JP2011219428A - Medical material, production process therefor, and medicinal composition containing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a medical material containing a drug inclusion carrier or fine particles to form a cell carrier in a readily injectable form, a method of producing the medical material, and a medicinal composition including the medical material.SOLUTION: The medical material includes particles including low-molecular heparin and protamine, and dextran. Preferably, the particles are formed by dropping a protamine solution into a low molecular heparin solution. The medicinal composition includes the medical material and a medicine carried on the medical material.

Description

  The present invention relates to a medical material useful as a drug carrier, a method for producing the same, and a pharmaceutical composition using the medical material. More specifically, the present invention relates to preparation of nanoparticles and microparticles that can be controlled in particle size and can be formulated as a lyophilized product, and its medical application, and comprises low molecular weight heparin (fragmin) and protamine. The present invention relates to nano-microparticles and their use in medicine as heparin-binding growth factor-carrying substrates such as human platelet-rich plasma (hPRP platelet-rich-plasma: hereinafter referred to as hPRP) and FGF-2 using the microparticles as a carrier.

  Multi-electrolyte complexes produced by intermolecular electrostatic reactions with opposite charges provide a model for studying the in vivo behavior of charged biopolymers. Furthermore, the multi-electrolyte complex consisting of a unique composition and structure forms soluble nanoparticles, composite microparticles (coacervation), amorphous precipitates, etc. Applicable. For example, the formation of a complex consisting of a protein and a nucleic acid is thought to affect the transcription process. In addition, a DNA / chitosan complex and a chitosan / chondroitin sulfate complex were described as a gene transport substrate and a drug transport substrate, respectively.

  In recent years, angiogenesis therapy and refractory skin wound therapy as regenerative medicine, which are being actively studied, aim to induce new blood vessels by injecting factors that promote angiogenesis and granulation from the outside into the target site Is the method. In addition, cell introduction methods aiming to induce tissue regeneration by engrafting the introduced cells themselves and injecting and proliferating the factors generated by the introduced cells into the target site to induce tissue regeneration. For example, vascular endothelial growth factor (VEGF) gene and vascular endothelial progenitor cells (EPC) are used as factors that promote angiogenesis. Is raised. However, there are concerns about safety and cost-effectiveness, so it has not been put into practical use.

  By the way, basic fibroblast growth factor (FGF-2), one of the heparin-binding growth factors originally present in the living body, is a recombinant protein for treating pressure ulcers and skin ulcers (product) Name: Fiblast, Kaken Pharmaceutical Co., Ltd.) is a spray-type topical medicine and there are few concerns about safety. In addition, an angiogenic effect has been confirmed in animal experiments. However, there is no application as an internal medicine such as angiogenesis therapy, and application of intravenous injection administration to humans is difficult.

  In addition, hPRP, which has been attracting attention in recent years, contains various growth factors and effective substances such as cytokines that promote cell growth including FGF-2, and various applied researches are being promoted in many fields such as regenerative medicine. Yes. These platelets carry over 20 growth factors, including platelet-derived growth factors (PDGFs), fibroblast growth factors (FGFs), hepatocyte growth factors (HGF), transforming growth factors (TGFs), and vascular endothelial growth factors. (VEGFs) etc., all of which are known to be heparin binding growth factors. In the field of plastic surgery, a wide range of clinical applications such as wound healing, deformation improvement (including wrinkle removal) and hair growth are expected, and it is also applied in clinical settings. However, although its effectiveness is recognized, active molecules such as heparin-binding growth factor contained in hPRP, which is a valuable patient's own blood product, can be retained more effectively locally while maintaining its activity. The emergence of safe and effective drug carriers (drug carriers) that can be gradually released over time has been demanded.

  In the present inventors, photocurable chitosan hydrogel (Patent Document 1, Non-Patent Document 1) and 6-O-position desulfated heparin hydrogel (Patent Document 2, Non-Patent Document 2) are basic fibroblasts. It has been reported that it protects the activity of various growth factors such as growth factor (FGF-2) and is effective as a carrier for sustained release. It has been demonstrated that FGF-2, which is protected and retains activity in the hydrogel, is gradually released to the surrounding site as the hydrogel biodegrades, contributing to the promotion of angiogenesis and granulation in vivo. . Furthermore, by using a combination of low molecular weight heparin (fragmin) and protamine, it is possible to easily produce fine particles having an average particle size of about 3 μm (0.5 μm or more and less than 5 μm), and the obtained fine particles are used for sustained-release drug inclusion. It has been found that it can be used as a carrier or a cell carrier (Non-patent Document 3).

International Publication No. 2003/090765 International Publication No. 2005/025538

Biomaterials, 24, 3437-3444,2003. J. Biomed. Mater. Res. (A78), 364-371, 2006. Akio Katagiri, Kayo Hashiya, Shingo Nakamura, Hidemi Hattori, Atsuko Kishimoto, Masaaki Maehara, Masayuki Ishihara. Examination of the effect of FGF-2-containing fragmin protamine microcarriers (F / PMPs) on the improvement of ischemia. Defense Hygiene 56 (1), 17-24, 2009.

  However, photocurable chitosan hydrogels and 6-O-position desulfated heparin hydrogels are intended to be applied to trauma sites such as skin folds and skin ulcers to prevent dripping and the like. It was designed to have a high viscosity. Therefore, when administered through a syringe or a catheter, clogging may occur, causing a problem that local injection administration is difficult. Moreover, if the viscosity is lowered by simply lowering the concentration of chitosan or the like, the amount of drug that can be carried is limited, and the desired effect may not be obtained. In addition, microparticles produced by combining low molecular weight heparin and protamine are unstable substances that become large particles (> 20 μm) due to excessive concentration and long-term fusion and eventually become a paste. It became clear. In particular, when a freeze-dried product is produced for a medical preparation, it becomes a cotton-like insoluble crystal. Moreover, it was not possible to produce nanoparticles (particle diameter of about 100 nm) that are highly useful as fine particles.

  In view of the above-described background art, the present invention provides a medical material containing microparticles (micro / nanoparticles) that can be administered by injection and can be stored in a freeze-dried form, or a microparticle (micro / nanoparticle) serving as a cell carrier. It is an object of the present invention to provide a production method and a pharmaceutical composition using the medical material.

As a result of intensive studies by the inventors, the present invention as described below has been completed.
(1) A medical material containing particles containing low molecular weight heparin and protamine, and dextran.
(2) The medical material according to (1), wherein the particles are obtained by dropping a protamine solution into a low molecular weight heparin solution.
(3) The medical material according to (2), wherein the protamine solution is dropped so that 0.2 to 1 part by weight of protamine is added to 1 part by weight of the low molecular weight heparin contained in the low molecular weight heparin solution.
(4) The medical material according to any one of (1) to (3), wherein the low molecular weight heparin has a number average molecular weight of 1,000 to 10,000.
(5) The medical material according to any one of (1) to (4), wherein the average particle size of the particles measured by electron microscope image analysis is 50 nm to 10 μm.
(6) The medical material according to any one of (1) to (4), wherein the average particle diameter of the particles measured by electron microscope image analysis is 0.5 to 5 μm.
(7) The medical material according to any one of (1) to (4), wherein the average particle diameter of the particles measured by electron microscope image analysis is 50 nm to 200 nm.
(8) A method for producing a medical material, in which a protamine solution is dropped into a low molecular weight heparin solution to form particles, and dextran is further added to the solution containing the particles.
(9) A pharmaceutical composition comprising the medical material of any one of (1) to (7) and a drug carried on the medical material.
(10) The drug is selected from the group consisting of human platelet-rich plasma (hPRP), a pharmaceutical composing a growth factor secreted from human adipose tissue-derived mesenchymal cells, heparin-binding growth factor, and cytokine. 9) The composition.

  The medical material of the present invention can carry, for example, many heparin-binding growth factors contained in hPRP, and protects the carried growth factors from inactivation factors caused by proteolytic enzymes such as heat and trypsin. It was confirmed that the maintenance was extended. This is equivalent to a characteristic function of a natural heparin-like molecule (such as heparan sulfate) exerted by binding to a heparin-binding growth factor.

  When the medical material of the present invention is used as a drug carrier, the average particle size of the particles is in the form of microparticles in the micron unit or nanoparticles in the nano unit, so that the stable composition is dispersed in an appropriate medium. Has a low viscosity, can be easily handled even with a syringe equipped with a fine needle, and is very easy to operate. In addition, since the drug inclusion carrier of the present invention is biodegraded in vivo, a carrier drug such as heparin-binding growth factor contained in hPRP can be gradually released in vivo along with the biodegradation.

  Low molecular weight heparin and protamine such as dalteparin (fragmin) used in the present invention are commercially available materials, and their safety is ensured. hPRP is prepared from the patient's own peripheral blood and has already been clinically applied. The drug inclusion carrier of the present invention carrying a heparin-binding growth factor and the like contained in hPRP is used for angiogenesis and granulation therapy for beauty formation such as hair-restoring agents and hair removal and for treatment of lower limb ischemia. Therefore, it is promising as a safe and effective new biomaterial. Microparticles and nanoparticles composed of low molecular weight heparin and protamine are not only hPRP growth factors, but also heparin-binding growth factors secreted from human adipose tissue-derived mesenchymal cells (PDGFs, FGFs, It can be used as a drug inclusion carrier for medicinal products (MesoSkin) that make up KGF, TGFs, HGF, VEGFs, etc., and FGF-2 preparations (fiblasts) marketed as medicinal products, as well as micro or nanoparticle formation and stability It is considered that molecules that do not bind to heparin can be included. That is, the heparin-binding property is not essential for the drug carried on the drug inclusion carrier of the present invention. The formation of micro or nano particles by low molecular weight heparin and protamine is due to the polyion complex due to the mutual negative charge and positive charge. For example, unless it is a very high molecular weight substance, it has a negative charge or a positive charge. Medical products can be included.

(A) is an electron micrograph of particles obtained according to the present invention, and (B) shows the particle size distribution. It is a plot of turbidity against volume ratio of low molecular weight heparin solution and protamine solution. (A) is an electron micrograph of particles obtained when various low molecular weight heparin solutions and protamine solutions are diluted. (B) shows their particle size distribution. The time-dependent change of FGF-2 activity by the medical material (LH / P NPs) of this invention is shown. The vertical axis of each graph indicates the degree of proliferation of FGF-2 sensitive cells (hMVEC), and each symbol in the figure is LH / PNPs (●), Fragmin (△), dextran (□), and control (○). . It is a figure which shows the protective effect of FGF-2 activity from the heat | fever by the medical material (LH / P NPs) of this invention. The vertical axis of each graph indicates the degree of proliferation of FGF-2 sensitive cells (hMVEC), and shows the change in FGF-2 activity at each heating temperature for 30 minutes. Each symbol in the figure is LH / PNPs (●), fragmin (Δ), dextran (□), and control (◯). It is a figure which shows the protective effect of FGF-2 activity from the light white degradation enzyme (trypsin) by the medical material (LH / P NPs) of this invention. The vertical axis of each graph indicates the degree of proliferation of FGF-2 sensitive cells (hMVEC), and shows the time course of FGF-2 activity due to trypsin treatment. Each symbol in the figure is LH / PNPs (●), fragmin (Δ), dextran (□), and control (◯). It is a figure which shows the acceleration | stimulation of granulation tissue formation of a nude mouse by hPRP containing LH / P MPs with time. ** P <0.001, * P <0.01. It is a figure which shows the acceleration | stimulation of epithelial tissue formation of a nude mouse by hPRP containing LH / P MPs with time. ** P <0.001, * P <0.01. It is a figure which shows the time-dependent change of the number of new blood vessels of a nude mouse by hPRP containing LH / P MPs. ** P <0.001, * P <0.01. It is a figure which shows the time-dependent change of the hair follicle number of a nude mouse by hPRP containing LH / P MPs. * P <0.01. It is a figure which shows the effect of the hind limb ischemia model of a nude mouse by hPRP containing LH / P NPs. The photograph is an appearance photograph one week after administration. FIG. 3 is a photograph showing hair-growth effect by scalp subcutaneous injection of self-PRP-containing LH / P MPs and a graph of hair cross-section (diameter) and number of hairs per unit surface area. * P> 0.01vs PRP alone (n = 13), + P> 0.005 vs control (n = 13)

  The medical material of the present invention includes particles and dextran. The particles contain low molecular weight heparin and protamine. The average particle diameter of the particles is preferably 50 nm to 10 μm, and more preferable upper limit values include, for example, 5 μm, 300 nm, 200 nm, 50 nm, and another preferable range is 0.5 to 50 μm. Can be mentioned.

In the present invention, as the average particle size of the particles, the value of the average particle size _Rave measured by analyzing an image obtained by electron microscope observation as follows is used. First, a solution obtained by the present invention on a smooth glass thin plate, a resin thin plate, a metal thin plate, etc., more preferably a solution obtained by subjecting the solution obtained by the present invention to a desalting treatment by dialysis, ultrafiltration, etc. After dripping and drying, it is coated with a conductive material such as gold, platinum, gold-palladium alloy, osmium, etc., and used as a sample for electron microscope observation. Subsequently, the secondary electron image of the sample is observed and photographed with a scanning electron microscope, and the particle diameter is measured by performing the following image processing on the obtained electron micrograph. The algorithm for calculating the particle size first detects an edge after binarizing the image, discriminates a circular one as one particle, and performs extraction for each particle. The number of pixels is measured for all the extracted particles, and this number of pixels is defined as _Ni . The number of particles to be extracted for particle size measurement is usually 100 or more, preferably 500 or more, more preferably 1000 or more. Since the particle shape is assumed to be circular, the particle size R _i of the i-th particle is expressed by the following formula 1.
Where R _{i is the} particle size (m), N _{i is the} number of pixels (pixel), and S _{p is} the area per pixel (m ² / pixel).

Using the R _i value obtained above, the average value of all the extracted particle diameters expressed by the following formula 2 is calculated and set as the average particle diameter R _ave .

  According to the present invention, low molecular weight heparin is generally low molecular weight heparin obtained by depolymerizing natural heparin (molecular weight of about 15,000 to 20000 Da). The upper limit of the number average molecular weight is only required to be such that fine particles can be formed by mixing with protamine, and is generally 10,000, preferably 9000, more preferably 8000, and still more preferably 6000. The lower limit of the molecular weight is usually 1000, preferably 3000, more preferably 4000. In addition, it is sufficient if the number average molecular weight can be distinguished from normal (natural) heparin. For example, even if a fraction having a molecular weight of 10,000 or more, or 20,000 or more is included, the average molecular weight is less than about 10,000 as a whole. It can be used as a low molecular weight heparin in the present invention. In the present invention, the number average molecular weight measured by gel permeation chromatography using a standard substance having a known molecular weight is used as the average molecular weight of heparin.

  For example, depolymerized heparin (dalteparin) obtained by nitrous acid decomposition of heparin derived from porcine small intestinal mucosa is commercially available as Fragmin (trade name) and has an average molecular weight of 4000 to 6000. Similarly, depolymerized heparin (leviparin) obtained by nitrous acid decomposition of heparin derived from porcine small intestinal mucosa is commercially available as lomorin (trade name), and heparin derived from bovine or porcine intestinal mucosa is converted to hydrogen peroxide and acetic acid. Depolymerized heparin (parnaparin) obtained by decomposition with cupric is commercially available as Lohepa (trade name), and any of these can be used as the low molecular weight heparin in the present invention.

  Heparin alone does not have an anticoagulant effect and exerts its effect by binding to plasma ATIII, and coagulation enzymes such as factor IIa, factor XIIa, factor XIa, factor Xa, factor IXa Inhibits and inactivates. On the other hand, although low molecular weight heparin used in the present invention has anti-factor XIIa and anti-factor Xa activities, it has been clarified as a pharmaceutical that its inhibitory activity against factor IIa, factor XIa and factor IXa is slight. Therefore, even if it is injected into the wound site, it can be used without promoting the bleeding tendency at the site.

  As the low molecular weight heparin, the above-mentioned commercially available one may be used, or heparin having a low molecular weight by periodate oxidation, specific desulfated heparin, or the like can be suitably used. By using such low molecular weight heparin, suitable fine particles can be obtained when mixed with protamine.

  The protamine used in the present invention is known as a highly basic protein that is present in the sperm nucleus of an animal by binding to DNA. Generally, it is a low molecular weight protein consisting of 27 to 65 residues, and it is said that arginine occupies 40 to 70% of amino acids. Protamine is also commercially available as a pharmaceutical. In the present invention, commercially available protamine can be used as it is.

  In a preferred embodiment of the present invention, a protamine solution is dropped into a low molecular weight heparin solution to produce particles containing low molecular weight heparin and protamine. For example, a protamine aqueous solution is dropped into an aqueous solution of a low molecular weight heparin such as dalteparin at a ratio described later, and particles are obtained by further stirring with a vortex or the like. The concentrations of low molecular weight heparin and protamine can be set respectively, and are preferably 0.01 to 30 mg / ml, respectively. At this time, the size of the obtained particles can be controlled by adjusting the concentrations of low molecular weight heparin and protamine. For example, when a solution having low molecular weight heparin and protamine concentrations of about 1 to 20 mg / ml is used, particles having an average particle diameter of about 0.5 to 10 μm can be obtained. When a solution of about 0.05 to 0.5 mg / ml is used, particles having an average particle diameter of about 50 to 200 nm can be obtained.

  Regarding the mixing weight ratio of the low molecular weight heparin and protamine, it is preferable from the viewpoint of improving the yield of the particles that the weight of the low molecular weight heparin with respect to the protamine is equal or slightly excessive, and the presence of the excessive protamine is an insoluble paste. There is concern about the formation of precipitates. Therefore, when the protamine solution is dropped into the low molecular weight heparin solution, the weight ratio of the low molecular weight heparin / protamine in the dropped solution is preferably 1/1 to 5/1, more preferably 1 / 1 to 2/1. That is, the protamine solution is added such that 0.2 to 1 part by weight, more preferably 0.5 to 1 part by weight of protamine is added to 1 part by weight of the low molecular weight heparin contained in the low molecular weight heparin solution. Is dripped.

  The solvent in the low molecular weight heparin solution and the protamine solution can be arbitrarily selected from distilled water, saline, phosphate buffered saline, aqueous glucose solution, 0-4 infusion, high calorie infusion, and the like.

  According to the present invention, dextran is also included in the pharmaceutical material in consideration of dispersibility after lyophilization and long-term storage. In the absence of dextran, the particles aggregate after lyophilization or long-term storage, resulting in an insoluble cottony precipitate. The amount of dextran is not particularly limited. When particles are produced from a solution as described above, dextran can be allowed to coexist in the solution, preferably at a concentration of 0.05 to 1%. Suitable lower limits of the concentration of dextran in this solution include 0.05%, 0.1% and 0.2%, and preferred upper limits are 1%, 0.5% and 0.3%. Is mentioned. Dextran is preferably added to the solution after particles containing low molecular weight heparin and protamine are formed. It is considered that the added dextran adheres to or binds to the particles containing low molecular weight heparin and protamine, and contributes to the storage stability of the particles and the prevention of aggregation after lyophilization. The type of dextran is not particularly limited, and the number average molecular weight is preferably 100 to 300 kDa, more preferably 150 to 250 kDa, and still more preferably 178 to 217 kDa. As medical use, MRC polysaccharide stock Commercially available products from a company (Tokyo), Meito Sangyo Co., Ltd. (Nagoya City), etc. can be used as appropriate.

  Recently, a combination of hyaluronic acid, which is one of glycosaminoglycans and protamine, is mixed with interleukin 11 (IL-11) in the same manner as heparin. Producing is proposed (Japanese Patent Laid-Open No. 2006-9675). However, it is said that the hyaluronic acid used in this document preferably has a molecular weight of 60,000 to 2,000,000. Carboxymethyldextran, which can be used in place of hyaluronic acid, also has a molecular weight of 20,000 to 80,000. . When such a high molecular weight hyaluronic acid and protamine are combined, it is difficult to obtain stable and freeze-driable fine particles (μm or nm order) as obtained in the present invention. In the literature, a mixture of hyaluronic acid, protamine and IL-11 is lyophilized to a powder form, and a compression molded pellet is embedded subcutaneously in the back of the rat. On the other hand, in a composition for intravenous injection, only hyaluronic acid (and IL-11) is used, and no protamine is added.

  When high molecular weight heparin, hyaluronic acid, or chondroitin sulfate is used instead of low molecular weight heparin, it is not possible to obtain stable and freeze-driable nano / microparticles as obtained in the present invention. In the present invention, by adopting a unique combination of low molecular weight heparin and protamine which has already been approved as a pharmaceutical product, microparticles having an average particle size of less than 10 μm, further 0.5 μm or more, and less than 200 nm, further 50 nm Nanoparticles having the above average particle diameter could be obtained.

  According to the present invention, the above-mentioned medical materials can be used in various fields in the medical field, but in particular, they are suitably used as drug carriers. The medical material of the present invention is preferably a drug carrier, more preferably a drug carrier for injectable preparations, and still more preferably a drug carrier for freeze-dried preparations. That is, a pharmaceutical composition including the above-described medical material and a drug carried on the medical material is also one embodiment of the present invention.

  For example, the medical material of the present invention can be used as a human platelet-rich plasma (hPRP) inclusion carrier. Since this hPRP inclusion carrier (nano / microcarrier) has a morphological feature of fine particles in the order of μm to nm, it is particularly suitable for use as a pharmaceutical composition to be injected into the body via a syringe or a catheter. Yes. In addition to the heparin-binding growth factor contained in hPRP, the medical material of the present invention contains growth factors (PDGFs, FGFs, KGF, TGFs, HGF, VEGFs, etc.) secreted from human adipose tissue-derived mesenchymal cells. It can also be applied as an inclusion carrier for the constituent pharmaceutical (MesoSkin) and FGF-2 (Fiblast) marketed as a pharmaceutical.

  In the present invention, the drug is not particularly limited, and is preferably a drug that is easily carried on the above-described medical material and desired to be sustainedly released in the body. Further, since the medical material of the present invention has particles containing low molecular weight heparin and protamine, the drug has high affinity with low molecular weight heparin or protamine, and is selected from, for example, heparin-binding growth factor or cytokine. It is preferable that the active substance is contained. In particular, hPRP can be prepared from blood collected from the patient itself, and most of the growth factors contained therein are known to be heparin-binding and are particularly preferred. The medium in the pharmaceutical composition of the present invention is not particularly limited, and an aqueous medium is preferably used. The pharmaceutical composition of the present invention is preferably an injectable preparation, more preferably a lyophilized preparation.

  The medical material of the present invention has an effect of favorably protecting the carried drug. For example, when FGF-2 contained in hPRP is supported, the decrease in FGF-2 activity over time and the decrease in activity due to external stimuli such as temperature change and enzymatic degradation are prevented. The activity is maintained for a long time. Furthermore, since LH / P MPs and LH / P NPs are biodegraded in vivo, it is possible to release FGF-2 and the like that are carried along with it. Therefore, by using the pharmaceutical composition of the present invention as well as a traumatic site such as conventional chitosan hydrogel, a drug such as FGF-2 is delivered to a target local site in the deep part of the body and sustainedly released at the site. And angiogenesis therapy can be performed.

  Examples according to the present invention will be described below. However, the present invention is not limited to the embodiments described in these examples.

(Example 1)
Preparation of low molecular weight heparin / protamine microparticles (LH / P MPs) Fragmin injection (trade name) (6.4), a commercially available dalteparin, as low molecular weight heparin
mg / ml, 1000 IU / ml, the number average molecular weight of heparin was about 5000, Kissei Pharmaceutical Co., Ltd.). A commercially available protamine injection solution (10 mg / ml; Mochida Pharmaceutical Co., Ltd.) was added dropwise to the low molecular weight heparin solution with vortexing until a volume ratio of 7: 3 was reached. FIG. 1A shows the electron microscope appearance of the obtained fine particle dispersion. Particle size distribution F / P MPs (Fig. 1) can be obtained from electron micrographs using particle size analysis software (LabVIEW (Ver 8.5) with the Vision Development / Module (National Instrument Co., Austin, TX, USA)). Measured. As a result, the generated F / P MPs was 2.93 ± 1.11 μm. These F / P MPs are stabilized without long-term fusion by adding 0.5% dextran (MW: 178-217 kDa; MRC Polysaccharide Corp., Tokyo, Japan), and are resuspendable even after freeze-drying after dialysis And the particle size was maintained.

  Fig. 2 shows the results of measurement of white turbidity generated when a protamine injection solution (10 mg / ml) was added dropwise to a fragmin injection solution (6.4 mg / ml) as a low molecular weight heparin solution so as to have a volume ratio shown. It is. When the drop volume ratio of protamine is 0.3 to 0.4, the turbidity is highest and the yield of microparticles is also high. However, when the volume fraction of protamine reached 0.45 or more, the turbidity decreased rapidly and a paste-like insoluble precipitate was formed. Further, when the fragmin injection solution was dropped into the protamine injection solution, a paste-like insoluble precipitate was immediately formed, and the paste-like insoluble precipitate was not dissolved even when the volume ratio of fragmin was 0.6 or more.

(Example 2)
Preparation of low-molecular-weight heparin / protamine nanoparticles (LH / P NPs) Low-molecular-weight heparin solution (6.4 mg / ml) and protamine solution (10 mg / ml) are equally 20 times, 50 times, 100 times physiological saline And then mixed according to the procedure for producing microparticles of Example 1 (volume ratio (7: 3)) to obtain nanoparticles having average particle diameters of 112.5 ± 46.1, 95.0 ± 27.0, and 84.6 ± 26.8 nm, respectively. (Fig. 3). The low molecular weight heparin solution (6.4 mg / ml) and the protamine solution (10 mg / ml) were the same commercial products as in Example 1. In the LH / P NPs, microparticles having a particle size of 1 μm or more are not initially observed, but the microparticles as shown in FIG. 1 appear by the fusion of nanoparticles after refrigerated storage for about one week. This LH / P NPs solution was stabilized without fusion for a long time by addition of 0.2% dextran, and maintained resolubility and nanoparticle size even after freeze-drying after dialysis.

(Example 3)
Effect of low molecular weight heparin / protamine nanoparticles (LH / P NPs) on FGF-2 activity It is known that the LH / P NPs DMEM medium solution prepared in Example 2 contains human platelet rich plasma (hPRP). FGF-2 (Fiblast; Kaken Pharmaceutical Co., Ltd.) was added to prepare a stock solution, and a stability test was performed under the following experimental conditions. The stock solution used in this experiment was LH / P NPs stock solution: 10 μg / ml FGF-2, 3.14 mg / ml F / P NPs, 20 mg / ml dextran in Medium-199, low molecular weight heparin stock solution: 10 μg / ml ml FGF-2, 1.6 mg / ml low molecular weight heparin, 20 mg / ml dextran in Medium-199, dextran stock solution: 10 μg / ml FGF-2, 20 mg / ml dextran in Medium-199, control stock solution: 10 μg / 4 ml FGF-2 in Medium-199. Thereafter, each preincubated stock solution was prepared to the indicated concentration and added to the culture medium of FGF-2 sensitive cells (human dermal micro-vascular endothelial cells; hMVEC). Cultivation for 3 days after addition (10% FBS / DMEM medium, 37 ° C., 5% CO ₂ atmosphere) was performed, and the activity of FGF-2 was evaluated by examining the degree of cell proliferation of hMVEC.

  The pre-incubation conditions in this experiment were pre-incubated for 0, 1, 3, and 7 days at 37 ° C. for each stock solution in the FGF-2 activity stability test over time. FIG. 4 shows the results of this experiment. In the control group and the dextran group, it was found that the cell proliferation decreased remarkably as the preincubation time was increased, and the FGF-2 activity decreased with time in these groups. On the other hand, in the LH / P NPs or low molecular weight heparin group, the activity of FGF-2 was maintained even after 7 days of pre-incubation.

  In the experiment for examining the protective effect of FGF-2 activity from heat, each stock solution was pre-incubated at 37, 44, 51, 58, 65, and 72 ° C. for 30 minutes. The results are shown in FIG. In the control group, it was found that the FGF-2 activity significantly decreased as the temperature increased from 37 ° C. On the other hand, the low molecular weight heparin group showed almost the same tendency as the LH / P NPs group, but the LH / P NPs group showed a slightly higher FGF-2 activity at the stage of exceeding 72 ° C. The dextran group showed an intermediate protective effect between the control group and the LH / P NPs group.

A trypsin solution (0.05% trypsin solution; Sigma) was added to each stock solution, and preincubation was performed at 37 ° C. for 10, 20, 30, 45, 60, 90, and 120 minutes, respectively. The results are shown in FIG. In the LH / P NPs group and the low molecular weight heparin group, FGF-2 activity was maintained at 70% or more after 2 hours of trypsin treatment. In the control group, it was found that FGF-2 activity decreased by 70% or more when treated with trypsin for 20 minutes. The dextran group showed an intermediate protective effect between the control group and the LH / P NPs group.
In addition, since low molecular weight heparin and dextran are completely soluble, they do not function as the carrier of FGF-2 intended by the present invention. Therefore, as a drug inclusion carrier, only LH / P NPs can maintain and prolong the activity by protecting FGF-2 from heat and proteolytic enzymes. Similarly, LH / P MPs have strong FGF-2 adsorption / retention / activity protection effects.

Example 4
The in vivo granulation, epidermis, hair follicle formation and angiogenic effects of hPRP-containing LH / P MPs were examined using nude mice. For hPRP, 40 ml of blood was collected from a volunteer using a blood collection tube containing 4 ml of 2% citric acid. The blood collection tube was centrifuged at 1700 rpm for 15 minutes with a table top refrigerated centroid separator (table top refrigerated centroid separator 2800, rotor: RS-240, Kubota Corporation, Tokyo). The platelet rich intermediate layer was separated and centrifuged at 3000 rpm for 5 minutes to further concentrate the platelets. To the obtained hPRP (about 4.5 ml), 2% calcium chloride solution (0.8 ml) was added, and about 35 mg of lyophilized product of LH / P MPs (containing dextran) was resuspended. Finally, about 5 ml of hPRP-containing LH / P MPs injection was obtained from 40 ml of peripheral blood.

  The above-prepared hPRP-containing LH / P MPs, hPRP alone, LH / P MPs alone, and control (saline) in nude mice (8 weeks old, male, BALB / c Slc-nu / nu; Japan SLC Co., Ltd.) The dorsal subcutaneous tissue was injected (200 μl) with a syringe with a 27-G needle (Nipro). At 1, 3, 5, 7, 14, and 30 days after injection, autopsy was performed to separate the tissue at the injection site, and the HE-stained specimen was prepared and observed with an optical microscope and photographed. For the results, the thickness of the granulation tissue formed (Fig. 7), the thickness of the epithelialized tissue formed (Fig. 8), the number of capillaries per field of view (Fig. 9), the number of hair follicles per field of view (Fig. Measurement was performed for 10).

  Injected hPRP-containing LH / P MPs or LH / P MPs were degraded in approximately one week and disappeared by gross findings. In the vicinity of the site where the hPRP-containing LH / P MPs injection was performed, it was observed that granulation and epithelial formation were promoted more strongly than the LH / P MPs single injection group or the hPRP single injection group. In addition, when the number of capillaries and hair follicles per field of microscopic observation was examined, the number of blood vessels and hair follicles in the hPRP-containing LH / P MPs injection group increased significantly compared to other control groups. The action continued for about one month. From the above, it was revealed that granulation and angiogenesis can be effectively induced by using hPRP-containing LH / P MPs.

(Example 5)
hPRP was prepared from volunteers according to Example 4 above. To the obtained hPRP (about 4.5 ml), 2% calcium chloride solution (0.8 ml) was added to dissolve about 35 mg of lyophilized powder of LH / P NPs. Finally, about 5 ml of hPRP-containing LH / P NPs injection was obtained from 40 ml of peripheral blood.
When the aorta and veins of the lower limbs are simultaneously occluded in nude mice (8 weeks old, male, BALB / c Slc-nu / nu; Japan SLC Co., Ltd.), they turn black and fall off in 1-2 weeks. In this model, HPRP-containing LH / P NPs prepared as described above, hPRP alone, LH / P NPs alone, and control (saline) were placed near the occlusion of nude mice, to a total of 10 locations in the thigh and shin. 27-G needle Each 30 μl was injected with a syringe attached (total 300 μl). Two weeks after the injection, the hPRP-containing LH / P NPs injection group maintained the lower extremities that maintained healthy blood flow in 5 out of 8 cases. In two cases of discoloration, there was only one drop-out limb. This improvement in blood flow was maintained for at least 4 weeks. On the other hand, all the LH / P NPs single group and control group dropped out. In the second week, the control group has dropped out or has turned black. In the hPRP alone group, there were 2 cases of discoloration, 6 cases of dropout limbs, and 0 remained healthy limbs (FIG. 11).

(Example 6)
Thirteen volunteers (9 males and 4 females) suffering from thinning hair with informed consent were directly injected subcutaneously into the scalp with autologous PRP-containing LH / P MPs by each blood sampling to examine the effects of hair growth and hair growth. . Treatment is subcutaneous injection of autologous PRP-containing LH / P MPs at 0, 2, 4, 7, and 10 weeks, and photographs are taken using Dermoscopic Degital Camera (Derma Medical, Kanagawa, Japan) at each week and 12 weeks. The hair cross-section (diameter) and the number of hairs per unit surface area were measured. This example was approved and implemented by the National Defense Medical College Hospital Ethics Committee. As a result, no increase in the number of hairs, that is, a significant increase in the number of hair growth, was observed, but in all 13 cases, a hair cross-section or hair growth promoting effect was observed. This hair growth-promoting effect was significantly promoted by PRP alone compared to the control, but the hair growth-promoting effect at the PRP + F / P MPs administration site was significantly higher at 4, 7, and 10 weeks than the PRP administration site (FIG. 12). In addition, all volunteers recognized the loss of hair loss after shampooing at the sites of PRP + F / P MPs and PRP. In particular, 8 women, including 3 women, showed remarkable effects (more than 75% increase in hair cross-section), no complaints of side effects, and very high satisfaction.

  The medical material of the present invention contains particles containing low-molecular-weight heparin and protamine that have been confirmed to be safe, and they are fused over time to become coarse particles or insoluble flocculent precipitates. Therefore, it was possible to prepare a re-dissolvable lyophilized preparation. Therefore, LH / P MPs and LH / P NPs can be stably supplied to medical applications administered via a syringe or a catheter. For example, it can be used as a drug delivery carrier for drug delivery that can stably carry active molecules such as heparin-binding growth factor contained in hPRP and can slowly release the active molecules carried at the target site. By applying this, in addition to being applied to cosmetic treatments such as hair restoration agents and wrinkle removal in the cosmetic plastic surgery field, medical applications as an angiogenesis-promoting preparation, an intractable wound healing promoter for skin, etc. are possible. It was.

Claims (10)

  Particles containing low molecular weight heparin and protamine, and medical materials containing dextran.   The medical material according to claim 1, wherein the particles are obtained by dropping a protamine solution into a low molecular weight heparin solution.   The medical material according to claim 2, wherein the protamine solution is dropped so that 0.2 to 1 part by weight of protamine is added to 1 part by weight of the low molecular weight heparin contained in the low molecular weight heparin solution.   The medical material according to any one of claims 1 to 3, wherein the low molecular weight heparin has a number average molecular weight of 1,000 to 10,000.   The medical material according to any one of claims 1 to 4, wherein an average particle diameter of the particles measured by electron microscope image analysis is 50 nm to 10 µm.   The medical material according to any one of claims 1 to 4, wherein an average particle diameter of the particles measured by electron microscope image analysis is 0.5 to 5 µm.   The medical material according to any one of claims 1 to 4, wherein an average particle diameter of the particles measured by electron microscope image analysis is 50 nm to 200 nm.   A method for producing a medical material, wherein particles are produced by dropping a protamine solution into a low molecular weight heparin solution, and dextran is further added to the solution containing the particles.   The pharmaceutical composition containing the medical material as described in any one of Claims 1-7, and the chemical | medical agent carry | supported by the said medical material.   10. The drug according to claim 9, wherein the drug is selected from the group consisting of human platelet-rich plasma (hPRP), a pharmaceutical constituting a growth factor secreted from human adipose tissue-derived mesenchymal cells, heparin-binding growth factor and cytokine. The composition as described.