perchloric acid aqueous solution = 35/65) was set in a column (manufactured by JASCO, Finepak SIL300C18T-5) at a flow rate of 1.5 mL. The peak of insulin was detected at a detection wavelength of 214 nm using a detector (manufactured by Shimadzu Corporation, SPD-6AV). The results are shown in Table 1.

  As is clear from Table 1, in the preparation of Example 2 that was complexed using a suspension of insulin in an aqueous trehalose solution, insulin was also encapsulated in the outer layer of trehalose. On the other hand, in the preparation of Comparative Example 1 complexed using an aqueous trehalose solution, insulin was encapsulated only inside the PLGA nanosphere. In Example 2, the amount of insulin charged was set so that insulin was encapsulated at a ratio of 1: 1 in the inner and outer layers of the nanosphere, but due to the loss of each component in the crystallization and freeze-drying processes, etc. The encapsulation ratio of insulin in the inner and outer layers was about 6: 4.

  In any nanocomposite preparation, the weight ratio of PLGA nanospheres to trehalose was approximately 1: 2. The insulin concentrations in the nanocomposite preparations of Example 2 and Comparative Example 1 having substantially the same dosage form size thus prepared were 0.41 and 0.25 (units / mg), respectively. Therefore, it was confirmed that, under the conditions in which the same unit was administered, the formulation of Example 2 in which insulin was also encapsulated in the outer layer could be made smaller in size than Comparative Example 1.

[Particle size distribution during re-dispersion of insulin-encapsulated PLGA nanocomposite formulation]
The particle size distribution of the PLGA nanosphere particles when the PLGA nanocomposite preparation obtained in Comparative Example 1 was redispersed in water was measured by a dynamic light scattering method. The results are shown in FIG. As is clear from FIG. 4, the redispersed PLGA nanosphere particles have a particle size distributed in the range of 100 nm to 600 nm, and the average particle size is 215 nm. In addition, when the average particle diameter when the nanocomposite preparation of Example 2 and Comparative Example 2 was redispersed by the same method was measured, it was 217 nm and 210 nm, respectively. I was not able to admit.

  The PLGA nanocomposite preparations prepared in Example 2 and Comparative Examples 1 and 2 were administered via pulmonary to beagle dogs having similar pharmacokinetics to humans, and changes in blood glucose concentration were measured every predetermined time.

  In addition, because the inspiratory volume per individual beagle dog is small and the respiratory timing cannot be controlled, it is judged that pulmonary administration of the preparation powder is difficult, and the nanocomposite preparation is dispersed in a small amount of physiological saline. Then, a method of delivering a mist by an ultrasonic nebulizer into the lungs by spontaneous breathing of a beagle dog through a tracheal insertion catheter was used. When the nanocomposite preparation is transpulmonary administered as a dry powder, it is thought that it absorbs a minute amount of water in the lung and is redispersed into the nanosphere. However, according to the above delivery method, it was redispersed in the nanosphere in advance. Therefore, it was judged that an evaluation result close to the actual one could be obtained.

  Insulin has a solubility and a degree of polymerization that change depending on the pH of the dispersion solvent, and it may affect the absorbability after administration. Therefore, for the purpose of minimizing such fluctuations, insulin has a pH of 6.0. When each preparation was dispersed in physiological saline at the time of use, it was confirmed that there was almost no change in pH. The preparation concentrations of Example 2 and Comparative Example 1 were 1.83 mg / mL and 3.01 mg / mL, respectively, so that the insulin concentration was constant (0.054 mg / mL).

[Method of administration of nanocomposite preparation]
A schematic diagram of the inhalation system is shown in FIG. The dispersion of each preparation was charged into an ultrasonic nebulizer 20 (NE-U17, manufactured by OMRON Healthcare), and mist was generated at a constant flow rate of 1.0 mL / min. This mist was mixed into the main pipe 22 (Teflon (trademark) hose having an inner diameter of 15 mm subjected to antistatic treatment) through which an air flow of 12.0 L / min was circulated by the exhaust pump 21. Part of the mist in the main pipe 22 flows into the bypass pipe 23 (antistatic treated polyethylene hose with an inner diameter of 19 mm) by spontaneous breathing of the beagle dog, and further, the catheter 24 inserted into the bronchus of the beagle dog (with an inner diameter of 8 mm chloride). It was delivered into the lung via a vinyl low-pressure cuffed endotracheal tube.

  In addition, in order to perform stable inhalation with little pressure fluctuation due to spontaneous breathing of the beagle dog, a check valve 25 is provided in the main pipe 22 after branching, and auxiliary air is taken in to maintain a constant exhaust amount of the inhalation system. did. On the other hand, surplus mist that was not inhaled by the beagle dog was collected by a recovery trap 26 and a recovery filter 27 (glass fiber filter GB-100R, manufactured by Advantech Toyo) installed in the main pipe 22.

  Based on human clinical trial data (0.15 units / kg), the dose of insulin was set to 1.5 units with the average body weight of beagle dogs set to 10 kg. With administration using the above inhalation system, it is estimated that 20% of the dosage is delivered into the lungs of beagle dogs, and saline is added to 18.3 mg of the preparation of Example 2 and 30.1 mg of the preparation of Comparative Example 1. In addition, a dispersion having a total amount of 10 mL was used. In Comparative Example 2, as in Comparative Example 1, 30.1 mg of the preparation was added with physiological saline to make a total volume of 10 mL.

  Further, as a comparative control example for evaluating the difference depending on the administration route, an insulin suspension (pH = 6.0, insulin concentration 0.054 mg / mL) by adding physiological saline to 5.4 mg of insulin to make a total volume of 100 mL. ) Was injected subcutaneously in the back of a beagle dog as a subcutaneous injection preparation (0.054 mg = 1.5 units as insulin dose). Three beagle dogs (female, 12-16 months old, body weight 7.25 kg, 11.10 kg, 10.10 kg) were used, and the drugs were administered in the order of subcutaneous insulin injection, Comparative Example 2, Comparative Example 1, and Example 2. . After the administration of each preparation, a 2-week drug holiday was provided.

[Measurement of blood glucose level]
FIG. 6 shows the administration of the insulin preparation and the blood collection pattern. Twenty-five minutes before administration of the insulin preparation, 1.0 mL of blood was collected from the cephalic vein of the beagle dog, and the measured blood glucose concentration was defined as the standard blood glucose concentration (= 100). Thereafter, pentobarbital sodium (Nembutal, manufactured by Dainippon Pharmaceutical Co., Ltd.) is intravenously administered and anesthetized, and the beagle dog (16-18 hours before administration and fasted for 24 hours after administration) is lateralized and the catheter is intubated into the trachea. Connected with inhalation system. After inhaling mist for 10 minutes, blood is collected at regular intervals (0.17, 0.5, 2, 4, 8, 12, 24 hours after the end of administration), and the serum obtained by centrifugation is automatically analyzed by blood. The ratio (blood glucose level) of the blood glucose concentration to the standard blood glucose concentration was measured by an enzyme method (HK-G6PDH method) using an apparatus (TBA-200FR, manufactured by Toshiba). For subcutaneous administration, blood was collected under the same conditions as inhalation administration, and blood glucose level was measured. The measurement results are shown in FIG.

  As is clear from FIG. 7, in the case of administration by subcutaneous injection of insulin, insulin is rapidly absorbed from the subcutaneous, and thus a decrease in blood glucose level is observed from 10 minutes after administration, and 75% of the initial value at 4 hours after administration. The effect disappeared when 8 hours passed after administration.

  On the other hand, in the administration by inhalation of the insulin preparations of Comparative Example 1 and Example 2, the blood glucose level decreased from 10 minutes after administration, as in the case of subcutaneous injection. In the preparation of Comparative Example 1, the blood glucose level immediately after administration decreased faster than subcutaneous injection, and in the preparation of Example 2, the immediate effect was almost the same as that of the subcutaneous injection, both of which were equal to or higher than that of the subcutaneous injection. Was recognized. Furthermore, the effects of the insulin preparations of Comparative Example 1 and Example 2 lasted for 12 hours, and both were significantly more persistent than subcutaneous injection.

  In particular, the blood glucose level from 30 minutes to 10 hours after administration is stable with little fluctuation compared to subcutaneous injection. From these results, it is possible to control the release of insulin in the alveolar region by encapsulating insulin in PLGA nanospheres, and to promote the sustained release of insulin and the level of basal insulin levels by optimizing the formulation. It was suggested that it could contribute to establishment. In addition, in administration by inhalation of the preparation of Comparative Example 2 containing no insulin, almost no change in blood glucose level with time was observed. From this, it was confirmed that the inhalation action of PLGA nanocomposite particles themselves and the PLGA nanosphere as a base do not affect the fluctuation of blood glucose concentration.

[Measurement of mist diameter distribution of nanocomposite preparations]
In order to evaluate the mist diameter inhaled by the beagle dog, a cascade impactor (ITP02-105, manufactured by In Tox Products) is attached to the tip of the catheter 24 of the inhalation system (see FIG. 5) under the same conditions as the inhalation test of the beagle dog. ) Was generated, and a mist of the dispersion liquid of each preparation described above was generated and classified and collected in seven stages (inhalation flow rate 500 mL / min, collection time 5 minutes). A glass fiber filter (GB-100R, manufactured by Advantech Toyo) was used to collect the mist, and the weight of the collected product at each stage was determined from the weight difference of the dried filter before and after the collection, and the aerodynamic diameter was evaluated. . The measurement results are shown in Table 2 and FIG.

  As apparent from Table 2 and FIG. 8, the mist frequency of each preparation recovered after the second stage corresponding to the bronchus (aerodynamic diameter of 4.0 μm) is as high as 74.7 to 87.6%. showed that. Moreover, the average aerodynamic diameter of each preparation was as small as 1.50 to 2.51 μm, and the delivery rate to the bronchus was not significantly different between the preparations. On the other hand, the arrival rate after the fifth stage (1.05 μm in aerodynamic diameter) corresponding to the alveoli is 21.8 to 46.3%, and the delivery rate to the alveoli is that of Comparative Examples 1 and 2. The formulation was almost equivalent, and the formulation of Example 2 was about half the value.

  This is because, in the nanocomposite preparation of Example 2, the set preparation concentration in the dispersion is 1.83 mg / mL, whereas in the nanocomposite preparations of Comparative Examples 1 and 2, 3.01 mg / mL. Since the formulation concentration is 1.64 times higher, the PLGA nanosphere content in the dispersion is increased by that amount, and it is estimated that the tension at the gas-liquid interface is reduced. It is thought that it contributes to the generation of finer mist at the liquid interface and increases the rate of arrival in the alveoli.

  Further, considering the blood glucose level lowering effect in Example 5 based on the above results, the nanocomposite preparation of Comparative Example 1 is more delivered to the alveolar part where insulin is easily transferred into the blood. Therefore, it was estimated that the insulin encapsulated near the surface of the PLGA nanosphere was eluted immediately and absorbed rapidly into the blood. On the other hand, in the nanocomposite preparation of Example 2, the ratio of the mist delivered to the alveoli and later is about half of that in Comparative Example 1, but the insulin impregnated in the outer layer of PLGA nanosphere is adsorbed on the surface of PLGA nanosphere. It was estimated that this adsorbed insulin was accompanied by nanospheres and delivered to the alveoli, contributing to immediate effect.

  Insulin is adsorbed on the surface of PLGA nanospheres. Insulin is added to and dispersed in a physiological saline suspension of PLGA nanospheres not encapsulated with insulin, and after ultrasonic treatment, PLGA nanospheres are isolated by centrifugation. The nanosphere powder obtained by re-dispersing in saline and then freeze-dried was treated by the same method as in Example 3 and analyzed by HPLC, which was confirmed by the detection of insulin.

[Measurement of inhalation volume of nanocomposite preparations by beagle dogs]
In order to evaluate the inhalation amount of the nanocomposite formulation of the beagle dog, a laboratory animal respirator (SN-480-3, manufactured by Shinano Seisakusho) was placed on the catheter 24 of the inhalation system (see FIG. 5) and a glass fiber filter (GB-100R). The inhalation system was operated under the same conditions as the beagle inhalation test. The operating conditions of the ventilator were set to the number of breaths under anesthesia (12 times / minute) and the amount of breathing (208 mL / time) of a beagle dog, and mist was generated. The simulated inhalation operation for 10 minutes was performed, and the estimated inhalation amount of the beagle dog was calculated from the collected weight of the preparation by the glass fiber filter. The inhalation amount of the nanocomposite preparations of Example 2 and Comparative Example 1 was 0.064 mg, respectively. / Min and 0.116 mg / min.

[Pharmacological effects of insulin-encapsulated PLGA nanocomposite preparation]
The total area AAC (area under the time concentration curve; here, the area above the line graph in FIG. 7 and below the blood glucose level 100) showing the blood glucose concentration lowering action is calculated, and this is the beagle dog Using the value (% × time / units) divided by the amount of insulin ingested as an index, the pharmacological effects of inhalation administration of the insulin preparation of Example 2 and Comparative Example 1 and subcutaneous administration as a comparative control example were evaluated. In addition, in order to evaluate the relative pharmacological effect of each preparation, the subcutaneous administration value of insulin was used as a reference (= 100). The results are shown in Table 3.

  From the results of Example 7, since the inhalation rates of the insulin preparations of Example 2 and Comparative Example 1 are 0.064 mg / min and 0.116 mg / min, respectively, effective insulin that the beagle dog ingested in the 10-minute inhalation test The amounts were 0.26 units and 0.29 units, respectively. The relative pharmacological effects obtained by dividing the AAC value obtained from FIG. 7 by these unit values and further indicating the result of subcutaneous injection of insulin as 100 were 347 in Example 2 and 383 in Comparative Example 1, both of which were subcutaneously injected. Greatly exceeded the results. This is because, as described above, PLGA nanospheres are delivered to the alveoli and their residence time is extended, so that the sustained release effect of the drug and the inhibitory effect on enzymatic degradation appear significantly compared to the subcutaneous administration method. it is conceivable that.

  In addition, the insulin preparation of Example 2 showed almost the same pharmacological effect as Comparative Example 1 although the PLGA nanosphere content was lower than that of Comparative Example 1. From the evaluation result of the mist diameter distribution of Example 6, this is the second stage or later corresponding to the bronchi although the frequency of the PLGA nanosphere particles reaching the alveoli is lower in the insulin preparation of Example 2 than in Comparative Example 1. Therefore, it is considered that the insulin impregnated in the outer layer of the PLGA nanospheres flies as a slightly large mist in the dispersion and deposits in the bronchi and bronchioles.

  Although these sites are thought to have a slower absorption rate than the alveoli, the reaching action site of insulin is enlarged as compared with Comparative Example 1, and thus the pharmacological effect is obtained by transferring from a wide range of tissue sites into the blood. Is estimated to have improved. Furthermore, since the insulin preparation of Example 2 can be formulated with a high content of insulin in the outer layer of the PLGA nanosphere, it has a high pharmacological effect and can be made smaller in dosage form than Comparative Example 1, and can be applied to clinical trials. This is a useful particle design method for realizing a practical transpulmonary dose.

[Preparation of calcitonin-encapsulated PLGA nanospheres]
250 mg of calcitonin (manufactured by Biosciences) was dissolved in 50 mL of methanol. After dissolving 5 g of lactic acid / glycolic acid copolymer (PLGA: PLGA7520 manufactured by Wako Pure Chemical Industries), which is a biocompatible polymer, in 100 mL of acetone, the methanol solution of calcitonin was added and mixed. This mixed solution was dropped at a constant rate (4 mL / min) with stirring at 40 ° C. and 400 rpm in 500 mL of a 2 wt% polyvinyl alcohol (PVA: manufactured by Kuraray Co., Ltd.) aqueous solution prepared as a poor solvent. A suspension of calcitonin-encapsulated PLGA nanospheres was obtained by diffusion into the solvent.

  Subsequently, stirring was continued at 40 ° C. and 100 rpm under reduced pressure, and after removing the organic solvent for 2 hours, excess PVA on the calcitonin-encapsulated PLGA nanosphere surface was removed by centrifugation (20,000 rpm, −20 ° C., 20 minutes). After removing the supernatant, the precipitate was recovered. The precipitate was resuspended in purified water and centrifuged again to remove excess polyvinyl alcohol. The supernatant was removed, and the resulting precipitate was resuspended in 150 mL of purified water, and then freeze-dried at -45 ° C. for 1 day to obtain a calcitonin-encapsulated PLGA nanosphere powder with good redispersibility in water. .

[Preparation of calcitonin-encapsulated PLGA nanocomposite preparation]
In 150 mL of purified water, 1.4 g of trehalose (manufactured by Hayashibara) was dissolved, and 1.4 mg of calcitonin (manufactured by Biosciences) was further dissolved. This calcitonin solution was added to a suspension obtained by resuspending 700 mg of the nanosphere powder obtained in Example 9 in 100 mL of purified water, mixed uniformly, and then freeze-dried at −45 ° C. overnight. Thus, a PLGA nanocomposite preparation of the present invention in which calcitonin was encapsulated in the inner and outer layers of the nanoparticles was obtained.

  Using a calcitonin solution in which 1.4 g of trehalose (manufactured by Hayashibara) was dissolved in 150 mL of purified water and 2.8 mg of calcitonin (manufactured by Biosciences) was dissolved, the inner and outer layers of the nanoparticles were obtained in the same manner as in Example 10. A PLGA nanocomposite preparation of the present invention in which calcitonin was encapsulated was obtained.

  Using a calcitonin solution in which 1.4 g of trehalose (manufactured by Hayashibara) was dissolved in 150 mL of purified water and 4.2 mg of calcitonin (manufactured by Biosciences) was dissolved, the inner and outer layers of the nanoparticles were obtained in the same manner as in Example 10. A PLGA nanocomposite preparation of the present invention in which calcitonin was encapsulated was obtained.

Comparative Example 3

  In place of the calcitonin solution of Example 10, a trehalose aqueous solution in which 1.4 g of trehalose (manufactured by Hayashibara) was dissolved in 150 mL of purified water was used, and calcitonin was enclosed only inside the nanoparticles by the same method as in Example 10. A PLGA nanocomposite formulation was obtained.

[Evaluation of particle size distribution and calcitonin content during re-dispersion of calcitonin-encapsulated PLGA nanocomposite]
When the nanocomposite formulations obtained in Examples 10 to 12 and Comparative Example 3 were redispersed in water and the average particle size was measured by the dynamic light scattering method in the same manner as in Example 4, all the formulations were from 215 nm to The range was 230 nm, and the particle size distribution was not significantly different among the preparations. Moreover, the calcitonin content rate of each formulation was quantified in the same procedure as Example 3 using HPLC. Table 4 shows PLGA nanospheres in each preparation, the outer layer (trehalose), the weight ratio of calcitonin contained in the inner and outer layers, and the amount of calcitonin (units / mg) in each preparation.

  As is apparent from Table 4, the weight ratio of cartonin in each preparation was 0.20% by weight, 0.26% by weight, 0.33% by weight, and 0.002% in the order of Examples 10-12 and Comparative Example 3, respectively. It was 13% by weight. In the preparations of Examples 10 to 12, which were combined using a solution obtained by dissolving calcitonin in an aqueous trehalose solution, calcitonin was also encapsulated in the outer layer of trehalose. On the other hand, in the preparation of Comparative Example 3 complexed with an aqueous trehalose solution, calcitonin was encapsulated only inside the PLGA nanosphere. In Examples 10 to 12, the amount of calcitonin charged was set so that calcitonin was encapsulated at a ratio of 2: 1, 1: 1, and 2: 3 in the inner and outer layers of the nanosphere. The ratio was almost consistent with the theoretical value.

  In any nanocomposite preparation, the weight ratio of PLGA nanospheres to trehalose was approximately 1: 2. The calcitonin concentrations in the nanocomposite preparations of Examples 10 to 12 and Comparative Example 3 thus prepared were 14.0, 18.62, 23.26, and 9.32 (units / mg), respectively. Therefore, under the conditions in which the same unit of calcitonin is administered, the dosage forms of Examples 10-12 in which calcitonin is also encapsulated in the outer layer can be reduced in size in the order of Examples 10-12 as compared with Comparative Example 3. .

[Pharmacological effects of calcitonin-encapsulated PLGA nanocomposite preparation]
The pharmacological effects of the calcitonin-encapsulated PLGA nanocomposite preparations prepared in Examples 10 to 12 and Comparative Example 3 were evaluated by rats. The evaluation procedure is shown below.
1) Rat trachea and jugular vein were exposed 3 hours before administration.
2) Blood was collected 0.5 hours before administration, and this blood calcium concentration was used as a reference (= 100).
3) A No. 7 cannula was inserted into the bronchus, and the nanocomposite preparations prepared in Examples 10 to 12 and Comparative Example 3 were filled in the bronchi so that the dose of calcitonin was 100 units per kg body weight of the rat. The attached tube was attached. A three-way stopcock was attached to this tube, and a syringe was further attached. The nanocomposite particles were administered into the rat lungs by injecting air into a syringe, compressing it, and then releasing the three-way stopcock.
4) After administration, blood was collected every 0.5, 1, 2, 4, 8, 12, and 24 hours. Each blood was centrifuged, and the calcium concentration in the obtained serum was measured using an automatic blood analyzer. The ratio of blood calcium concentration at each time (blood calcium level) to the standard blood calcium concentration was calculated.
The results are shown in FIG.

  When the preparation of Comparative Example 3 containing no calcitonin in the outer layer was administered, the blood calcium level gradually decreased until 4 hours after administration, whereas the preparations of Examples 10-12 in which the outer layer was impregnated with calcitonin were: An immediate effect was observed within 0.5 to 2 hours after administration. It was considered that this was because the outer layer calcitonin was absorbed more quickly from the alveoli with higher absorption efficiency.

  As for persistence, the preparation of Comparative Example 3 was observed to have a blood calcium level lowering effect for 12 hours after administration, and the preparations of Examples 10 to 11 were confirmed to have the same degree of sustainability as Comparative Example 3. In contrast, the formulation of Example 12 returned to a blood calcium level comparable to that before administration 8 hours after administration. This is because as the amount of nanospheres encapsulating calcitonin increases, calcitonin is less susceptible to enzymatic degradation, and PLGA nanospheres that remain sufficiently long in the absorption site gradually release calcitonin encapsulated by hydrolysis over a long period of time. It was thought that it was because it was absorbed inside. Further, when the pharmacological effect (AAC; area under the time concentration curve) of the preparation of Comparative Example 3 is set to 100, the relative pharmacological effects of the preparations are 85, 70, and 45 in the order of Examples 10 to 12, respectively. It was.

  From the above results, the preparations of Examples 10 and 11 in which the mixing ratio of calcitonin encapsulated in nanospheres and calcitonin in the outer layer was designed to be 1: 0.5 to 1: 1 were comparative examples in which the outer layer was not impregnated with calcitonin. Compared with the preparation of No. 3, it was proved that it had the same immediate effect and sustainability, suggesting that the particle design is advantageous for miniaturization of the dosage form.

Pharmaceutical formulations of the present invention comprising a peptide hormone encapsulated composite particles peptide hormone loaded nanoparticles to each other formed by complexing increases the immediate by peptide hormone encapsulated in the outer layer of the composite particles was and enclosed inside the nanoparticles Since the persistence can be increased by the peptide hormone, it is possible to provide a pharmaceutical preparation having a balance between immediate effect and sustainability. In addition, since peptide hormone is encapsulated in the outer layer of the composite particle in addition to the inside of the nanoparticle, the peptide hormone content in the preparation can be increased and the dosage form can be downsized. In particular, it is suitable for transpulmonary administration formulations using insulin, which is a therapeutic agent for diabetes, and calcitonin, which is a therapeutic agent for osteoporosis, as peptide hormones.

  In addition, as a material that forms nanoparticles, biocompatible polymers that are low in irritation and toxicity to the living body and are decomposed and metabolized after administration are used, ensuring safety to the human body and the efficacy of peptide hormones. Can be stored for a long period of time, and the peptide hormone can be released gradually by degrading the biocompatible polymer. In particular, it is suitable when PGA, PLA, or PLGA is used as the biocompatible polymer.

  Further, if peptide hormone-containing nanoparticles are complexed using a binder, peptide hormones can be encapsulated in the outer layer of the nanoparticles by a simple method. Furthermore, it becomes an agglomerated particle that is easy to handle at the time of filling into a container and is redispersible at the time of use. In particular, when sugar alcohol is used as the binder, the dispersibility and heat resistance of the composite nanoparticles can be improved, and re-leakage of the peptide hormone once encapsulated in the nanoparticles to the particle surface can be prevented.

  In addition, the method for producing a pharmaceutical preparation of the present invention in which nanoparticles are formed using a spherical crystallization method, the concentration of an aqueous polyvinyl alcohol solution to which a mixed solution of a peptide hormone solution and an organic solvent solution of a biocompatible polymer is added is 0. .5% by weight or less eliminates the need for washing the nanoparticles by centrifugation, as in the case of using a high-concentration polyvinyl alcohol aqueous solution. Become.

  On the other hand, when forming a nanoparticle using a high-concentration polyvinyl alcohol aqueous solution and then providing a step of removing the excess polyvinyl alcohol by washing the nanoparticle by centrifugation or the like, the peptide hormone encapsulated in the nanoparticle The encapsulation rate of can be stabilized. At this time, by setting the concentration of the polyvinyl alcohol aqueous solution to 0.1 wt% or more and 10 wt% or less, the viscosity of the poor solvent can be suppressed to an extent that does not affect the diffusion of the good solvent.

  In addition, since acetone, ethanol or the like having little influence on the human body and the environment is used as a good solvent, it is highly safe as a raw material for pharmaceutical preparations to be directly administered to the human body. If the compounding step is performed by freeze drying or spray drying fluidized bed granulation, the nanoparticles can be compounded satisfactorily and efficiently.

These are the schematic diagrams which show the structure of the peptide hormone containing nanocomposite used for the pharmaceutical formulation of this invention. These are the schematic diagrams which show an example of the composite process of the nanoparticle by a spray-drying type fluidized-bed granulation method. FIG. 2 is a schematic diagram showing the structure of peptide hormone-containing nanoparticles having a core-shell structure. These are the graphs which show the result of having measured the particle size distribution of the PLGA nanosphere particle | grains at the time of re-dispersing the PLGA nanocomposite formulation in water in Example 4 by the dynamic light scattering method. FIG. 1 is a schematic view of an inhalation system that delivers a mist of a dispersion of a nanocomposite formulation into the lungs by spontaneous breathing in a beagle dog. These are the figures which show the administration of the insulin formulation in Example 5, and a blood collection pattern. These are the graphs which show the measurement result of the blood glucose level in Example 5. These are the graphs which show the result of having measured the aerodynamic diameter of the mist which the beagle dog inhales in Example 6. These are the graphs which show the measurement results of blood calcium level in Example 14.

Explanation of symbols

1 Nanocomposite 2 Biocompatible nanoparticles (nanospheres)
3 Binder (outer layer)
4 Peptide Hormone 5 Powder Processing Device 6 Fluidized Bed Space 8 Spray Nozzle 8a Spray Zone 9a Bag Filter 9b Back Filter 10 Composite Particle 11 Composite Particle 12 Hydrophobic Polymer Block 13 Hydrophilic Polymer Block 14 Block Copolymer 15 Shell Portion 16 Core 20 Ultrasonic nebulizer 21 Exhaust pump 22 Main pipe 23 Bypass pipe 24 Catheter 25 Check valve 26 Collection trap 27 Collection filter

Claims (11)

Polylactic acid, polyglycolic acid, or is formed in one of a lactic acid-glycolic acid copolymer, biocompatible nanoparticles together encapsulating peptide hormone therein is complexed by the binding agent consisting of a sugar alcohol or sucrose In addition, a pharmaceutical preparation comprising peptide hormone-encapsulated composite particles formed by encapsulating peptide hormone in an outer layer formed of the binder. The pharmaceutical preparation according to claim 1, wherein the peptide hormone is insulin.   The pharmaceutical preparation according to claim 1, wherein the peptide hormone is calcitonin. The pharmaceutical preparation according to any one of claims 1 to 3, which is used for transpulmonary administration . A peptide hormone in which the peptide hormone is encapsulated in the biocompatible polymer by adding a mixed solution of at least a peptide hormone solution and a solution in which a biocompatible polymer is dissolved in an organic solvent to an aqueous polyvinyl alcohol solution A nanoparticle formation process for producing a suspension of encapsulated nanoparticles;
A solvent distillation step of distilling off the organic solvent from the suspension of peptide hormone-encapsulated nanoparticles;
And decryption steps of complexing the binding agent comprising the peptide hormone containing nanoparticles among which the organic solvent is distilled off from the peptide hormone include sugar alcohols or sucrose,
A method for producing a pharmaceutical preparation containing peptide hormone-encapsulated composite particles, comprising : 6. The method for producing a pharmaceutical preparation according to claim 5, wherein the complexing step is performed by lyophilization . The method for producing a pharmaceutical preparation according to claim 5 , wherein the complexing step is performed by a spray drying fluidized bed granulation method . The method for producing a pharmaceutical preparation according to any one of claims 5 to 7, wherein the polyvinyl alcohol concentration in the aqueous polyvinyl alcohol solution is less than 0.5 wt% . The pharmaceutical according to any one of claims 5 to 8 , further comprising a removal step of removing polyvinyl alcohol from the suspension of peptide hormone-encapsulated nanoparticles after the solvent distillation step. Preparation method of the preparation. The method for producing a pharmaceutical preparation according to claim 9 , wherein the polyvinyl alcohol concentration in the aqueous polyvinyl alcohol solution is 0.1 wt% or more and 10 wt% or less . The method for producing a pharmaceutical preparation according to any one of claims 5 to 10, wherein the organic solvent contains at least acetone .

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