JP5569783B2 - pH-responsive drug sustained-release carrier and method for producing the same - Google Patents

Description

The present invention relates to a pH-responsive drug sustained-release carrier using water-soluble elastin and a method for producing the same.

The drug delivery system (DDS) is intended to “activate drugs in the required amount at the required place when needed” for efficient use of drugs, reduction of side effects, and improvement of patient QOL. It is also expected to play a very important role. Currently, technological development is progressing in various administration forms such as oral, intravenous injection, transdermal, and transpulmonary.

For example, pulmonary administration is a route that can be directly administered to lung cancer, pulmonary infection, allergic pulmonary disease, etc., and can increase the effectiveness of the drug and reduce systemic exposure. Exfera, an insulin powder inhaler from Pfizer, is a non-porous spray-dried product in which human insulin optimized for a particle size of about 3 μm is packaged as a formulation with mannitol, glycine and sodium citrate, respectively. It is. It was launched in 2006 as the world's first systemic inhaler, but was discontinued in just two years. This highlighted the problems associated with pulmonary administration, such as high cost, difficulty in proper use of the device, and ensuring dispersibility of the formulation.

Transdermal absorption is an expected dosage form because of its large application area. This is the first blood circulation, and can bypass the liver, so that the medicinal ingredients are not broken and can be used as effectively as intravenous injection, without pain like injection. A typical example is nitroderm TTS. This can prevent an angina attack by applying a patch containing nitroglycerin that spreads the coronary artery to the chest and arms. However, since there is a time delay in the drug's skin absorption due to resistance by the stratum corneum, there is no immediate effect like injection, so it is effective in preventing seizures, but has little effect after the seizure occurs. In addition, since an additive for promoting skin absorption such as a permeation enhancer is used in the percutaneous absorption preparation, a person with sensitive skin may receive skin irritation by these substances.

All of the above administration routes are considered to have much room for improvement from the viewpoint of improvement in patient QOL and economical cost. Among them, oral administration is the most widely used administration route because of its high convenience and has the advantage of being safe and not costly. It is considered the most desirable route of administration compared to the route of administration. Oral administration has the advantage that it often causes problems with administration by injection of insulin and the like, and hyperinsulinemia due to eradication is less likely to occur. However, there are also problems such as controlled release in the digestive tract, protection from digestive enzymes, and improved absorption. It is necessary to eliminate such problems and to prepare a carrier that has the property of being able to reach the small intestine without being decomposed in the stomach and being decomposed in the small intestine to absorb the drug.

On the other hand, the present inventors have focused on elastin, a biopolymer, and have studied its application as a drug carrier. Elastin is a major constituent of animals such as the aorta, ligaments and skin of animals. Elastin is insoluble but can be solubilized by acid or alkali treatment. The solubilized elastin causes a phenomenon called coacervation in an aqueous solution. This is a reversible series of solutions in which an elastin aqueous solution becomes cloudy when heated to near body temperature, and when left as it is, it is separated into two layers of a transparent equilibrium solution and a pale yellow highly viscous coacervate, and when cooled, it returns to the original homogeneous solution. This refers to a phenomenon (see Patent Documents 1 and 2). The present inventors paid attention to the coacervate droplets formed during the coacervation, and succeeded in producing more stable nanoparticles by irradiating them with γ rays. The inventors have also sought to apply and develop such nanoparticle production technology.

JP 2007-45722 A JP 2009-219422 A

The object of the present invention is to further develop the technology of coacervate droplets of water-soluble elastin, the ability to reach the small intestine as it is without being decomposed in the stomach, and the property that the drug is released and absorbed in the small intestine The object is to provide a carrier having the property of being absorbed into the small intestine as it is.

The present inventors prepared nanoparticles having different particle sizes by irradiating water-soluble elastin coacervate droplets under various conditions, and examined the influence of the difference in γ-ray irradiation conditions. The range of particle sizes obtained by this method was 10 to 1000 nm. Also, acylation of water-soluble elastin, for example, acetylation, shifts the isoelectric point to the strong acid side, and produces N-acetyl water-soluble elastin that self-assembles under stronger acid conditions and is stable by γ-ray irradiation Nanoparticles were obtained. Then, using the nanoparticles, particle size measurement and dye sustained release test were performed under the conditions of pH 1.2 and pH 7.4, and application to a pH-responsive drug sustained release carrier was studied, and the present invention was completed. .

That is, the present invention relates to a pH-responsive drug sustained-release carrier comprising nanoparticles obtained by crosslinking N-acyl water-soluble elastin by γ-ray irradiation , the particle size of which changes in response to pH change, and It is a pH-responsive drug sustained-release carrier comprising nanoparticles obtained by crosslinking N-acyl water-soluble elastin by γ-ray irradiation , which regulates drug release in response to pH change.

Another embodiment of the present invention relates to a method for producing such a carrier, wherein a coacervate droplet of N-acyl water-soluble elastin is irradiated with radiation, for example, γ-rays to form the droplet into nanoparticles. This is a method for producing a pH-responsive drug sustained-release carrier.

Intravenous administration is painful for patients, and recently, various administration techniques such as oral, transdermal, and transpulmonary have been developed. Oral administration is administered in the same manner as normal drug intake. It is the most desired administration method because it is convenient and highly convenient. The pH-responsive drug sustained-release carrier of the present invention is a carrier utilizing biodegradability of a biopolymer material, a simple and sterilized nanoparticle production method using a γ-ray irradiation device, and can be administered orally. It can greatly contribute to the medical field in terms of improving the patient's QOL and economical cost. In addition to oral administration, the pH-responsive drug sustained-release carrier of the present invention can be applied to administration techniques such as intravenous injection, percutaneous, and transpulmonary that require sustained drug release corresponding to pH response.

The figure which shows the particle size of the nanoparticle obtained by gamma-ray irradiation on the conditions which changed the density | concentration. The figure which shows the particle size of the nanoparticle obtained by gamma-ray irradiation on the conditions which changed the temperature increase rate. The figure which shows the particle size of the nanoparticle obtained by carrying out the gamma ray irradiation on the conditions which changed irradiation amount. The figure which shows the turbidity curve of the acetyl water-soluble elastin under different pH conditions. The figure which shows the result of the pigment | dye sustained release from the nanoparticle measured on condition of pH1.2 and 7.4. The figure which shows the result of the pigment | dye sustained release from the nanoparticle at the time of changing from pH1.2 to 7.4.

In the present invention, using water-soluble elastin derived from a living body, first, under the same conditions as in the acidity of the stomach, the drug shrinks by self-assembly and does not release the drug. Designed to swell by releasing self-assembly and to release drug, water-soluble elastin is chemically modified to produce N-acyl water-soluble elastin, and formed by the resulting N-acyl water-soluble elastin Nanoparticles are produced from coacervate droplets by a radiation irradiation technique such as γ-rays. The obtained N-acyl water-soluble elastin nanoparticles do not release a drug under the same acidity as in the stomach, but are a carrier having a property of releasing the drug in a weak alkali similar to that in the small intestine. In addition, when this carrier is orally administered, it swells under the acidity of the stomach, reaches the small intestine without undergoing enzymatic degradation by pepsin, etc., and then swells under a weak alkali in the small intestine, thereby causing an enzyme such as elastase. It is a carrier that has the property of undergoing decomposition. Furthermore, this carrier is also a carrier having a property that a part of the carrier is absorbed in the small intestine as nanoparticles before being decomposed in the small intestine.

The N-acyl water-soluble elastin of the present invention can be obtained by N-acylating part or all of the primary amine and the secondary amine contained in the high molecular weight water-soluble elastin molecule. The N-terminus of water-soluble elastin and the amino groups of the side chains of amino acid residues such as lysine and arginine are acylated. N-acylation includes N-formylation, N-acetylation, N-benzoylation, etc., and N-acetylation is preferred. Moreover, you may use a urethane type and an alkyl type.

Various methods and means for obtaining water-soluble elastin have been proposed. Preferable is the following method proposed by the present inventor (see Japanese Patent Application Laid-Open No. 2007-45722 (Patent Document 1)).

In the first method, insoluble elastin is obtained by removing collagen and other unnecessary proteins from animal living tissue, and then this insoluble elastin is immersed and dissolved in a solubilizing solution such as oxalic acid, To manufacture. Collagen and other unwanted protein removal treatment is an alkaline solution containing at least one of sodium hydroxide, potassium hydroxide, calcium hydroxide, and barium hydroxide, and sodium hydroxide added to the alkaline solution. It is performed by immersing the animal living tissue for 10 to 20 minutes in an alkaline solution in which the total amount of potassium hydroxide, calcium hydroxide and barium hydroxide is 90 to 105 ° C at 0.05 to 0.15 mol per liter. Is preferred. In addition, when removing collagen and other unnecessary proteins, prior to treatment with an alkaline solution, animal biological tissue is placed in a salt solution containing at least one of sodium chloride, potassium chloride, calcium chloride, and barium chloride. It is also preferable to perform an immersion treatment (pretreatment) for immersion.

The animal living tissue is not particularly limited, but it is preferable to use a ligament or aortic blood vessel obtained from mammals such as pigs, horses, cows, sheep, etc. in terms of a high content of elastin. Alternatively, fish arterial spheres containing a large amount of elastin may be used. Animal biological tissue is preferably homogenized first using a homogenizer. For homogenization, any animal tissue such as a mixer or meat chopper can be shredded, and preferably a tool capable of shredding into 3 mm square or less, more preferably paste-like. It is preferable that the size of the shredded animal biological tissue is smaller because the efficiency of removing collagen and other unnecessary proteins can be increased. The homogenized animal biological tissue may be degreased by boiling it with hot water or a hot dilute alkaline aqueous solution, or treating it with an organic solvent, for example.

Examples of the solubilized solution include oxalic acid, formic acid, acetic acid, succinic acid, malic acid, tartaric acid, citric acid, benzoic acid, betaine, difluoroacetic acid, trifluoroacetic acid, phosphoric acid, sulfamic acid, perchloric acid, and trichloroacetic acid. An acidic solution containing at least one of them is used. The total amount of acid in this acidic solution is preferably 0.1 to 0.5 mol per liter, and the liquid temperature is preferably 90 to 105 ° C.

The solubilizing solution may also be an alkaline solution containing at least one of sodium hydroxide, potassium hydroxide, calcium hydroxide, and barium hydroxide. The total amount of sodium hydroxide, potassium hydroxide, calcium hydroxide and barium hydroxide added to this alkaline solution is 0.05 to 0.5 mol per liter, and the alkaline solution has a liquid temperature of 90 to 105 ° C. It is preferable to do this.

The second method includes a pretreatment step including at least one of removal treatment of unnecessary parts of animal biological tissue, degreasing treatment of animal biological tissue, and shredding treatment of animal biological tissue; Alkaline dissolution process of immersing animal biological tissue in alkaline solution and filtering, alkali dissolution process is repeated a predetermined number of times, filtrate recovery process to obtain filtrate containing water-soluble elastin by filtration, and water-soluble elastin is generated from filtrate The water-soluble elastin production step is sequentially performed to produce water-soluble elastin. The alkali used in the alkali dissolution step is preferably any one of sodium hydroxide, potassium hydroxide, calcium hydroxide, barium hydroxide, or a mixture.

This operation is different from the first method in which collagen and other unwanted proteins are removed from the tissue to obtain insoluble elastin, and then the insoluble elastin is solubilized to obtain water-soluble elastin. This is a method for directly obtaining water-soluble elastin without obtaining insoluble elastin. That is, degreased and shredded animal living tissue is immersed for 10 to 20 minutes in an alkaline solution of 0.05 to 0.15 mol per liter at 90 to 105 ° C. to remove collagen and unnecessary proteins other than elastin. The treated tissue was then obtained, and the treated tissue was then added at 0.05 to 0.5 mol per liter (higher concentration of alkaline solution) in an alkaline solution at 90 to 105 ° C. for 60 to 240 minutes (longer time). This is a method of obtaining water-soluble elastin by immersing and dissolving.

As described above, the water-soluble elastin obtained by the first or second method is then made into a water-soluble elastin having a molecular weight of about 8000 or more by removing the low-molecular-weight one by, for example, dialysis. The Particularly preferably used is a high molecular weight water-soluble elastin having an average molecular weight of about 200,000.

In the production of the N-acetyl water-soluble elastin of the present invention, first, part or all of the primary amine and the secondary amine contained in the water-soluble elastin molecule are N-acylated, preferably N-acetylated. Thus, N-acetylated water-soluble elastin is obtained. Among amino acid residues constituting elastin, examples of amino acids having a reactive primary amine or secondary amine (basic amino acids) include lysine, arginine and histidine. In the molecule of high molecular weight water-soluble elastin, The primary amine contained in includes a terminal amino group.

In the present invention, some or all of the primary amine and secondary amine contained in the water-soluble elastin molecule are preferably N-acetylated by an acetylating reagent such as acetic anhydride. The degree of conversion is preferably 95% or more as a modification rate represented by the following formula.
Modification rate (%) = (1−B / A) × 100
A represents a value obtained by subtracting the average value of the absorbance of the blank from the average value of the absorbance (wavelength 400 nm) of the water-soluble elastin. B represents a value obtained by subtracting the average value of the absorbance of the blank from the average value of the absorbance (wavelength 400 nm) of the N-acetylated water-soluble elastin.

In the present invention, N-acetyl water-soluble elastin is obtained by acylating, for example, acetylating water-soluble elastin to shift the isoelectric point to the strong acid side and self-assemble under stronger acid conditions. Next, a coacervate droplet of N-acetyl water-soluble elastin is prepared, and a stable nanoparticle is obtained by irradiating the droplet with radiation such as an electron beam or γ-ray. The obtained nanoparticles can be used for, for example, particle size measurement under a condition of pH 1.2 and pH 7.4 and a dye sustained release test.

The obtained N-acetyl water-soluble elastin was adjusted with a solvent of a broad buffer solution (pH 1.0 to 11.0) in a concentration range of 0.1 to 60 mg / ml, and the temperature rising rate was 0.1 to 40 ° C. Coacervate droplets can be formed by heating at / min. And the production | generation of a coacervate droplet can be confirmed by measuring the turbidity of the aqueous solution of N-acetyl water-soluble elastin in the temperature range of 5-80 degreeC.

The coacervate droplets obtained as described above may be irradiated with radiation, for example, γ rays by a Co-60 γ ray irradiation apparatus. As irradiation conditions, an irradiation temperature of 20 to 80 ° C. and an irradiation amount of about 5 to 60 kGy are appropriate.

Hereinafter, the present invention will be described in detail by way of examples.

[Preparation of water-soluble elastin from pig]
1) Isolation of porcine-derived insoluble elastin NaCl-soluble and NaOH-soluble insoluble proteins were extracted from porcine aortic defatted tissue according to the following procedure.

Using porcine aortic defatted tissue (living tissue), remove the unnecessary part by scraping off the low elastin content parts such as fat and muscle attached as pretreatment with a blade etc., and then living tissue Was shredded by homogenizing using a homogenizer. The homogenized living tissue was degreased by adding 1M sodium chloride of about 10 times the weight and stirring at room temperature for 1 hour. This operation was repeated 5 times, then washed with distilled water, and drained by centrifugation (3000 rpm, 5 minutes).

Add about 10 times volume (10 ml per gram) of 0.1N sodium hydroxide aqueous solution to the weight of the defatted biological tissue as described above, stir at 100 ° C. for 15 minutes, collagen other than elastin and unnecessary protein The process which removes was performed. Then, the biological tissue and the alkaline solution were separated. For the separated alkaline solution, the total protein was quantified by, for example, the burette method, and this operation was repeated 5 times until the total protein amount contained in the alkaline solution was 0.1 mg / mL or less. Thereafter, acetic acid was added for neutralization, washing was performed by centrifugation (5000 rpm, 20 minutes), and the residue was dried to obtain insoluble elastin.

2) Preparation of porcine-derived water-soluble elastin 0.5N sodium hydroxide having a volume 10 times the dry weight of porcine-derived insoluble elastin was added and stirred at 100 ° C. for 30 minutes. After the reaction, the solution was quickly cooled with ice and neutralized with acetic acid. Then, it dialyzed for 1 week using the dialysis membrane which fractionates molecular weight 6,000-8,000 or more. Then, it lyophilized | freeze-dried and obtained high molecular weight pig origin water-soluble elastin.

[Example 1]
Preparation of nanoparticles of water-soluble elastin γ-ray irradiation was performed using a Co-60 γ-ray irradiation facility. The irradiation conditions are 10 kGy and 30 kGy, the water-soluble elastin concentration is 2, 5, 10 mg / ml, the heating rate is 2.0, 10.0, 20.0 ° C / min, and the irradiation temperature is 60 ° C. It was. These conditions were determined from the results of turbidity measurement. NICOMP380ZLS was used to measure the particle size of the nanoparticles after γ-ray irradiation. Each sample was placed in a 3 ml cell and measured for 2 minutes × 3 times. The measurement temperatures are 4 ° C, 37 ° C and 60 ° C.

Figure 1 (a) Effect of water-soluble elastin concentration, Figure 2 (b) Effect of temperature rise rate, Figure 3 (c) The particle size (measured at 4 ° C) of nanoparticles irradiated with γ-rays under different conditions. ) Effect of γ-ray irradiation amount. From FIG. 1 to FIG. 3 , it was confirmed that the difference between the concentration and the heating rate greatly affects the particle size. In particular, the particle size at a concentration of 10 mg / ml was nearly five times larger than the particle size at 2 mg / ml. This suggests that in order to produce particles with a small particle size, it is necessary to perform γ-ray irradiation under conditions where the concentration is low and the heating rate is high.

[Example 2]
Preparation of N-acetyl water-soluble elastin nanoparticles

1) Preparation of N-acetyl water-soluble elastin TFE was added to and dissolved in the porcine-derived water-soluble elastin obtained above, and then pyridine and acetic anhydride were added in this order, followed by stirring and reaction at 4 ° C for 24 hours. After the reaction, a color reaction with ninhydrin was performed to confirm that the amino group was acetylated almost quantitatively. Thereafter, the solvent was removed by concentration under reduced pressure. Then, it was redissolved in distilled water, dialyzed at 4 ° C. for 1 week, and freeze-dried to obtain N-acetyl water-soluble elastin. The yield of N-acetyl water-soluble elastin was 80.6%, and the N-acetylation modification rate was 96.8%.

2) Turbidity measurement of N-acetyl water-soluble elastin (coacervate droplet adjustment)
The N-acetyl water-soluble elastin obtained above was adjusted with a solvent having a concentration of 1.0 mg / ml and a broad buffer solution (pH 2.0 to 8.0), respectively, and a spectrophotometer with a Peltier temperature controller (JASCO: V-560) was used to measure turbidity under a nitrogen stream. The wavelength was 400 nm, the temperature range was 5 to 65 ° C, and the temperature increase rate was 0.5 ° C / min.

The turbidity curves at different pH conditions of N- acetyl-soluble elastin produced are shown in FIG. The produced N-acetyl water-soluble elastin started turbidity at around 20 ° C. in a strong acid having a pH of 2 to 4, and reached a peak at around 37 ° C. which is a physiological temperature. On the other hand, it was confirmed that turbidity started from around 40 ° C. at pH 5-8.

3) γ-ray irradiation of N-acetyl water-soluble elastin γ-ray irradiation was performed using a Co-60 γ-ray irradiation facility. The irradiation conditions were an irradiation amount of 10 kGy, an N-acetyl water-soluble elastin concentration of 2.0 mg / ml, a temperature increase rate of 20 ° C./min, and an irradiation temperature of 60 ° C. Nanoparticles were generated by TEM observation. That is, 5 μl of a suspension (1.0 mg / ml) of the obtained N-acetyl water-soluble elastin nanoparticles (AcENPs) was placed on a grid, and the excess sample was blotted with filter paper and left in front of the lamp for several tens of seconds. And dried and observed by TEM. As a result, it was confirmed from the TEM image of cENPs prepared by γ-ray irradiation (conditions: concentration 2 mg / ml, temperature rising rate 20 ° C./min, irradiation dose 10 kGy) that AcENPs were spherical nanoparticles. .

[Example 3]
1) Dye sustained release test of N-acetyl water-soluble elastin nanoparticles

The dye sustained release test was conducted in the following two ways.
(1) 300 μl of 0.5 mM · PonceauBS was added to 1 mg of AcENPs and allowed to stand at 4 ° C. for 24 hours and then at 37 ° C. for 24 hours to carry the dye. The supernatant was collected by centrifugation (10,000 rpm, 5 min, 37 ° C.), and 300 μl of 0.1 M HCl (pH 1.2) or PBS (pH 7.4) was added. Subsequently, the mixture was shaken at 37 ° C. for 30 minutes, centrifuged, and the supernatant was collected. Then, 300 μl of 0.1M HCl (pH 1.2) or PBS (pH 7.4) was added again. The same operation was repeated until the sustained release was completed, and the collected supernatant was subjected to absorbance measurement (505 nm) using a spectrophotometer.

(2) After loading the dye in the same manner as in 1) above, 300 μl of 0.1 M HCl (pH 1.2) was added. The mixture was shaken at 37 ° C. for 30 minutes, and then centrifuged. The supernatant was collected, and 300 μl of 0.1M HCl (pH 1.2) was added again. This operation was further repeated 3 times, and then the solvent was changed to PBS (pH 7.4) and the same operation was repeated 8 times. The collected supernatant was subjected to absorbance measurement (505 nm) using a spectrophotometer.

FIG. 5 shows the results of the sustained release of the dye measured under the conditions of pH 1.2 and 7.4 because the stomach pH is pH 1-2 and the small intestine pH is 7-8. Although the sustained release of the dye was less than 10% at pH 1.2, it was confirmed that the release was nearly 80% at pH 7.4.

Next, the actual digestive organs (pH 1-2 in the stomach, pH 7-8 in the small intestine, that is, pH 1.2 to 7.4 assuming a sustained release in the process of reaching the small intestine through the stomach). 6 shows the result of the sustained release of the dye when the pH is changed to Fig. 6. Similar to Fig. 5 , the dye is hardly released at pH 1.2, but when the pH is changed to 7.4, rapid sustained release starts. confirmed.

2) Particle size measurement of N-acetyl water-soluble elastin nanoparticles
AcENPs were dissolved in a 0.1M HCl solution at pH 1.2 and a PBS solution at pH 7.4 so that the concentration was 2 mg / ml, and the particle size was measured using NICOMP380ZLS. The particle size of the nanoparticles in different pH environments (pH 1.2, pH 7.4) is 279.7 ± 57.0 nm at pH 1.2 and 579.6 ± 98.9 nm at pH 7.4. It was confirmed that the particle size at pH 7.4 was nearly twice as large as that at pH 1.2.

Claims (4)

A pH-responsive drug sustained-release carrier comprising nanoparticles obtained by crosslinking N-acyl water-soluble elastin by γ-ray irradiation , the particle size of which changes in response to pH change. A pH-responsive sustained-release carrier comprising nanoparticles obtained by crosslinking N-acyl water-soluble elastin by γ-ray irradiation , which regulates drug release in response to pH change. A method for producing a pH-responsive drug sustained-release carrier, characterized in that a coacervate droplet of N-acyl water-soluble elastin is irradiated with radiation to form nanoparticles. In the method for producing nanoparticles, a particle having a nano-order particle size is obtained by irradiating a coacervate droplet of high molecular weight water-soluble elastin with radiation .
Using a high molecular weight water-soluble elastin having a molecular weight of 8000 or more,
The coacervate droplets are formed by heating a solution of the high molecular weight water-soluble elastin having a concentration of 0.1 to 60 mg / ml at a heating rate of 0.1 to 40 ° C./min,
A method for producing nanoparticles , wherein the irradiation is performed at an irradiation temperature of 20 to 80 ° C. and an irradiation amount of 5 to 60 kGy .

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