JP2005206491A - Pulmonary emphysema-treating transpulmonary administration preparation - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a useful pulmonary emphysema-treating transpulmonary administration preparation by forming the preparation which can efficiently transport a neovascularization action-having medicine such as a neovascularization proliferation factor into bronchi. <P>SOLUTION: This pulmonary emphysema-treating preparation comprises gelatin microspheres containing a neovascularization action-having medicine. The neovascularization action-having medicine includes basic fibroblast growth factors, hepatocyte growth factors, other FGF, endothelial cell growth factors, platelet-originated growth factors, transforming growth factors, cell growth factors such as angiopoietin, angiostatin, and adrenomedulin, interleukin, kemokine, and genes encoding them, physiologically active low molecular substances, and medicines for inducing the above-mentioned neovascularization-promoting action-having substances. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a preparation for transpulmonary administration for treating emphysema.

  Emphysema is a condition in which the peripheral alveolar wall is diffusely destroyed from the terminal bronchiole, and is a disease that is common in the elderly. Even in Japan, about 50,000 people receive home oxygen therapy due to this disease, but including those with mild pathologies, it can be said that about 3 million people are a reserve population for emphysema. Currently, there is no effective treatment other than home oxygen therapy, and there is no way to delay the progression of this condition. In addition, although there are reports that tissue regeneration of pulmonary emphysema has been recognized by ATRA (all-trans retinoic acid) or matrix metalloproteinase inhibitors, it is still in the stage of animal experiments at this stage, and various investigations are still underway There is a need.

  In pulmonary emphysema, the respiratory bronchiole wall and alveolar wall are destroyed and the alveoli are dilated and cannot exhale sufficiently. Therefore, if the regeneration of the alveoli can be induced, it will be a radical treatment. A treatment using bone marrow-derived cells having high differentiation potential has already been attempted. Another method is to induce regeneration using a cell growth factor. For example, angiogenic growth factors are used to promote angiogenesis, thereby inducing key cells to the site or supplying humoral factors necessary for regeneration from the bloodstream, resulting in alveolar By promoting regeneration, it may be possible to delay the progression of emphysema and improve symptoms. Examples of such angiogenic growth factors include basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), other FGFs, vascular endothelial growth factor ( VEGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF), cell growth factors such as angiopoietin, angiostatin, adrenomedullin, physiologically active proteins and peptides such as interleukins and chemokines, and prostaglandins And other biologically active low molecular weight substances. Alternatively, genes encoding these angiogenic factors are also included. In addition, a drug having an action of promoting angiogenesis or a drug having an action of enhancing production and secretion of angiogenic factors in the body can be used. Examples of such drugs include protein drugs and genes related to the production and secretion of angiogenic factors in addition to low molecular weight drugs.

  bFGF is the first growth factor identified from the pituitary gland and brain as a factor that promotes the proliferation of fibroblasts (3T3) (Rifkin and Moscatelli, J. Cell Biol. 109, 1-6, 1989). bFGF has been found to have a wide variety of functions in various tissues and organs such as angiogenesis, smooth muscle cell growth, wound healing, tissue repair, hematopoiesis, neuronal differentiation (Bikfalvi, et al.,). Endocrine Rev., 18, 26-45, 1997).

  International Publication WO94 / 27630 discloses a sustained-release preparation in which bFGF is contained in a crosslinked gelatin gel. Japanese Patent Application Laid-Open No. 2001-172203 discloses a pharmaceutical composition for pulmonary administration comprising a polymer drug and cationized gelatin fine particles. However, this composition is intended to improve the pulmonary absorption of the high molecular drug by using cationized gelatin and to transfer the high molecular drug to the systemic blood flow. There is no mention of releasing.

  Recently, Goto et al. Observed that microspheres were present in the pulmonary artery by administering bFGF sustained-release gelatin microspheres into the pulmonary artery, and pulmonary alveolar structures were observed histologically. However, there is no evidence of alveoli, and the recovery of pulmonary function, which is most important in the treatment of emphysema, has not been investigated (Journal of Japanese Thoracic Surgery, 2003 October Vol 51, p281). When bFGF-impregnated gelatin is administered into the pulmonary artery, small sized particles do not stay in the artery, but pass through the lungs and physically clog blood vessels of other organs, which may impair the organ function. In addition, the pulmonary artery may become clogged, and conversely, lung function may be worsened. In particular, when the particles embolize the cerebral blood vessels, they cause cerebral infarction. Furthermore, it has been reported that some drugs are effective when administered via pulmonary artery, but are not effective when administered via airway. In particular, protein drugs and nucleic acid drugs are decomposed and deactivated more rapidly than oxygen in blood vessels due to oxygen in the respiratory tract, so that therapeutic effects cannot be expected. In addition, since arterial administration requires skilled medical personnel, development of a simpler administration method is required.

Prior art document information related to the present invention includes the following.
WO94 / 27630 JP 2001-172203 A

  Accordingly, an object of the present invention is to provide a useful therapeutic agent for pulmonary emphysema for transpulmonary administration by forming a preparation capable of efficiently transporting an angiogenic drug such as angiogenic growth factor into the bronchi. And

  The present inventors have found that a preparation for transpulmonary administration of bFGF produced using gelatin microspheres has a significant therapeutic effect in a canine pulmonary emphysema model, and completed the present invention. That is, the present invention provides a pulmonary emphysema therapeutic agent comprising a drug having an angiogenic action and gelatin microspheres. Preferably, the drug having angiogenic activity is basic fibroblast growth factor, hepatocyte growth factor, other FGF, vascular endothelial growth factor, platelet-derived growth factor, transforming growth factor, angiopoietin, angiostatin, address It is selected from the group consisting of cell growth factors such as nomedulin, interleukins, chemokines, and genes encoding them, bioactive low molecular substances, and drugs that induce the above-described substances that promote angiogenesis. Particularly preferably, the drug having an angiogenic action is basic fibroblast growth factor.

  As shown in the following examples, according to the present invention, gelatin microspheres containing a drug having an angiogenic action are administered by pulmonary administration and sustained release, thereby improving blood flow and improving the effect of treating emphysema. It became clear that it was obtained. In pulmonary administration, there is no risk of clogging of microspheres into the blood vessels of pulmonary arteries or other organs, embolization of cerebral blood vessels, and the administration method is simple and the patient himself can perform drug administration operations. It is also possible to apply to.

  Gelatin used in the present invention is collagen that can be collected from any part of the body such as skin, bone, tendon of various animal species including cattle, pigs, fish, etc., or a substance used as collagen, It can be obtained by modification by various treatments such as alkaline hydrolysis, acid hydrolysis, and enzymatic decomposition. Gelatin, which is a modified form of recombinant collagen, may be used. The properties of gelatin vary depending on the material and processing method used, and gelatin having any of these properties can be used as a hydrogel material for sustained release of a drug having an angiogenic action in the present invention.

  The gelatin microsphere used in the present invention is a fine particle gelatin hydrogel obtained by forming chemical cross-links between various chemical cross-linking agents and gelatin molecules using the above-mentioned gelatin. As the chemical cross-linking agent, a condensing agent that forms a chemical bond between water-soluble carbodiimide such as EDC, propylene oxide, diepoxy compound, hydroxyl group, carboxyl group, amino group, thiol group, and imidazole group can be used. Preference is given to glutaraldehyde. Gelatin can also be chemically crosslinked by heat treatment, ultraviolet irradiation, electron beam irradiation, and the like. These crosslinking treatments can be used in combination. Furthermore, it is also possible to produce a hydrogel by physical crosslinking using salt crosslinking, electrostatic interaction, hydrogen bonding, hydrophobic interaction, and the like.

  The degree of crosslinking of gelatin can be appropriately selected according to the desired water content, that is, the level of bioabsorbability of the hydrogel. The preferred ranges of the concentration of gelatin and the crosslinking agent in preparing the gelatin hydrogel are a gelatin concentration of 1 to 20 w / w% and a crosslinking agent concentration of 0.01 to 1 w / w%. The crosslinking reaction conditions are not particularly limited, but can be performed, for example, at 0 to 40 ° C. for 1 to 48 hours. In general, as the concentration of gelatin and the crosslinking agent and the crosslinking time increase, the degree of crosslinking of the hydrogel increases and the bioabsorbability decreases.

  Gelatin microspheres, for example, are equipped with a stirring motor (for example, Shinto Kagaku Co., Ltd., Three One Motor, EYELA miniD. C. Stirrer, etc.) fixed to a three-necked round bottom flask and a Teflon (registered trademark) propeller. Put the gelatin solution in the fixed device together, add oil such as olive oil and stir at a speed of about 200-600 rpm to make a W / O type emulsion, add the cross-linking agent aqueous solution to this, or add the gelatin aqueous solution Pre-emulsified in olive oil (for example, using vortex mixer Advantec TME-21, homogenizer, polytron PT10-35, etc.) is dropped into olive oil to prepare finely divided W / O emulsion Then, an aqueous solution of a crosslinking agent was added thereto to cause a crosslinking reaction, and after collecting gelatin microspheres by centrifugation, Tons, washed with ethyl acetate and the like, further 2-propanol, by stopping immersed in a crosslinking reaction such as ethanol, can be prepared. The obtained gelatin microspheres are sequentially washed with distilled water containing 2-propanol, Tween 80, distilled water, etc., and used for preparation of the preparation. When gelatin microspheres aggregate, for example, a surfactant or the like may be added or sonication (preferably within about 1 minute under cooling) may be performed.

  The average particle diameter of the obtained gelatin microsphere varies depending on the gelatin concentration at the time of preparing the above-mentioned particles, the volume ratio of the gelatin aqueous solution and olive oil, the stirring speed, and the like. In general, the particle diameter is 500 nm to 1000 μm, and particles having a necessary size may be appropriately screened and used according to the purpose. Furthermore, by pre-emulsification, fine-particle gelatin microspheres having a particle size of 50 nm to 20 μm or less can be obtained. Furthermore, particles having a size of 50 nm to 1 μm can be obtained by causing phase separation from the aqueous solution state and self-assembling. Phase separation is achieved by a known technique such as addition of a second component, change in pH of an aqueous solution, ionic strength, and the like. The preferred particle size in the present invention is about 50 nm-100 μm, more preferably about 100 nm-20 μm.

  Another method for preparing gelatin microspheres includes the following method. Olive oil is put into the same apparatus as the above method, and stirred at a speed of about 200 to 600 rpm. A gelatin aqueous solution is added dropwise to prepare a W / O type emulsion. After cooling this, acetone, ethyl acetate, etc. are added. In addition, the mixture is stirred and uncrosslinked gelatin particles are recovered by centrifugation. The collected gelatin particles are further washed with acetone, ethyl acetate, etc., then 2-propanol, ethanol, etc., and then dried. The dried gelatin particles are suspended in an aqueous solution of a crosslinking agent containing 0.1% Tween 80 and allowed to undergo a crosslinking reaction with gentle stirring. Depending on the crosslinking agent used, a 100 mM glycine aqueous solution containing 0.1% Tween 80 or 0.1% Gelatin microspheres can be prepared by washing with 0.004N HC1 or the like containing% Tween 80 and stopping the crosslinking reaction. The average particle size of the gelatin microspheres obtained by this method is the same as in the above method.

  The gelatin microspheres of the present invention can be used after lyophilization and sterilization. Freeze-drying is performed, for example, by putting gelatin microspheres in distilled water, freezing them in liquid nitrogen for 30 minutes or more, or at -80 ° C for 1 hour or more, and then drying them in a freeze dryer for 1 to 3 days. Can do.

  In the present invention, the drug having an angiogenic action is preferably a basic fibroblast growth factor, hepatocyte growth factor, other FGF, vascular endothelial growth factor, platelet-derived growth factor, transforming growth factor, angiopoietin , Cell growth factors such as angiostatin and adrenomedullin, interleukins, chemokines, and genes encoding these, bioactive low molecular weight substances, and drugs that induce the above-mentioned substances that promote angiogenesis, Selected. Particularly preferably, the drug having an angiogenic action is basic fibroblast growth factor. Any drug used in the present invention is a known substance, and any substance prepared by various methods can be used as long as it is purified to the extent that it can be used as a medicine. May be used. When the drug having an angiogenic action is a protein such as angiogenic growth factor, for example, primary cultured cells or established cells that produce angiogenic growth factor are cultured, separated from the culture supernatant, etc. Angiogenic growth factors can be obtained. Alternatively, a gene encoding an angiogenic growth factor can be incorporated into an appropriate vector by genetic engineering techniques, inserted into an appropriate host, transformed, and the desired recombinant angiogenesis from the culture supernatant of the transformant. Growth factors can also be obtained. The host cell is not particularly limited, and various host cells conventionally used in genetic engineering techniques such as Escherichia coli, yeast, insects, silkworms, or animal cells can be used. As long as the angiogenic growth factor thus obtained has substantially the same action as the natural angiogenic growth factor, one or more amino acids in the amino acid sequence are substituted, deleted and / or added. Similarly, sugar chains may be substituted, deleted, and / or added.

  The weight ratio of the drug having an angiogenic action to gelatin is preferably about 5 times or less. More preferably, the weight ratio is about 0.1 to about 3 times that of gelatin.

  Since the gelatin microsphere containing the drug having an angiogenic action of the present invention has a sustained release effect and a stabilizing effect of the drug, the function of the drug can be exerted for a long time with a small amount. Therefore, the improvement of blood flow by a drug having an angiogenic action is effectively exhibited at a locally administered site.

  The mechanism of sustained release in gelatin hydrogel is based on the fact that a drug having an angiogenic action is physically immobilized on gelatin in the hydrogel. The inventors of the present invention have so far attempted sustained release using bioabsorbable polymer hydrogels such as growth factors, cytokines, monokines, lymphokines, and other physiologically active substances, thereby preventing physiology that cannot be achieved by other materials. We have succeeded in sustained release of active ingredients with activity and control of the sustained release period. In the present invention, it is considered that a drug having an angiogenic action is gradually released from the hydrogel by the same mechanism. In a state where the gel is immobilized on the gelatin hydrogel and the hydrogel is insoluble in water, the drug is not released from the hydrogel. As the hydrogel is degraded in the living body, the gelatin molecule becomes water-soluble, and accordingly, the drug immobilized on the gelatin molecule is released from the hydrogel. That is, the sustained release of the drug can be controlled by hydrogel decomposition. The degradability of the hydrogel can be changed by adjusting the degree of crosslinking during the preparation of the hydrogel. In addition, when the drug interacts with gelatin, the in vivo stability, such as resistance to enzymatic degradation, is improved.

  The water content is an index of the degradation rate of the gelatin microsphere of the present invention. This is the weight percent of water in the gelatin hydrogel relative to the weight of the swollen gelatin hydrogel. If the water content is large, the degree of crosslinking of the hydrogel is low and it is easily decomposed. That is, this moisture content value determines the sustained release of the drug. Since the degradation period of the hydrogel coincides with the sustained release period of the drug, the sustained release period in this case also varies from 5 days to 3 months. The water content showing a preferable sustained-release effect is about 80 to 99 w / w%, and more preferably about 95 to 98 w / w%.

  The gelatin microsphere preparation containing a drug having an angiogenic action of the present invention can be administered by bronchial inhalation. Administration by inhalation can be performed using any of the inhalation devices and inhalation methods currently used clinically. For example, a drug-containing gelatin microsphere formulation is applied from a nebulizer to an aerosol spray using a propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, and a suitable powder base such as lactose or starch. Can be performed by transporting to the lung in the form of In addition, transpulmonary administration may be performed using a bronchoscope.

  The dose of the drug having an angiogenic action in the preparation of the present invention can be appropriately adjusted depending on the severity of the disease, the age of the patient, the body weight, etc., but is usually in the range of about 0.01 to about 500 μg per adult patient, Preferably, the dosage is selected from the range of about 0.1 to about 200 μg. Moreover, when the effect is insufficient with a single administration, the administration can be performed a plurality of times.

  EXAMPLES The present invention will be described below in more detail with reference to examples, but the present invention is not limited to these examples.

Preparation of bFGF-containing gelatin microspheres 375 ml of olive oil was added to a 1000 ml round bottom flask, and a Teflon (registered trademark) stirring propeller was attached to a fixed stirring motor (manufactured by Shinto Kagaku Co., Ltd., Three-One Motor). Fixed. While stirring the olive oil at 37 ° C. and 420 rpm, 10 ml of an alkali-treated gelatin aqueous solution (concentration: about 10%) having an isoelectric point of 4.9 was added dropwise to prepare a W / O type emulsion. After stirring for 10 minutes, the flask was cooled to 4 ° C. and stirred for 30 minutes. After cooling, 100 ml of acetone was added thereto and stirred for 1 hour, and then gelatin microspheres were recovered by centrifugation. The recovered microspheres were washed with acetone and further washed with 2-propanol (hereinafter abbreviated as IPA) to obtain uncrosslinked gelatin microspheres. The microspheres were dried and stored at 4 ° C. 500 mg of dried uncrosslinked gelatin microspheres were suspended in 100 ml of an aqueous solution of glutaraldehyde (0.05%) containing 0.1% Tween 80, and gelatin was crosslinked by gently stirring at 4 ° C. for 24 hours. After completion of the reaction, the crosslinked gelatin microspheres were collected by centrifugation, and the crosslinking reaction was stopped by washing with a 100 mM glycine aqueous solution at 37 ° C. for 1 hour. After the reaction was stopped, the crosslinked gelatin microspheres were washed three times with distilled water and then lyophilized to obtain dried crosslinked gelatin gel microspheres (average particle size 40 μm, water content 95%). To 2 mg of the obtained cross-linked gelatin gel microsphere, 20 μl of 0.1 M phosphate buffer (pH 7.4) containing 50 μg of bFGF was dropped, and left at 25 ° C. for 1 hour, whereby the bFGF aqueous solution was placed in the microsphere. Penetration and gelatin microspheres containing bFGF were prepared.

すなわち、ｂＦＧＦをゼラチンマクロスフェアに含浸させて投与することによって、より多く、しかもより長くｂＦＧＦを投与部位周辺にとどめておくことが可能となった。 In vivo stability of bFGF-containing gelatin microspheres
¹²⁵ I-labeled microspheres and bFGF were administered subcutaneously to the back of the mice. The time course of disappearance of microspheres and bFGF in vivo was evaluated by measuring the residual radioactivity over time. The change in radioactivity when the residual radioactivity after administration is defined as 100 is shown in the following table.

That is, bFGF was impregnated into gelatin macrospheres and administered, so that it was possible to keep bFGF around the administration site more and longer.

Production of Beagle Dog Model A beagle dog (body weight: 9 to 14 kg) was anesthetized by subcutaneous injection of 1 to 3 mg / kg of xylazine (ceractal) and 5 to 10 mg / kg of ketamine hydrochloride (ketalal). An endotracheal intubation was performed and a bronchoscope was inserted into the trans-airway. 50 mg (3000 units) of porcine pancreatic elastase (NAKARAI TESQUE) was dissolved in 5 ml of physiological saline, and a nebulization catheter (OLYMPUS) was taken to the area bronchus and dispersed in the left lung in 10 times. The thing which passed 28 days after spraying was utilized as a chronic obstructive pulmonary disease model. The right lung was used as a control.

Administration of bFGF-containing gelatin microspheres to a beagle dog model In a beagle dog model in which 200 μg of bFGF-containing gelatin microspheres were agitated in 5 ml of physiological saline and the left lung was made into an emphysema model with elastase, a bronchoscope was used. In 10 times, it was sprayed diffusely. The average particle size of gelatin microspheres was adjusted to 10 μm. As a control, the same treatment was performed at the same dose or three times the dose using bFGF aqueous solution. Blood flow was measured by MRI 28 days after spraying elastase emphysema. As a control, a normal model dog and an elastase emphysema model dog were used.

  Using a 1.5T Vision MRI manufactured by Siemens, the beagle dog is anesthetized and then photographed in the supine position. A venous route was secured in the right internal carotid artery using a 16 gauge venous catheter, and MRI imaging was started after injecting 3 ml of dimeglumine gadopentetate (Magnevist, Nippon Schering). About 120 seconds after the injection of contrast medium, 170 coronal sections were imaged, and signal values in a limited area of the lung parenchyma were continuously measured and graphed. The results are shown in Fig. 1-3. When the ratio of the left and right signal values was measured from these graphs, it was 0.95-1 in the normal model, 0.6-0.8 in the elastase model, and 0.8-0.95 in the bFGF-administered elastase model. . That is, significant improvement in blood flow was observed by administering bFGF-containing gelatin microspheres. In the bFGF aqueous solution administration, even when 3 times the dose was administered, no improvement effect was observed compared to the bFGF-containing microsphere administration group.

FIG. 1 shows MRI signal values of left and right lung parenchyma in a normal model dog. FIG. 2 shows MRI signal values of left and right lung parenchyma in an elastase emphysema model dog. FIG. 3 shows MRI signal values of left and right lung parenchyma in an elastase emphysema model dog administered with bFGF-containing gelatin microspheres.

Claims (3)

A preparation for transpulmonary administration for treating emphysema, comprising gelatin microspheres containing a drug having an angiogenic action. Drugs with angiogenic activity include basic fibroblast growth factor, hepatocyte growth factor, other FGF, vascular endothelial growth factor, platelet-derived growth factor, transforming growth factor, angiopoietin, angiostatin, adrenomedullin, etc. The cell growth factor, interleukin, chemokine, and a gene encoding these, a biologically active low molecular weight substance, and a drug that induces angiogenesis-promoting drug are selected from the group consisting of Formulation for pulmonary administration. The preparation for transpulmonary administration according to claim 1, wherein the drug having an angiogenic action is a basic fibroblast growth factor.

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